RU2780425C2 - Neisseria meningitidis compositions and methods - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and is an immunogenic composition capable of causing an immune response against a strain of Neisseria meningitidis serological group A, Neisseria meningitidis serological group C, Neisseria meningitidis serological group W and Neisseria meningitidis serological group Y in humans, containing: a) a liquid composition containing: (i) the first lipidized polypeptide comprising the amino acid sequence presented in SEQ ID NO: 1; and (ii) a second lipidized polypeptide comprising the amino acid sequence presented in SEQ ID NO: 2 and aluminum; and (b) a lyophilized composition containing: i) Neisseria meningitidis serological group A (MenA) capsular saccharide conjugated with adipic acid dihydroside linker (ADH) by 1-cyano-4 tetrafluoroborate-dimethylaminopyridinium, where the specified linker is conjugated with tetanus toxoid (TT) by a carbodiimide chemical method (MenAAH-TT conjugate); ii)capsular saccharide of Neisseria meningitidis of serological group C (MEPs) conjugated with ADH-linker by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, where said linker is conjugated with tetanus toxoid (TT) by carbodiimide chemical method (MenCAH-TT conjugate); iii) capsular saccharide of Neisseria meningitidis of serological groups W135 (MenW), directly conjugated with tetanus toxoid (TT) by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate in the absence of a linker (MenW-TT conjugate); and iv) capsular saccharide of Neisseria meningitidis of serological group Y (MenY), directly conjugated with tetanus toxoid (TT) by means of 1-cyano-4-dimethylaminopyridinium tetrafluoroborate in the absence of a linker (MenY-TT conjugate), where the lyophilized composition is diluted with a liquid composition.

EFFECT: invention makes it possible to effectively induce an immune response against the strain of Neisseria meningitidis serological group A, Neisseria meningitidis serological group C, Neisseria meningitidis serological group W and Neisseria meningitidis serological group Y in humans.

15 cl, 16 dwg, 31 tbl, 22 ex

Description

Cross-reference to related applications

The present application claims priority to U.S. Provisional Application 62/452,963, filed January 31, 2017; U.S. provisional application 62/503295, filed May 6, 2017; 62/613945, filed January 5, 2018, and U.S. Provisional Application 62/623233, filed January 29, 2018. All of the above applications are hereby incorporated by reference in their entirety.

Field of invention

The present invention relates to Neisseria meningitidis compositions and methods.

Prerequisites for the creation of the invention

Neisseria meningitidis is a gram-negative encapsulated bacterium that can cause sepsis, meningitis, and death. N. meningitidis can be classified into at least 12 serogroups (including serogroups A, B, C, 29E, H, I, K, L, W-135 (now often referred to as W), X, Y, and Z ) based on chemically and antigenically distinct polysaccharide capsules. Strains with five serogroups (A, B, C, Y and W135) are responsible for the development of most diseases.

Meningococcal meningitis is a widespread disease that can kill children and young people within hours, despite the availability of antibiotics. Therefore, it is necessary to improve immunogenic compositions against meningococcal serogroups A, B, C, Y and W135 and/or X.

Currently, a cross-protective vaccine or composition effective against a wide range of MnB and isolates of meningococcal serogroups A, C, Y and W and/or X is not yet commercially available. For example, currently published data relating to a licensed multicomponent composition for protection against a disease associated with serogroup B did not show a direct bactericidal immune response against a variety of meningococcal strains that express capsular polysaccharides that are not serogroup B at least measure in adolescents. Accordingly, there is a need for a cross-protective vaccine or composition that is effective against various MnB and isolates of meningococcal serogroups A, C, Y and W and/or X and that has been tested worldwide as a vaccine against panel different or heterologous meningococcal strains (eg appropriate for different geographic regions).

Another object of the present invention is the development of improved regimens for the administration of meningococcal vaccine, in particular to children. Although the incidence of invasive meningococcal disease (IMD) varies with age, however, in most cases, it is highest in children aged 1 month to 1 year and reaches a second peak in young people. In the United States, between 1998 and 2007, the total incidence of meningococcal disease in children under 2 years of age was 3.9 per 100,000. In children aged 2 to 10 years, the incidence was 0.68 per 100,000, with 41% of cases in this age group were in children aged 2 to 3 years. Data from the Australian National Audit Office showed a peak incidence in children aged 4 years or less, and a second peak in adolescents and young adults, with approximately 85% of all cases related to serogroup B disease.

The essence of the invention

To meet these and other requirements, the present invention relates to the compositions of Neisseria meningitidis and to methods of their use.

The present inventors unexpectedly provided a composition comprising at least one factor H binding protein (fHBP) and at least one N. meningitidis capsular saccharide conjugate. This composition was surprisingly stable and elicited an immune response against strains expressing fHBP variants that are homologous to the fHBP variant in the multicomponent composition and an immune response against strains expressing fHBP variants that are heterologous to the fHBP variant in the multicomponent composition.

The composition contains the first lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1; a second lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2; Neisseria meningitidis serogroup A (MenA) capsular saccharide conjugated to a tetanus toxoid (TT) carrier protein; Neisseria meningitidis serogroup C (MenC) capsular saccharide conjugated to a tetanus toxoid (TT) carrier protein; Neisseria meningitidis serogroup W135 (MenW) capsular saccharide conjugated to a tetanus toxoid (TT) carrier protein; and Neisseria meningitidis serogroup Y (MenY) capsular saccharide conjugated to a tetanus toxoid (TT) carrier protein.

In one embodiment, the composition comprises a Neisseria meningitidis serogroup A (MenA) capsular saccharide conjugated to an adipic acid dihydrozide (ADH) linker via the chemical compound 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, wherein said linker is conjugated to a tetanus toxoid carrier protein. toxoid (TT) by carbodiimide chemistry (MenA _AH -TT conjugate); Neisseria meningitidis serogroup C (MenC) capsular saccharide conjugated to an ADH linker via the chemical compound 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, wherein said linker is conjugated to a tetanus toxoid (TT) carrier protein via a carbodiimide chemical method (MenC _AH conjugate - TT); Neisseria meningitidis serogroup W ₁₃₅ (MenW) capsular saccharide directly conjugated to a tetanus toxoid (TT) carrier protein via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry in the absence of a linker (MenW-TT conjugate); and Neisseria meningitidis serogroup Y (MenY) capsular saccharide directly conjugated to a tetanus toxoid (TT) carrier protein via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry in the absence of a linker (MenY-TT conjugate).

In one of its aspects, the present invention provides a kit comprising (a) a first composition comprising a lipidized MenB rLP2086 subfamily A polypeptide and a lipidized MenB rLP2086 subfamily B polypeptide; and (b) a second composition comprising a Neisseria meningitidis serogroup A (MenA) capsular saccharide conjugated to a tetanus toxoid (TT) carrier protein; Neisseria meningitidis serogroup C (MenC) capsular saccharide conjugated to a tetanus toxoid (TT) carrier protein; Neisseria meningitidis serogroup W ₁₃₅ (MenW) capsular saccharide conjugated to a tetanus toxoid (TT) carrier protein; and Neisseria meningitidis serogroup Y (MenY) capsular saccharide conjugated to a tetanus toxoid (TT) carrier protein. In one embodiment of the invention, the first composition is a liquid composition and the second composition is a lyophilized composition. In another embodiment of the invention, this kit also does not include any of the following immunogenic compositions: MENACTRA(R), MENVEO(R), ADACEL(R), HAVRIX(R), GARDASIL(R), REPEVAX, or any combinations thereof. In one of the embodiments of the invention, the set includes any of ibuprofen, paracetamol and amoxicillin.

In one of its aspects, the present invention relates to an immunogenic composition comprising a liquid composition containing: (i) a first lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1; and (ii) a second lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2; and a lyophilized composition comprising: Neisseria meningitidis serogroup A (MenA) capsular saccharide conjugated to an adipic acid dihydrozide (ADH) linker via the chemical compound 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, wherein said linker is conjugated to a tetanus toxoid (TT) carrier protein ) by carbodiimide chemistry (MenA _AH -TT conjugate); Neisseria meningitidis serogroup C (MenC) capsular saccharide conjugated to an ADH linker via the chemical compound 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, wherein said linker is conjugated to a tetanus toxoid (TT) carrier protein via a carbodiimide chemical method (MenC _AH conjugate - TT); Neisseria meningitidis serogroup W ₁₃₅ (MenW) capsular saccharide directly conjugated to a tetanus toxoid (TT) carrier protein via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry in the absence of a linker (MenW-TT conjugate); and Neisseria meningitidis serogroup Y (MenY) capsular saccharide directly conjugated to a tetanus toxoid (TT) carrier protein via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry in the absence of a linker (MenY-TT conjugate). In one of the embodiments of the invention, the lyophilized composition is diluted with a liquid composition.

In another aspect, the present invention provides a method for inducing a bactericidal immune response against a strain of Neisseria meningitidis serogroup X. In some embodiments, the method comprises administering to a human a composition comprising an fHBP protein. In some embodiments, the method includes administering to a human a composition comprising a polypeptide comprising an amino acid sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% , 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99%, or 99.9% identical to the amino acid sequence shown in any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61 and SEQ ID NO: 62. In some embodiments, the method includes administering to a human a composition comprising a first lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1; and a second lipidized polypeptide containing the amino acid sequence shown in SEQ ID NO: 2.

In another aspect, the present invention relates to a method of inducing a bactericidal immune response against a strain of Neisseria meningitidis serogroup X. The method includes administering to a human a composition that contains a first lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1; a second lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2; Neisseria meningitidis serogroup A (MenA) capsular saccharide conjugated to a tetanus toxoid (TT) carrier protein; Neisseria meningitidis serogroup C (MenC) capsular saccharide conjugated to a tetanus toxoid (TT) carrier protein; Neisseria meningitidis serogroup W135 (MenW) capsular saccharide conjugated to a tetanus toxoid (TT) carrier protein; and Neisseria meningitidis serogroup Y (MenY) capsular saccharide conjugated to a tetanus toxoid (TT) carrier protein.

In one of its aspects, the present invention relates to a method for generating an immune response in a patient of any age. The method includes administering to a human a composition that contains a first lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1; a second lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2. In one embodiment, the composition also comprises polysorbate-80. In one of the embodiments of the invention, the composition also includes aluminum. In one of the embodiments of the invention, the composition also includes histidine. In one of the embodiments of the invention, the composition also includes sodium chloride. In one of the embodiments of the invention, the composition also includes polysorbate-80, aluminum, histidine and sodium chloride. In another embodiment of the invention, the composition also includes a capsular saccharideNeisseria meningitidis serogroup A (MenA) conjugated to a tetanus toxoid (TT) carrier protein; capsular saccharideNeisseria meningitidis serogroup C (MenC) conjugated to a tetanus toxoid (TT) carrier protein; capsular saccharideNeisseria meningitidis serogroup W135 (MenW) conjugated to a tetanus toxoid (TT) carrier protein; and capsular saccharideNeisseria meningitidis serogroup Y (MenY) conjugated to a tetanus toxoid carrier protein (TT).

Brief description of the drawings

Fig. 1 - Combined IOC-WEC chromatograms for the bivalent composition MnB rLP2086 in the absence and presence of the composition MenACWY-TT, as described in Example 6.

Fig. 2 - Combined RP-HPLC chromatograms showing that the presence of the MenACWY-TT composition does not affect the purity evaluation of the bivalent MnB rLP2086 composition as described in Example 7.

Fig. 3 - Combined RP-HPLC chromatograms showing the ratio of purity and peaks of the rLP2086 protein in the combined MenABCWY formulation as described in Example 14.

Fig. 4A - Primary amino acid sequence of the MnB protein rLP2086 subfamily A A05 and

Fig. 4B - Primary structure of the MnB rLP2086 subfamily A A05 protein.

Fig. 5A - Primary amino acid sequence of the MnB protein rLP2086 subfamily B B01 and

Fig. 5B - Primary structure of the MnB protein rLP2086 subfamily B B01.

Fig. 6A - Amino acid sequences of factor H binding protein (fHBP) B16 (SEQ ID NO: 26) of N. meningitidis serogroup A strain (fHBP B16 variant (PMB3257, MenA);

Fig. 6B - fHBP A10 (SEQ ID NO: 27) strain of N. meningitidis serogroup C (variant fHBP A10 (PMB5208, MenC and PMB5523, MenW);

Fig. 6C - fHBP A19 (SEQ ID NO: 28) strain of N. meningitidis serogroup W (variant fHBP A19 (PMB5248, MenW);

Fig. 6D - fHBP A10 (SEQ ID NO: 27) strain of N. meningitidis serogroup W (variant A10 (PMB5523, MenW);

Fig. 6E - fHBP B47 (SEQ ID NO: 29) strain of N. meningitidis serogroup Y (variant B47 (PMB5187, MenY);

Fig. 6F - fHBP B49 (SEQ ID NO: 30) strain of N. meningitidis serogroup X (variant B49 (PMB5540, MenX).

Fig. 7 - Serum bactericidal activity correlated with protection against meningococcal disease. Titer ≥1:4 in serum bactericidal activity tests using human complement (hSBA) is an established correlate of protection against meningococcal disease.

Fig. 8 - Selection of test strains MenA, C, W, Y and X.

Fig. 9 - Schematic representation of the relevant groups participating in the clinical trial, from which a subpopulation of test sera was randomly selected.

Fig. ten -Distribution expression levels of surface FHbp (MFI) determined in experiments by flow cytometry using mAb MN 994-11 that reacts with FHbp. FHbp surface expression for each of the strains of a particular serogroup is shown as black dots, and FHbp surface expression levels for selected test strains of each serogroup are shown with a colored asterisk.

Fig. 11 - hSBA response rate (percentage of individuals with hSBA titers ≥1:8) for MenA PMB3257 (B16). Response rates and 95% confidence intervals are shown for sera collected before immunization (month 0) and 1 month after administration of 1, 2 and 3 doses of MenBFHbp. The geometric mean of the obtained titers (GMT) were 2, 3, 4 and 5, respectively. The response rates for individuals in the positive control group were 3% before vaccination and 97% one month after MCV4 administration. GMT for the positive control group were 2 and 95, respectively.

Fig. 12 - hSBA response rate (percentage of individuals with hSBA titers ≥1:8) for MenC PMB5208 (A10). Response rates and 95% confidence intervals are shown for sera collected before immunization (month 0) and 1 month after administration of 1, 2, and 3 doses of MenBFHbp. The resulting GMTs were 4, 8, 12 and 29, respectively. The response rates for individuals in the positive control group were 20% before vaccination and 90% 1 month after MCV4 administration. GMT for the positive control group were 3 and 119, respectively.

Fig. 13 - hSBA response rate (percentage of individuals with hSBA titers ≥1:8) for MenW PMB5248 (A19). Response rates and 95% confidence intervals are shown for sera collected before immunization (month 0) and 1 month after administration of 1, 2 and 3 doses of MenBFHbp. The resulting GMTs were 4, 18, 47 and 77, respectively. The response rates for individuals in the positive control group were 40% before vaccination and 97% 1 month after MCV4 administration. GMT for the positive control group were 5 and 88, respectively.

Fig. fourteen- hSBA response rate (percentage of individuals with hSBA titers ≥1:8) for MenW PMB5523 (A10). Response rates and 95% confidence intervals are shown for sera collected before immunization (month 0) and 1 month after administration of 1, 2 and 3 doses of MenBFHbp. The resulting GMTs were 7, 15, 21 and 42, respectively. The response rates for individuals in the positive control group were 55% before vaccination and 97% 1 month after MCV4 administration. GMT for the positive control group were 8 and 60, respectively.

Fig. 15 - hSBA response rate (percentage of individuals with hSBA titers ≥1:8) for MenY PMB5187 (B47). Response rates and 95% confidence intervals are shown for sera collected before immunization (month 0) and 1 month after administration of 1, 2 and 3 doses of MenBFHbp. The resulting GMTs were 3, 7, 31 and 58, respectively. The response rates for individuals in the positive control group were 13% before vaccination and 97% 1 month after MCV4 administration. GMT for the positive control group were 3 and 79, respectively.

Fig. 16 - hSBA response rate (percentage of individuals with hSBA titers ≥1:8) for MenX PMB5540 (B49). Response rates and 95% confidence intervals are shown for sera collected before immunization (month 0) and 1 month after administration of 1, 2 and 3 doses of MenBFHbp. The resulting GMTs were 2, 3, 7 and 20, respectively. The response rates for individuals in the positive control group were 0% before vaccination and 0% one month after MCV4 administration. The GMT for the positive control group were 2 and 2, respectively.

Sequence IDs

SEQ ID NO: 1 is the amino acid sequence for N. meningitidis recombinant polypeptide antigen, serogroup B, variant 2086 A05.

SEQ ID NO: 2 is the amino acid sequence for N. meningitidis recombinant polypeptide antigen, serogroup B, variant 2086 B01.

SEQ ID NO: 3 is the amino acid residues at positions 1-4 of SEQ ID NO: 1 and SEQ ID NO: 2.

SEQ ID NO: 4 is the amino acid sequence of the N-terminal recombinant Neisseria subfamily A polypeptide LP2086 (rLP2086) (A05).

SEQ ID NO: 5 is the amino acid sequence of the N-terminal polypeptide of Neisseria subfamily A LP2086 M98250771 (A05).

SEQ ID NO: 6 is the amino acid sequence of N. meningitidis , serogroup B, variant 2086 B153.

SEQ ID NO: 7 is the amino acid sequence of N. meningitidis , serogroup B, variant 2086 A04.

SEQ ID NO: 8 is the amino acid sequence of N. meningitidis , serogroup B, variant 2086 A05.

SEQ ID NO: 9 is the amino acid sequence of N. meningitidis , serogroup B, variant 2086 A12.

SEQ ID NO: 10 is the amino acid sequence of N. meningitidis , serogroup B, variant 2086 A22.

SEQ ID NO: 11 is the amino acid sequence of N. meningitidis , serogroup B, variant 2086 B02.

SEQ ID NO: 12 is the amino acid sequence of N. meningitidis , serogroup B, variant 2086 B03.

SEQ ID NO: 13 is the amino acid sequence of N. meningitidis , serogroup B, variant 2086 B09.

SEQ ID NO: 14 is the amino acid sequence of N. meningitidis , serogroup B, variant 2086 B22.

SEQ ID NO: 15 is the amino acid sequence of N. meningitidis , serogroup B, variant 2086 B24.

SEQ ID NO: 16 is the amino acid sequence of N. meningitidis , serogroup B, variant 2086 B44.

SEQ ID NO: 17 is the amino acid sequence of N. meningitidis , serogroup B, variant 2086 B16.

SEQ ID NO: 18 is the amino acid sequence of N. meningitidis , serogroup B, variant 2086 A07.

SEQ ID NO: 19 is the amino acid sequence of N. meningitidis , serogroup B, variant 2086 A19.

SEQ ID NO: 20 is the amino acid sequence of N. meningitidis , serogroup B, variant 2086 A06.

SEQ ID NO: 21 is the amino acid sequence of N. meningitidis , serogroup B, variant 2086 A15.

SEQ ID NO: 22 is the amino acid sequence of N. meningitidis , serogroup B, variant 2086 A29.

SEQ ID NO: 23 is the amino acid sequence of N. meningitidis , serogroup B, variant 2086 B15.

SEQ ID NO: 24 is the amino acid sequence of the N-terminal recombinant Neisseria subfamily A polypeptide LP2086 (rLP2086) (B01).

SEQ ID NO: 25 is the amino acid sequence of the N-terminal polypeptide of Neisseria subfamily B LP2086 CDC-1573 (B01).

SEQ ID NO: 26 is the amino acid sequence of serogroup A N. meningitidis strain expressing factor H binding protein (fHBP) B16.

SEQ ID NO: 27 is the amino acid sequence of a N. meningitidis serogroup C strain expressing fHBP A10. SEQ ID NO: 27 is also the amino acid sequence of a N. meningitidis serogroup W strain expressing fHBP A10.

SEQ ID NO: 28 is the amino acid sequence of a N. meningitidis strain of serogroup W expressing fHBP A19.

SEQ ID NO: 29 is the amino acid sequence of serogroup Y N. meningitidis strain expressing fHBP B47.

SEQ ID NO: 30 is the amino acid sequence of a N. meningitidis strain of serogroup X expressing fHBP B49.

SEQ ID NO: 31 is the amino acid sequence of non-lipidized N. meningitidis , serogroup B, variant 2086 B16.

SEQ ID NO: 32 is the amino acid sequence of non-lipidized N. meningitidis , serogroup B, variant 2086 A07.

SEQ ID NO: 33 is the amino acid sequence of non-lipidized N. meningitidis , serogroup B, variant 2086 A19.

SEQ ID NO: 34 is the amino acid sequence of non-lipidized N. meningitidis , serogroup B, variant 2086 A06.

SEQ ID NO: 35 is the amino acid sequence of non-lipidized N. meningitidis , serogroup B, variant 2086 A15.

SEQ ID NO: 36 is the amino acid sequence of non-lipidized N. meningitidis , serogroup B, variant 2086 A29.

SEQ ID NO: 37 is the amino acid sequence of non-lipidized N. meningitidis , serogroup B, variant 2086 B15.

SEQ ID NO: 38 is the amino acid sequence of a non-lipidized serogroup A strain of N. meningitidis expressing factor H binding protein (fHBP) B16.

SEQ ID NO: 39 is the amino acid sequence of a non-lipidized N. meningitidis strain of serogroup C expressing fHBP A10. SEQ ID NO: 39 is also the amino acid sequence of a non-lipidized N. meningitidis strain of serogroup W expressing fHBP A10.

SEQ ID NO: 40 is the amino acid sequence of a non-lipidized N. meningitidis strain of serogroup W expressing fHBP A19.

SEQ ID NO: 41 is the amino acid sequence of a non-lipidized serogroup Y strain of N. meningitidis expressing fHBP B47.

SEQ ID NO: 42 is the amino acid sequence of a non-lipidized N. meningitidis strain of serogroup X expressing fHBP B49.

SEQ ID NO: 43 is the amino acid sequence of non-lipidized N. meningitidis , serogroup B, variant 2086 B44.

SEQ ID NO: 44 is the amino acid sequence of non-lipidized N. meningitidis , serogroup B, variant 2086 B09.

SEQ ID NO: 45 is the amino acid sequence of N. meningitidis , serogroup B, variant 2086 B09.

SEQ ID NO: 46 is the amino acid sequence of non-lipidized N. meningitidis , serogroup B, variant 2086 A05.

SEQ ID NO: 47 is the amino acid sequence of non-lipidized N. meningitidis , serogroup B, variant 2086 B01.

SEQ ID NO: 48 is the amino acid sequence of N. meningitidis , serogroup B, variant 2086 B01, which includes the N-terminal Cys at amino acid position 1.

SEQ ID NO: 49 is the amino acid sequence of N. meningitidis , serogroup B, variant 2086 B15, which includes the N-terminal Cys at amino acid position 1.

SEQ ID NO: 50 is the amino acid sequence of N. meningitidis , serogroup B, variant 2086 B16, which includes the N-terminal Cys at amino acid position 1.

SEQ ID NO: 51 is the amino acid sequence of N. meningitidis , serogroup B, variant 2086 B22.

SEQ ID NO: 52 is the amino acid sequence of N. meningitidis , serogroup B, variant 2086 A22.

SEQ ID NO: 53 is the amino acid sequence of non-lipidized N. meningitidis , serogroup B, variant 2086 A12.

SEQ ID NO: 54 is the amino acid sequence of non-lipidized N. meningitidis , serogroup B, variant 2086 A22.

SEQ ID NO: 55 is the amino acid sequence of N. meningitidis , serogroup B, variant 2086 A62, which includes the N-terminal Cys at amino acid position 1.

SEQ ID NO: 56 is the amino acid sequence of non-lipidized N. meningitidis , serogroup B, variant 2086 A62.

SEQ ID NO: 57 is the amino acid sequence of N. meningitidis , serogroup B, variant 2086 A29, which includes the N-terminal Cys at amino acid position 1.

SEQ ID NO: 58 is the amino acid sequence of non-lipidized N. meningitidis , serogroup B, variant 2086 B22.

SEQ ID NO: 59 is the amino acid sequence of N. meningitidis , serogroup B, variant 2086 A05.

SEQ ID NO: 60 is the amino acid sequence of non-lipidized N. meningitidis , serogroup B, variant 2086 A05.

SEQ ID NO: 61 is the amino acid sequence of N. meningitidis , serogroup B, variant 2086 B24.

SEQ ID NO: 62 is the amino acid sequence of N. meningitidis , serogroup B, variant 2086 B24.

Detailed description of the invention

The present inventors unexpectedly provided a composition comprising at least one factor H binding protein (fHBP) and at least one N. meningitidis capsular saccharide conjugate. This composition was surprisingly stable and elicited an immune response against strains expressing fHBP variants that are homologous to the fHBP variant in the multicomponent composition and an immune response against strains expressing fHBP variants that are heterologous to the fHBP variant in the multicomponent composition. The inventors of the present invention have also unexpectedly found that the fHBP polypeptide effectively elicits an immune response in children, for example, in children aged 12 months and above. In addition, the authors unexpectedly found that the fHBP polypeptide effectively elicits an immune response against a strain of N. meningitidis serogroup X.

The authors of the present invention unexpectedly obtained a composition comprising (a) a first lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1; (b) a second lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2; (c) Neisseria meningitidis serogroup A (MenA) capsular saccharide conjugated to an adipic acid dihydrazide (ADH) linker via the chemical compound 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, wherein said linker is conjugated to a tetanus toxoid (TT) carrier protein via a carbodiimide chemical method (MenA _AH -TT conjugate); (d) Neisseria meningitidis serogroup C (MenC) capsular saccharide conjugated to an ADH linker via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry, wherein said linker is conjugated to a tetanus toxoid (TT) carrier protein via a carbodiimide chemistry (conjugate _MenCAH -TT); (e) Neisseria meningitidis serogroup W ₁₃₅ (MenW) capsular saccharide directly conjugated to a tetanus toxoid (TT) carrier protein via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry in the absence of a linker (MenW-TT conjugate); (f) Neisseria meningitidis serogroup Y (MenY) capsular saccharide directly conjugated to a tetanus toxoid (TT) carrier protein via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry in the absence of a linker (MenY-TT conjugate). The composition includes a lyophilized composition of MenACWY-TT, which, as was unexpectedly found, can be easily diluted with a liquid bivalent composition of MnB rLP2086, where such a composition is contained in one vessel. The present inventors found that the MenACWY-TT lyophilized formulation and the rLP2086 liquid divalent MnB formulation were compatible and stable after reconstitution for at least 24 hours at room temperature.

In addition, the inventors of the present invention have also found that the bivalent composition of MnB rLP2086 produces bactericidal antibodies not only against N. meningitidis serogroup B, but also against N. meningitidis of all serogroups except B. For example, the bivalent composition of MnB rLP2086 produces bactericidal antibodies against N. meningitidis serogroups of at least A, C, W, Y, and X. The bivalent composition of MnB rLP2086 was unexpectedly found to produce bactericidal antibodies against N. meningitidis serogroup X, indicating that the bivalent the MnB rLP2086 composition elicits a broad spectrum cross-reactive bactericidal immune response in humans against Neisseria meningitidis of at least two different serogroups.

In addition, the present inventors surprisingly detected an immune response assessed by a serum bactericidal assay using human complement (hSBA) performed using 4 primary test strains of Neisseria meningitidis serogroup B (MnB), 2 of which expressed the subfamily protein A LP2086 and 2 expressed the LP2086 subfamily B protein, and this analysis was performed 1 month after the third vaccination with divalent rLP2086 in healthy individuals aged ≥24 months to <4 years at baseline. The present inventors unexpectedly detected an immune response assessed by an hSBA assay performed using 4 primary MnB strains tested, 2 of which expressed the LP2086 subfamily A protein and 2 of which expressed the LP2086 subfamily B protein, and this analysis was performed after 1 month after the third vaccination with bivalent rLP2086 in healthy subjects aged ≥4 years to <10 years at baseline. The present inventors also unexpectedly detected an immune response assessed by hSBA using 4 primary test MnB strains, 2 of which expressed LP2086 subfamily A protein and 2 expressed LP2086 subfamily B protein, and this analysis was performed after 1 month after the third vaccination with bivalent rLP2086 in healthy individuals aged ≥24 months to <10 years at the beginning of the study (ie, in the pooled age stratified group). The present inventors also unexpectedly detected an immune response assessed by hSBA using 4 primary MnB strains tested, 2 of which expressed LP2086 subfamily A protein and 2 expressed LP2086 subfamily B protein, and this analysis was performed 1 month after the second vaccination and 6 months after the third vaccination with bivalent rLP2086, in healthy individuals aged ≥24 months to <4 years at baseline, in healthy individuals aged ≥4 years to <10 years at baseline, and in a pooled age stratified group . The immune response was also described by additional endpoints assessed by hSBA conducted using 4 primary MnB strains tested, 2 of which expressed the LP2086 subfamily A protein and 2 expressed the LP2086 subfamily B protein, and these analyzes were performed at specific time points in healthy individuals aged ≥24 months to <4 years at study entry, in healthy individuals aged ≥4 years to <10 years at baseline, and in a pooled age stratified group.

In addition, the present inventors surprisingly detected an immune response assessed by a serum bactericidal assay using human complement (hSBA) performed using 4 primary test strains of Neisseria meningitidis serogroup B (MnB), 2 of which expressed the subfamily protein A LP2086 and 2 expressed the LP2086 subfamily B protein, and this analysis was performed 1 month after the third vaccination with bivalent rLP2086 in healthy children aged 12 to <18 months at baseline. The present inventors also unexpectedly detected an immune response assessed by hSBA using 4 primary MnB strains tested, 2 of which expressed the LP2086 subfamily A protein and 2 of which expressed the LP2086 subfamily B protein, and this analysis was performed 1 month after the third vaccination with bivalent rLP2086 in healthy children aged 18 to <24 months at baseline. The present inventors also unexpectedly detected an immune response assessed by hSBA using 4 primary MnB strains tested, 2 of which expressed the LP2086 subfamily A protein and 2 of which expressed the LP2086 subfamily B protein, and this analysis was performed 1 month after the third vaccination with bivalent rLP2086 in healthy children aged 12 to <24 months at baseline (i.e., in both pooled age stratified groups). The present inventors also unexpectedly detected an immune response assessed by hSBA using 4 primary MnB strains tested, 2 of which expressed LP2086 subfamily A protein and 2 expressed LP2086 subfamily B protein, and this analysis was performed 1 month after the second vaccination and at least 6 months after the third vaccination, in healthy children aged 12 to <18 months and 18 to <24 months at baseline and in both pooled age stratified groups. For example, hSBA can be given at any time, including 12, 24, 36, and 48 months after the third vaccination in healthy children aged 12 to <18 months and 18 to <24 months at baseline and both pooled age groups. stratified groups. The immune response was also described by additional endpoints assessed by hSBA performed using 4 primary MnB strains tested, 2 of which expressed LP2086 subfamily A protein and 2 expressed LP2086 subfamily B protein, and this analysis was performed 1 month after the second vaccination and at least 1 month after the third vaccination with bivalent rLP2086 in healthy children aged 12 to <18 months at baseline and 18 to <24 months at baseline and both pooled age stratified groups. For example, hSBA can be performed at any time, including 6, 12, 24, 36, and 48 months after the third vaccination with bivalent rLP2086 in healthy children aged 12 to <18 months and 18 to <24 months at baseline and in both combined age stratified groups. Immune response was also described by additional endpoints assessed by hSBA conducted using secondary test strains of MnB expressing LP2086 subfamily A and B proteins at 1 month after the second vaccination and at least 1 month after the third vaccination in healthy children aged 12 to <18 months and 18 to <24 months at baseline and in both pooled age stratified groups. For example, hSBA can be given at any time, including 6, 12, 24, 36, and 48 months after the third vaccination of healthy children aged 12 to <18 months and 18 to <24 months at baseline and in both pooled age stratified groups.

Accordingly, in one of its aspects, the present invention relates to a method for generating an immune response in a patient of any age. In some embodiments, the human is at least 4 weeks old, 5 weeks old, 6 weeks old, 7 weeks old, 8 weeks old, 9 weeks old, 10 weeks old, 11 weeks old, or 12 weeks old. For example, in preferred embodiments of the invention, the age of the person is at least 6 weeks. As known to those skilled in the art, a meningococcal group A, C, W-135 and Y conjugate vaccine such as NIMENRIX® is suitable for children at least six weeks of age and can be administered to anyone six weeks of age or older. In some embodiments of the invention, the age of the person is at least 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months. Thus, for example, in a preferred embodiment of the invention, the age of the person is at least 12 months. In one of the embodiments of the invention, the age of the person is at least 12 to 18 months. In another aspect, the present invention relates to a method for generating an immune response in a patient at least 18 months of age. In one of the embodiments of the invention, the age of the person is from 18 to 24 months. In another aspect, the present invention relates to a method for generating an immune response in a patient at least 24 months of age. In one of the embodiments of the invention, the age of the person is from 24 to 10 years. In another aspect, the present invention relates to a method for generating an immune response in a patient aged 10 years and above. In another aspect, the present invention relates to a method for generating an immune response in a patient aged 10 to 25 years. The method includes administering to a human a composition comprising a first lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1; and a second lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2. In one embodiment, the composition also comprises polysorbate-80. In one of the embodiments of the invention, the composition also includes aluminum. In one of the embodiments of the invention, the composition also includes histidine. In one of the embodiments of the invention, the composition also includes sodium chloride. In one of the embodiments of the invention, the composition also includes polysorbate-80, aluminum, histidine and sodium chloride. In another embodiment of the invention, the composition also includes a capsular saccharideNeisseria meningitidis serogroup A (MenA) conjugated to a tetanus toxoid (TT) carrier protein; capsular saccharideNeisseria meningitidis serogroup C (MenC) conjugated to a tetanus toxoid (TT) carrier protein; capsular saccharideNeisseria meningitidis serogroup W135 (MenW) conjugated to a tetanus toxoid (TT) carrier protein; and capsular saccharideNeisseria meningitidis serogroup Y (MenY) conjugated to a tetanus toxoid carrier protein (TT). As known to those skilled in the art, a meningococcal group A, C, W-135 and Y conjugate vaccine such as NIMENRIX® is suitable for children at least six weeks of age and can be administered to anyone six weeks of age or older.

Additional description of representative compositions follows.

Composition and vaccine

The present inventors have found that a composition comprising fHBP elicits an effective immune response in a human at least 12 months of age. The composition also elicits an immune response against a strain of N. meningitidis serogroup X. In addition, we unexpectedly obtained a composition comprising at least one factor H binding polypeptide (fHBP) and at least one N. meningitidis capsular saccharide conjugate. This composition was surprisingly stable and capable of generating an immune response against strains expressing fHBP variants that are homologous to the fHBP variant in the multicomponent composition and an immune response against strains expressing fHBP variants that are heterologous to the fHBP variant in the multicomponent composition. In one of the embodiments of the invention, the composition includes any fHBP, such as, for example, any of the following polypeptides: B24, B16, B44, A22, B03, B09, A12, A19, A05, A07, A06, A15, A29, B01, A62, B15 and any combination thereof. Preferably, the composition comprises a combination of A05 and B01 polypeptides. In another preferred embodiment of the invention, the composition comprises a combination of B24 and A05 polypeptides. In another embodiment of the invention, the composition comprises a combination of A05, A12, B09, and B44 polypeptides. In one of the embodiments of the invention, the composition includes lipidized fHBP. In one embodiment of the invention, the composition does not include non-lipidized fHBP.

In another embodiment, the composition comprises a non-lipidized fHBP, such as any of the non-lipidized fHBPs described in International Patent Application Publication No. WO2012/032489, US Patent Publication No. US20120093852, in International Patent Application Publication No. WO2013/132452 and US Patent Publication No. US20160030543, which are incorporated herein by reference in their entirety. In one embodiment, the composition comprises at least one non-lipidized fHBP and at least one lipidized fHBP.

In some embodiments of the invention, the composition includes a polypeptide, the amino acid sequence of which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99%, or 99.9% identical to the amino acid sequence shown in any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO : 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27 , SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO : 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO : 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61 and SEQ ID NO: 62.

The inventors of the present invention have also surprisingly found that the liquid divalent formulation of MnB rLP2086 can be readily reconstituted to form a lyophilized MenACWY-TT formulation and that the combined formulation is compatible and stable.

In one of its aspects, the present invention relates to compositions against Neisseria meningitidis . The composition includes (a) a first lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1; (b) a second lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2; (c) Neisseria meningitidis serogroup A (MenA) capsular saccharide conjugated to an adipic acid dihydrazide (ADH) linker via the chemical compound 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, wherein said linker is conjugated to a tetanus toxoid (TT) carrier protein via a carbodiimide chemical method (MenA _AH -TT conjugate); (d) Neisseria meningitidis serogroup C (MenC) capsular saccharide conjugated to an ADH linker via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry, wherein said linker is conjugated to a tetanus toxoid (TT) carrier protein via a carbodiimide chemistry (conjugate _MenCAH -TT); (e) Neisseria meningitidis serogroup W ₁₃₅ (MenW) capsular saccharide directly conjugated to a tetanus toxoid (TT) carrier protein via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry in the absence of a linker (MenW-TT conjugate); (f) Neisseria meningitidis serogroup Y (MenY) capsular saccharide directly conjugated to a tetanus toxoid (TT) carrier protein via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry in the absence of a linker (MenY-TT conjugate).

In another aspect, the present invention provides a composition comprising a combination of a divalent MnB rLP2086 composition and a MenACWY-TT composition. rLP2086 MnB bivalent composition means a composition comprising a single N. meningitidis polypeptide component that induces an effective broad spectrum protective immune response against a plurality of N. meningitidis serogroup B strains. In particular, in one embodiment, the rLP2086the bivalent MnB composition comprises a subfamily A MnB rLP2086 (SEQ ID NO: 1) and MnB subfamily B protein rLP2086 (SEQ ID NO: 2). In one embodiment of the invention, the composition does not include a fusion protein. In one of the embodiments of the invention, the composition does not include a chimeric protein. In one of the embodiments of the invention, the composition does not include a hybrid protein. In one of the embodiments of the invention, the composition also does not include a peptide fragment. In another embodiment, the composition also does not include a Neisserial.beta . polypeptide that is not an fHBP. Thus, for example, in one embodiment of the invention, the composition does not include the PorA protein. In another embodiment of the invention, the composition does not include the NadA protein. In another embodiment of the invention, the composition also does not include Neisserial heparin-binding antigen (NHBA). In another embodiment of the invention, the composition also does not include the outer membrane vesicle Neisserial (OMV). In a preferred embodiment of the invention, the composition also does not include antigens other than the first polypeptide and the second polypeptide. In a preferred embodiment of the invention, the divalent composition of MnB rLP2086 also includes polysorbate-80. In one of the embodiments of the invention, the divalent composition of MnB rLP2086 also includes a histidine buffer. In one of the embodiments of the invention, the divalent composition of MnB rLP2086 also includes sodium chloride. In one of the embodiments of the invention, the divalent composition of MnB rLP2086 also includes aluminum phosphate. In one embodiment, the bivalent MnB rLP2086 composition also includes polysorbate-80, histidine buffer, sodium chloride, and aluminum phosphate. Preferably, the rLP2086 bivalent MnB formulation is a liquid formulation wherein the polypeptides are formulated as 120 μg/mL/subfamily in 10 mM histidine buffer, pH 6.0, 150 mM sodium chloride (NaCl) with 0.5 mg/mL aluminum phosphate (AlPO ₄ ), and also includes 0.018 mg of polysorbate-80 at a dose of 0.5 ml.

Composition MenACWY-TT means a composition that includes purified capsular polysaccharides of Neisseria meningitidis serogroups A, C, W-135 and Y, each independently conjugated to TT in the ratios (TT:polysaccharide) ~3, ~3, ~1.5 and ~1.3, respectively. In particular, the composition comprises: (c) Neisseria meningitidis serogroup A (MenA) capsular saccharide conjugated to an adipic acid dihydrazide (ADH) linker via the chemical compound 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, wherein said linker is conjugated to a tetanus toxoid carrier protein. toxoid (TT) by carbodiimide chemistry (MenA _AH -TT conjugate); (d) Neisseria meningitidis serogroup C (MenC) capsular saccharide conjugated to an ADH linker via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry, wherein said linker is conjugated to a tetanus toxoid (TT) carrier protein via a carbodiimide chemistry (conjugate _MenCAH -TT); (e) Neisseria meningitidis serogroup W ₁₃₅ (MenW) capsular saccharide directly conjugated to a tetanus toxoid (TT) carrier protein via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry in the absence of a linker (MenW-TT conjugate); (f) Neisseria meningitidis serogroup Y (MenY) capsular saccharide directly conjugated to a tetanus toxoid (TT) carrier protein via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry in the absence of a linker (MenY-TT conjugate). Preferably, the MenACWY-TT composition is presented as a lyophilized powder.

The MenA _AH -TT, MenC _AH -TT, MenW-TT, and MenY-TT conjugates were prepared by the following steps: preparation of a polysaccharide drug intermediate, preparation of a drug intermediate with TT, microfluidization of the polysaccharide, derivatization of the polysaccharide (Methods for the preparation of MenAAH- TT and MenCAH-TT), further purification of TT and conjugation of individual polysaccharides with TT.

For the MenA _AH -TT conjugate, the MenA polysaccharide is first microfluidized to reduce the size and viscosity of the molecule, and then activated by cyanylation with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP). Activated MenA is derivatized with adipic acid dihydrazide (ADH) to form MenA _AH . MenA _AH and tetanus toxoid (TT) are coupled by carbodiimide-mediated condensation (according to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) coupling method) to give a MenA _AH -tetanus toxoid conjugate (MenA _AH - TT).

As for the MenC _AH -TT conjugate, the MenC polysaccharide is first microfluidized to reduce the size and viscosity of the molecule, and then activated by cyanylation with CDAP. Activated MenC is derivatized with adipic acid dihydrazide (ADH) to form MenC _AH . MenC _AH and TT are coupled by carbodiimide-mediated condensation according to the EDAC coupling procedure to give a MenC _AH -tetanus toxoid conjugate (MenC _AH -TT).

For the MenW-TT conjugate, the MenW polysaccharide is first subjected to microfluidization to reduce the size and viscosity of the molecule, and then activated by cyanylation with CDAP. Activated MenW is directly coupled to TT to produce MenW-tetanus toxoid (MenW-TT).

For the MenY-TT conjugate, the MenY polysaccharide is first subjected to microfluidization to reduce the size and viscosity of the molecule, and then activated by cyanylation with CDAP. Activated MenY is directly coupled to TT to produce MenY-tetanus toxoid (MenY-TT).

In accordance with another aspect of the invention, the authors have unexpectedly found that polypeptide antigens derived from a maximum of two strainsN. meningitidis serogroup B, induce a broad range of effective protective immune responses against multiple strainsN. meningitidis serogroup B. Accordingly, in one embodiment of the invention, the composition also does not include a polypeptide that is not derived from strain fHBP M98250771 subfamily AN. meningitidis serogroup B and/or from an fHBP strain CDC1573 subfamily BN. meningitidis serogroup B.

In one embodiment, the composition also does not include a polypeptide whose sequence is less than 100% identical to SEQ ID NO: 1. In another embodiment, the composition also does not include a polypeptide whose sequence is less than 100% identical to SEQ ID NO: 2. Thus, for example, the composition also does not include a polypeptide whose sequence is less than 100% identical to the full-length SEQ ID NO: 1 and/or SEQ ID NO: 2.

In one of the embodiments of the invention, the composition also includes polysorbate-80, aluminum, histidine and sodium chloride. In one embodiment, the composition comprises approximately 60 μg of a first lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1, approximately 60 μg of a second lipidized polypeptide containing the amino acid sequence shown in SEQ ID NO: 2, polysorbate-80 in a molar ratio of 2.8 to each polypeptide, 0.5 mg aluminum/ml as aluminum phosphate, 10 mM histidine and 150 mM sodium chloride, where the total volume of the composition is preferably about 0.5 ml.

In another aspect of the invention, the composition comprises about 120 μg/ml of the first lipidized polypeptide containing the amino acid sequence shown in SEQ ID NO: 1, about 120 μg/ml of the second lipidized polypeptide containing the amino acid sequence shown in SEQ ID NO: 2, polysorbate -80 in a molar ratio of 2.8 to each polypeptide, 0.5 mg aluminum/ml as aluminum phosphate, 10 mM histidine and 150 mM sodium chloride.

In another aspect of the invention, the composition comprises a) 60 μg of the first lipidized polypeptide containing the amino acid sequence shown in SEQ ID NO: 1; b) 60 μg of a second lipidized polypeptide containing the amino acid sequence shown in SEQ ID NO: 2, c) 18 μg of polysorbate-80; d) 250 µg aluminum, e) 780 µg histidine and f) 4380 µg sodium chloride.

In a representative embodiment of the invention, the composition comprises about 60 µg of a first lipidized polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, about 60 µg of a second lipidized polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, polysorbate- 80 in a molar ratio of 2.8 to the first lipidized polypeptide and to the second lipidized polypeptide, 0.5 mg aluminum/ml as aluminum phosphate, 10 mM histidine and 150 mM sodium chloride, where the total composition volume is preferably about 0.5 ml . In a representative embodiment of the invention, the composition is a sterile isotonic buffered liquid suspension. In a representative embodiment of the invention, the composition has a pH of 6.0. In a representative embodiment of the invention, the first polypeptide and the second polypeptide are adsorbed to aluminum.

In one embodiment, the composition comprises a MenA _AH -TT conjugate having an average TT/polysaccharide ratio of 3; a MenC _AH -TT conjugate having an average TT/polysaccharide ratio of 3; a MenW-TT conjugate having an average TT/polysaccharide ratio of 1.5; and a MenY-TT conjugate having an average TT/polysaccharide ratio of 1.3. In a preferred embodiment of the invention, the composition comprises a MenA _AH -TT conjugate having 5 μg MenA polysaccharide and ~15 μg TT; a MenC _AH -TT conjugate having 5 μg MenC polysaccharide and ~15 μg TT; a MenW-TT conjugate having 5 μg MenW polysaccharide and ~7.5 μg TT; and a MenY-TT conjugate having 5 μg MenY polysaccharide and ~6.5 μg TT. The composition may also include Tris-HCl, sucrose and sodium chloride.

In another embodiment of the invention, the composition comprises a MenA _AH -TT conjugate; conjugate MenC _AH -TT; conjugate MenW-TT; and the MenY-TT conjugate, which include the MenA polysaccharide; polysaccharide MenC; polysaccharide MenW; and the MenY polysaccharide and TT carrier protein. The composition may also include sucrose and tromethanol. Thus, for example, in one embodiment of the invention, the composition comprises 10 μg/ml MenA polysaccharide; 10 µg/ml MenC polysaccharide; 10 μg/ml MenW polysaccharide; and 10 μg/ml MenY polysaccharide; 88 μg/ml carrier protein TT; 164 mM sucrose; and 1.6 mM tromethanol.

In one of the embodiments of the invention, the composition has a total volume of approximately 0.5 ml. In one of the embodiments of the invention, the first dose of the composition has a total volume of approximately 0.5 ml. "First dose" means the dose of the composition administered on day 0. "Second dose" or "third dose" means the doses of the composition administered after the first dose, which may or may not be the same as the amount of the first dose.

In one of its aspects, the present invention relates to a liquid immunogenic composition obtained from a lyophilized composition of MenACWY-TT diluted with a liquid divalent composition of MnB rLP2086. Dilution means the conversion of a dry lyophilized composition into a liquid form by adding a liquid diluent. In one preferred embodiment of the invention, the rLP2086 MnB liquid divalent composition is not administered simultaneously, together with, or in combination with a MenACWY-TT lyophilized composition, wherein the MenACWY-TT lyophilized composition has been diluted with a liquid composition other than the rLP2086 liquid MnB divalent composition. Thus, for example, in one preferred embodiment of the invention, the lyophilized MenACWY-TT composition is not diluted with an aqueous diluent consisting of sodium chloride and water, and is not administered simultaneously, together with, or in combination with the liquid divalent composition of MnB rLP2086.

Conversely, in a preferred embodiment of the invention, the lyophilized MenACWY-TT composition is administered to a human together with the divalent MnB rLP2086 composition all at once, i.e., in one administration. Such single administration (eg, MenABCWY formulations) can be done by taking the divalent MnB rLP2086 formulation from the first container and mixing it with the lyophilized MenACWY-TT formulation from the second container. Alternatively, a single administration of the MenABCWY composition may be from a single container that includes the bivalent MnB rLP2086 composition and the lyophilized MenACWY-TT composition. Methods for delivering vaccines or immunogenic compositions are known to those skilled in the art. In one of the embodiments of the invention, the MenABCWY composition is administered simultaneously with any of such compounds as ibuprofen, paracetamol and amoxicillin.

The composition is immunogenic after the first dose is administered to a human. In one of the embodiments of the invention, the first dose is approximately 0.5 ml in total volume.

The composition induces a bactericidal serum immunoglobulin titer that, after administration of the first dose to a human, is at least more than 1-fold, and preferably at least 2-fold, of the bactericidal serum immunoglobulin titer prior to administration of the first dose to a human, if these titers are measured in identical conditions in the assay for bactericidal activity of serum using human complement (hSBA).

The bactericidal titer or bactericidal immune response is directed against N. meningitidis serogroup B. In a preferred embodiment, the bactericidal titer or bactericidal immune response is directed against N. meningitidis serogroup B subfamily A fHBP strain and N. meningitidis serogroup B subfamily B fHBP strain. Most preferably, the bactericidal titer or bactericidal immune response is directed at least against strain fHBP subfamily B N. meningitidis serogroup B, strain B01.

In one embodiment, the composition induces a bactericidal serum immunoglobulin titer that, upon administration of the composition to a human, is at least more than 1-fold, e.g., at least 1.01-fold, 1.1-fold, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times or 16 times the bactericidal serum titer immunoglobulin in humans prior to the indicated dose, if these titers are measured under identical conditions in a serum bactericidal assay using human complement.

In one of the embodiments of the invention, the composition is an immunogenic composition. In one of the embodiments of the invention, the composition is an immunogenic composition for humans. In another embodiment of the invention, the composition is a vaccine. "Vaccine" means a composition comprising an antigen containing at least one epitope that induces an immune response specific to that antigen. The vaccine may be administered to the individual directly by subcutaneous, oral, oronasal, or intranasal administration. Preferably, the vaccine is administered intramuscularly. In one embodiment of the invention, the composition is a human vaccine. In one embodiment, the composition is an anti- N. meningitidis immunogenic composition.

In one of the embodiments of the invention, the composition is a liquid composition. In a preferred embodiment of the invention, the composition is a liquid suspension composition. In another preferred embodiment of the invention, the composition is not lyophilized.

First polypeptide; subfamily A protein MNB RLP2086 (A05)

In one embodiment, the composition comprises a first polypeptide having the amino acid sequence shown in SEQ ID NO: 1. The polypeptide is a modified factor H binding protein (fHBP) of N. meningitidis strain M98250771. A description of fHBP is given in WO2012032489 and US Patent Application Publication 2012/0093852, each of which is incorporated herein by reference in its entirety. The polypeptide is lipidized at the N-terminus predominantly with three C16:0, C16:1 and C18:1 fatty acids covalently linked at three positions of the polypeptide. The first polypeptide includes a total of 258 amino acids.

A representative primary structure of the MnB rLP2086 A05 protein is shown in FIG. 4. The primary structure of a protein is illustrated in FIG. 4 single letter codes for all amino acids except for the N-terminal cysteine and glyceryl groups (which are represented by the full chemical formula). This structure includes the primary protein sequence structure in which the N-terminal cysteine residue has been lipidized. The amino group of the N-terminal cysteine residue at the N-terminus of the protein was attached to a fatty acid (R1) to form an amide bond, and the cysteinyl sulfhydryl group was attached to a glycerol group containing two ester-linked fatty acids (R2). The structure of R1 was found to be hexadecanoic acid (C16:0) and the structures of R2 varied depending on the MnB isoforms of rLP2086.

The first polypeptide includes two modifications introduced in the N-terminal region of the polypeptide compared to the corresponding sequence of the wild-type strain N. meningitidis M98250771. The glycine at the second position was attached after the introduction of the cloning site. The second modification involves the deletion of four amino acids. Accordingly, in one embodiment of the invention, the first polypeptide includes the sequence CGSS (SEQ ID NO: 3) at the N-terminus. See SEQ ID NO: 1, first four amino acid residues.

The N-terminal differences between the first polypeptide sequence and the wild-type Neisserial sequence are shown below. Accordingly, in one embodiment of the invention, the first polypeptide comprises at least the first 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 1. Preferably, the first polypeptide comprises at least the first 4, more preferably at least the first 6, and most preferably at least the first 8 amino acid residues of SEQ ID NO: 1.

Comparison of the predicted N-terminal sequences of the recombinant polypeptide and the Neisserial LP2086 subfamily A polypeptide

rLP2086 M98250771 CGSS-----GGGGVAAD (SEQ ID NO: 4) Neisserial LP2086 M98250771 C-SSGS-GSGGGGVAAD (SEQ ID NO: 5)

>A05 (SEQ ID NO: 1)

CGSSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAEKTFKVGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPSGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELASAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIREKVHEIGIAGKQ

In one embodiment, the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1. In one embodiment, the first polypeptide has a total of 258 amino acids. In one embodiment, the first polypeptide does not include an amino acid sequence that is less than 100% identical to SEQ ID NO: 1. In another embodiment, the first polypeptide consists of the amino acid sequence shown in SEQ ID NO: 1. B in another embodiment of the invention, the first polypeptide comprises the amino acid sequence of KDN. See, for example, amino acid residues 73-75 of SEQ ID NO: 1. In another embodiment of the invention, the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3 at the N-terminus of the polypeptide. In another embodiment of the invention, the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 4 at the N-terminus of the polypeptide.

In a preferred embodiment of the invention, the first polypeptide can be easily expressed in a recombinant host cell by standard methods known in the art. In another preferred embodiment, the first polypeptide comprises a bactericidal epitope on the N- and/or C-domain of SEQ ID NO: 1. In one embodiment, the first polypeptide comprises at least the first 4, 5, 6, 7, 8 , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 , 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 , 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83 , 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acid residues of the amino acid sequence shown in SEQ ID NO: 1. Preferably, the first the polypeptide comprises at least the first 2, more preferably at least the first 4, and most preferably at least the first 8 amino acid residues of SEQ ID NO: 1.

In another embodiment, the first polypeptide comprises at least the last 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 , 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 , 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 , 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 , 98, 99 or 100 amino acid residues of the amino acid sequence shown in SEQ ID NO: 1.

In one embodiment, the composition comprises approximately 30 μg/ml of the first polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1. In one preferred embodiment, the composition comprises approximately 60 μg/ml of the first polypeptide comprising the amino acid sequence, set forth in SEQ ID NO: 1. In one preferred embodiment of the invention, the composition comprises approximately 60 μg/ml of the first polypeptide containing the amino acid sequence set forth in SEQ ID NO: 1, where the composition preferably has a total volume of 0.5 ml. In another embodiment of the invention, the composition comprises approximately 120 μg/ml of the first polypeptide containing the amino acid sequence shown in SEQ ID NO: 1.

Second polypeptide; subfamily B protein MnB rLP2086 (B01)

In one embodiment, the composition comprises a second polypeptide having the amino acid sequence shown in SEQ ID NO: 2. The polypeptide is a factor H binding protein (fHBP) of N. meningitidis strain CDC1573. A description of fHBP is given in WO2012032489 and US Patent Application Publication 2012/0093852, each of which is incorporated herein by reference in its entirety. The polypeptide is lipidated at the N-terminus predominantly with three C16:0, C16:1 and C18:1 fatty acids covalently linked at three positions of the polypeptide. The first polypeptide includes a total of 261 amino acids.

A representative primary structure of the MnB rLP2086 B01 protein is shown in FIG. 5. The primary structure of a protein is illustrated in FIG. 5 single letter codes for all amino acids except for the N-terminal cysteine and glyceryl groups (which are represented by the full chemical formula). This structure includes the primary protein sequence structure in which the N-terminal cysteine residue has been lipidized. The amino group of the N-terminal cysteine residue at the N-terminus of the protein was attached to a fatty acid (R1) to form an amide bond, and the cysteinyl sulfhydryl group was attached to a glycerol group containing two ester-linked fatty acids (R2). The structure of R1 was found to be hexadecanoic acid (C16:0) and the structures of R2 varied depending on the isoforms of rLP2086.

The second polypeptide includes one modification introduced into the N-terminal region of the rLP2086 subfamily B protein compared to the corresponding sequence of the N. meningitidis CDC1573 wild-type strain. The glycine at the second position was attached after the introduction of the cloning site.

The N-terminal differences compared to the original Neisseria sequences are presented below.

Comparison of the predicted N-terminal sequences of the recombinant polypeptide and the Neisseria LP2086 subfamily B polypeptide

rLP2086 CDC-1573 CGSSGGGGSGGGGVTAD (SEQ ID NO: 24) Neisseria LP2086 CDC-1573 C-SSGGGGSGGGGVTAD (SEQ ID NO: 25)

In one of the embodiments of the invention, the second polypeptide includes the sequence C-G-S-S (SEQ ID NO: 3) at the N-terminus. See the first four amino acid residues of SEQ ID NO: 2.

>B01 (SEQ ID NO: 2)

CGSSGGGGSGGGGVTADIGTGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQEQDPEHSEKMVAKRRFRIGDIAGEHTSFDKLPKDVMATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAVAYIKPDEKHHAVISGSVLYNQDEKGSYSLGIFGEKAQEVAGSAEVETANGIHHIGLAAKQ

In one embodiment, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2. In one embodiment, the second polypeptide has a total of 261 amino acids. In one embodiment, the second polypeptide consists of the amino acid sequence shown in SEQ ID NO: 2. In another embodiment, the second polypeptide also does not include a polypeptide whose sequence is less than 100% identical to SEQ ID NO: 2. In a preferred embodiment of the invention, the first polypeptide and the second polypeptide include the sequence C-G-S-S (SEQ ID NO: 3) at the N-terminus of the respective polypeptide.

In a preferred embodiment of the invention, the second polypeptide can be easily expressed in the recombinant host cell by standard methods known in the art. In another preferred embodiment, the second polypeptide comprises a bactericidal epitope on the N- and/or C-domain of SEQ ID NO: 2. In one embodiment, the second polypeptide comprises at least the first 2, 3, 4, 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56 , 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acid residues of the amino acid sequence shown in SEQ ID NO: 2 Preferably, the second polypeptide comprises at least the first 2, more preferably at least the first 4, and most preferably at least the first 8 amino acid residues of SEQ ID NO: 2.

In another embodiment, the second polypeptide comprises at least the last 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 , 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 , 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 , 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 , 98, 99 or 100 amino acid residues of the amino acid sequence shown in SEQ ID NO: 2.

In one embodiment, the composition comprises approximately 30 μg/ml of a first polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2. In one preferred embodiment, the composition comprises approximately 60 μg/ml of a first polypeptide comprising the amino acid sequence, set forth in SEQ ID NO: 2. In one preferred embodiment of the invention, the composition comprises approximately 60 μg/ml of a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, wherein the composition preferably has a total volume of 0.5 ml. In another embodiment of the invention, the composition comprises approximately 120 μg/ml of a second polypeptide containing the amino acid sequence shown in SEQ ID NO: 2.

saccharides

The term "saccharide" as used herein can mean a polysaccharide or an oligosaccharide and includes both. The polysaccharides are isolated from bacteria or isolated from bacteria and corrected in size to a certain extent by known methods, and optionally by microfluidization. The size of the polysaccharides can be adjusted to reduce the viscosity in the polysaccharide samples and/or to improve the filterability of the conjugated products. Oligosaccharides have a low number of repeat units (typically 5-30 repeat units) and are usually hydrolyzed polysaccharides.

Each N. meningitidis capsular saccharide can be conjugated to a carrier protein independently selected from the group consisting of TT, DT, CRM197, TT fragment C, and protein D. Although one or more N. meningitidis capsular saccharides can be conjugated to carrier proteins , different from other proteins, however, in one embodiment of the invention, they are all conjugated to the same carrier protein. For example, they can all be conjugated to the same carrier protein selected from the group consisting of TT, DT, CRM197, TT fragment C, and protein D. In this context, CRM197 and DT can be considered as one and the same. same carrier protein as they differ in only one amino acid. In a preferred embodiment of the invention, all N. meningitidis capsular saccharides present are conjugated to TT.

If the carrier protein is the same for 2 or more saccharides in the composition, then that saccharide can be conjugated to the same carrier protein molecule (carrier molecules having 2 or more different saccharides conjugated to them) [see ., for example, WO 04/083251; for example, one carrier protein can be conjugated to MenA and MenC; MenA and MenW; MenA and MenY; MenC and MenW; MenC and MenY; Men W and Men Y; MenA, MenC and MenW; MenA, MenC and MenY; MenA, MenW and MenY; MenC, MenW and MenY; MenA, MenC, MenW and MenY. Alternatively, each saccharide can be individually conjugated to different carrier protein molecules (each carrier protein molecule has only one type of saccharide conjugated to it).

In one embodiment, at least 2 different saccharide conjugates are separately conjugated to carrier proteins of the same type, wherein one or more saccharides are conjugated to the carrier protein via a first type chemical group on the carrier protein, and one or more more saccharides are conjugated to the carrier protein via a second (different) type of chemical group on the carrier protein.

In one of the embodiments of the invention, 2 conjugates include the same saccharide associated with the same carrier, but using different chemical methods of conjugation. Alternatively, 2 different saccharides are conjugated to different groups on the carrier protein.

The term "conjugated separately to the same type of carrier protein" means that the saccharides are conjugated separately to the same carrier (for example, MenA is conjugated to tetanus toxoid via an amino group on the tetanus toxoid, and MenC is conjugated to tetanus toxoid via the group carboxylic acid on another tetanus toxoid molecule).

The capsular saccharide(s) may be conjugated to the same carrier protein independently selected from the group consisting of TT, DT, CRM197, TT fragment C, and protein D. A more complete list of proteins -carriers that can be used in the conjugates according to the invention are given below. In this context, CRM197 and DT can be considered the same carrier protein as they differ in only one amino acid. In one embodiment of the invention, all capsular saccharides present are conjugated to TT.

The saccharides may include any of the following: N. meningitidis serogroup A capsular saccharide (MenA), N. meningitidis serogroup C capsular saccharide (MenC), N. meningitidis serogroup Y capsular saccharide (MenY), and N capsular saccharide .meningitidis serogroup W (MenW), or any combination thereof.

The first and second chemical groups present on the carrier protein are different from each other and are ideal natural chemical groups that can be successfully used for conjugation purposes. These groups may be independently selected from the group consisting of: carboxyl groups, amino groups, sulfhydryl groups, hydroxyl groups, imidazolyl groups, guanidyl groups and indolyl groups. In one embodiment of the invention, the first chemical group is carboxyl and the second is amino, or vice versa. These groups are explained in more detail below.

In a specific embodiment of the invention, the immunogenic composition comprises at least 2 different N. meningitidis capsular saccharides, where one or more saccharides are selected from the first group consisting of MenA and MenC, which are conjugated to the carrier protein via a first type chemical group on the protein - carrier (eg, carboxyl) and one or more saccharides selected from the second group consisting of MenC, MenY and MenW, which are conjugated to the carrier protein via a second type chemical group on the carrier protein (eg, an amino group).

In another embodiment of the invention, the immunogenic composition of the invention comprises MenA conjugated through a first type chemical group (eg, carboxyl) and MenC conjugated through a second type chemical group (eg, an amino group).

In another embodiment of the invention, the immunogenic composition comprises MenC conjugated through a first type chemical group (eg, carboxyl) and MenY conjugated through a second type chemical group (eg, an amino group).

In another embodiment, the immunogenic composition comprises MenA conjugated through a first type chemical group (eg, carboxyl) and MenC, MenY and MenW conjugated through a second type chemical group (eg, amino group).

In another embodiment, the immunogenic composition comprises MenA and MenC conjugated through a first type chemical group (eg, carboxyl) and MenY and MenW conjugated through a second type chemical group (eg, an amino group).

The saccharides of the invention included in the pharmaceutical (immunogenic) compositions of the invention are conjugated to a carrier protein such as tetanus toxoid (TT), tetanus toxoid C fragment, tetanus toxoid non-toxic mutants [it should be noted that all such TT variants are considered to be proteins- carriers of the same type used for the purposes of the invention], diphtheria toxoid (DT), CRM197, other non-toxic diphtheria toxin mutants [such as CRM176, CRM 197, CRM228, CRM 45 (Uchida et al J. Biol. Chem. 218 ; 3838-3844, 1973); CRM 9, CRM 45, CRM102, CRM 103 and CRM107 and other mutations described by Nicholls and Youle in Genetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc, 1992; deletion or mutation of Glu-148 to Asp, Gln or Ser and/or Ala 158 to Gly and other mutations described in US Pat. Nos. 4,709,017 or 4,950,740; mutation of at least one or more residues of Lys 516, Lys 526, Phe 530 and/or Lys 534 and other mutations described in US patents 5917017 or 6455673; or a fragment as described in US Pat. -13), OMPC (meningococcal outer membrane protein, usually extracted from strain of N. meningitidis serogroup B - EP0372501), synthetic peptides (EP0378881, EP0427347), heat shock proteins (WO 93/17712, WO 94/03208), viral proteins pertussis (WO 98/58668, EP0471177), cytokines, lymphokines, growth factors or hormones (WO 91/01146), artificial proteins containing multiple human CD4 ⁺ T cell epitopes derived from various pathogenic antigens (Falugi et al (2001 ) Eur J Immunol 31; 3816-3824), such as N19 protein (Baraldoi et al (2004) Infect Immun 72; 4884-7), pneumococcal surface protein PspA (WO 02/091998), iron-absorbing proteins (WO 01/ 72337), C. difficile toxin A or B (WO 00/61761) or b trees D (EP594610 and WO 00/56360).

In one embodiment, the immunogenic composition of the invention uses carrier proteins of the same type (independently) in at least two, three, four, or each of the saccharides it contains.

In one embodiment, the immunogenic composition of the invention comprises a N. meningitidis saccharide conjugated to a carrier protein selected from the group consisting of TT, DT, CRM197, TT fragment C, and protein D.

The immunogenic composition of the invention optionally comprises at least one meningococcal saccharide conjugate (e.g., MenA; MenC; MenW; MenY; MenA and MenC; MenA and MenW; MenA and MenY; MenC and Men W; Men C and MenY; Men W and MenY; MenA, MenC and MenW; MenA, MenC and MenY; MenA, MenW and MenY; MenC, MenW and MenY or MenA, MenC, MenW and MenY) having a ratio of Men saccharide to carrier protein from 1:5 to 5:1, 1:2 to 5:1, 1:0.5 to 1:2.5, or 1:1.25 to 1:2.5 (w/w). In one preferred embodiment, the composition comprises MenA, MenC, MenW, and MenY, each of which is conjugated to tetanus toxoid in a (toxoid:polysaccharide) ratio of about 3, about 1.5, and about 1.3, respectively.

The ratio of saccharide to carrier protein (w/w) in the conjugate can be determined using the sterilized conjugate. The amount of protein is determined by Lowry analysis (see, for example, Lowry et al (1951) J. Biol. Chem. 193, 265-275 or Peterson et al Analytical Biochemistry 100, 201-220 (1979)), and the amount of saccharide is determined using ICP-OES (Inductively Coupled Plasma Optical Emission Spectroscopy) for MenA, DMAP analysis for MenC, and resorcinol analysis for MenW and MenY (Monsigny et al. (1988) Anal. Biochem. 175, 525-530).

In one embodiment, the immunogenic composition of the invention comprises N. meningitidis saccharide(s) conjugate(s), wherein the N. meningitidis saccharide(s) is conjugated to a carrier protein via a linker, eg, a bifunctional linker. The linker is optionally a heterobifunctional or homobifunctional linker having, for example, an amino reactive group and a carboxylic acid reactive group, 2 amino reactive groups, or two carboxylic acid reactive groups. The linker has, for example, 4 to 20, 4 to 12, 5 to 10 carbon atoms. A possible linker is ADH. Other linkers are B-propionamido (WO 00/10599), nitrophenylethylamine (Gever et al (1979) Med. Microbiol. Immunol. 165; 171-288), haloalkyl halides (US4057685), glycosidic linkages (US4673574, US4808700), hexanediamine and 6-aminocaproic acid (US4459286).

The saccharide conjugates present in the immunogenic compositions of the invention may be prepared by any known conjugation method. The conjugation method can be based on activation of the saccharide with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form a cyanate ester. Thus, the activated saccharide can be linked directly or via a spacer (linker) group to the amino group on the carrier protein. Thus, for example, the spacer may be cystamine or cysteamine, which forms a thiolated polysaccharide, which can be attached to the carrier via a thioether bond formed after an interaction reaction with a maleimide-activated carrier protein (for example, using GMBS), or to a holoacetylated protein - carrier (for example, using iodoacetamide or N-succinimidyl-bromoacetobromoacetate). A cyanate ester (obtained optionally using the CDAP chemistry) is optionally attached to hexanediamine or ADH and the amino-derivatized saccharide is conjugated to the carrier protein using a carbodiimide chemistry (e.g., EDAC or EDC) via a carboxyl group on the carrier protein. Such conjugates are described in PCT application WO 93/15760 of the University of Universal Technologies and in WO 95/08348 and WO 96/29094.

Other suitable methods use carbinides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU. Many of them are described in WO 98/42721. Conjugation can be carried out using a carbonyl linker, which can be formed by reacting the free hydroxyl group of the saccharide with CDI (Bethell et al J. Biol. Chem. 1979, 254; 2572-4, Hearn et al J. Chromatogr. 1981. 218; 509-18), followed by an interaction reaction with the protein to form a carbamate bond. Such a reaction can be accomplished by reducing the anomeric end to a primary hydroxyl group, optionally protecting/deprotecting the primary hydroxyl group with CDI to form a CDI carbamate intermediate, and coupling the CDI intermediate carbamate to an amino group on the protein.

Conjugates can also be prepared by direct reductive amination methods as described in US Pat. No. 4,365,170 (Jennings) and US Pat. No. 4,673,574 (Anderson). Other methods are described in EP-0-161-188, EP-208375 and EP-0-477508.

Another method involves coupling a cyanogen bromide activated (or CDAP) saccharide derivatized with adipic acid hydrazide (ADH) to a carrier protein via a carbodiimide condensation reaction (Chu C. et al Infect. Immunity, 1983 245 256), for example using EDAC.

In one embodiment, a hydroxyl group (optionally an activated hydroxyl group, e.g., a cyanate ester activated hydroxyl group) on the saccharide is linked to an amino or carboxyl group on the protein, either directly or indirectly (via a linker). If a linker is present, then the hydroxyl group on the saccharide is optionally linked to the amino group on the linker, for example by CDAP conjugation. Another amino group on the linker (eg ADH) can be conjugated to a carboxylic acid group on the protein, eg using a chemical carbodiimide technique, eg using EDAC. In one embodiment, the N. meningitidis capsular(s) saccharide(s) (or any saccharide in general) is conjugated to the linker prior to its contact with the carrier protein. Alternatively, the linker may be conjugated to the carrier prior to conjugation to the saccharide.

Typically, the following types of chemical groups on a carrier protein can be used to carry out a coupling/conjugation reaction:

A) Carboxyl (eg via aspartic acid or glutamic acid). In one of the embodiments of the invention, this group is attached to the amino groups on the saccharides directly or to the amino group on the linker using a carbodiimide chemical technique, for example, using EDAC.

B) Amino group (eg via lysine). In one embodiment of the invention, this group is attached to the carboxyl groups on the saccharides directly or to the carboxyl group on the linker using a carbodiimide chemical technique, for example using EDAC. In another embodiment of the invention, this group is attached to CDAP or CNBr activated hydroxyl groups on saccharides directly or to such hydroxyl groups on a linker; to saccharides or linkers having an aldehyde group; to saccharides or linkers having a succinimide ester group.

C) Sulfhydryl (eg via cysteine). In one embodiment of the invention, this group is attached to a bromo- or chloroacetylated saccharide or linker using a maleimide chemical technique. In one of the embodiments of the invention, this group is activated/modified with bis-diazobenzidine.

D) Hydroxyl group (eg via tyrosine). In one of the embodiments of the invention, this group is activated/modified with bis-diazobenzidine.

E) An imidazolyl group (eg via histidine). In one of the embodiments of the invention, this group is activated/modified with bis-diazobenzidine.

F) Guanidyl group (eg via arginine).

G) Indolyl group (eg via tryptophan).

On the saccharide, in general the following groups can be used for binding: OH, COOH or NH2. Aldehyde groups can be obtained after various treatments known to those skilled in the art, such as periodate treatment, acid hydrolysis, hydrogen peroxide treatment, and the like.

Direct linking methods:

Saccharide-OH+CNBr or CDAP -----> cyanate ester+NH2-Prot ----> conjugate

Saccharide-aldehyde+NH2-Prot ----> Schiff base+NaCNBH3 ----> conjugate

Saccharide-COOH+NH2-Prot+EDAC ----> conjugate

Saccharide-NH2+COOH-Prot+EDAC ----> conjugate

Indirect binding methods using a spacer (linker):

Saccharide-OH+CNBr or CDAP ---> cyanate ester+NH2----NH2 ----> saccharide----NH2+COOH-Prot+EDAC -----> conjugate

Saccharide-OH+CNBr or CDAP ----> cyanate ester+NH2-----SH -----> saccharide----SH+SH-Prot (native protein with unprotected cysteine or obtained after modification of amino groups protein via e.g. SPDP) -----> saccharide-S-S-Prot

Saccharide-OH+CNBr or CDAP ---> cyanate ester+NH2----SH -------> saccharide----SH+maleimide-Prot (modification of amino groups) ----> conjugate

Saccharide-COOH+EDAC+NH2-----NH2 ---> saccharide------NH2+EDAC+COOH-Prot ----> conjugate

Saccharide-COOH+EDAC+ NH2----SH -----> saccharide----SH+SH-Prot > saccharide-S-S-Prot

Saccharide-COOH+EDAC+ NH2----SH -----> saccharide----SH+maleimide-Prot (modification of amino groups) ----> conjugate

Saccharide--aldehyde+NH2-----NH2 ----> saccharide---NH2+EDAC+COOH-Prot ----> conjugate

Note: Any suitable carbodiimide may be used in place of the EDAC described above.

In general, these types of carrier protein chemical groups that are commonly used in saccharide coupling reactions are amino groups (for example, on lysine residues), COOH groups (for example, on aspartic and glutamic acid residues), and SH groups (if present). ) (for example, on cysteine residues).

In one embodiment, at least one N. meningitidis capsular saccharide (or any saccharide in general) is directly conjugated to a carrier protein; The Men W and/or MenY and/or MenC saccharide(s) are directly conjugated, optionally, to a carrier protein. So, for example MenW; MenY; Menc; MenW and MenY; MenW and MenC; MenY and MenC; or MenW, MenY and MenC are directly attached to the carrier protein. At least one of the N. meningitidis capsular saccharides is directly conjugated, optionally using CDAP. So, for example MenW; MenY; Menc; MenW and MenY; MenW and MenC; MenY and MenC; or MenW, MenY and MenC are directly attached to the carrier protein using CDAP (see WO 95/08348 and WO 96/29094). In one embodiment, all N. meningitidis capsular saccharides are conjugated to tetanus toxoid.

In one embodiment, the ratio of Men W and/or Y saccharide to carrier protein is from 1:0.5 to 1:2 (w/w) and/or the ratio of MenC saccharide to carrier protein is from 1: 0.5 to 1:4 or 1:0.5 to 1:1.5 (w/w), and in particular if these saccharides are directly linked to the protein, optionally via CDAP.

In one embodiment of the invention, at least one of the N. meningitidis capsular saccharides (or any saccharide in general) is conjugated to a carrier protein via a linker, such as a bifunctional linker. The linker is optionally a heterobifunctional or homobifunctional linker having, for example, an amino reactive group and a carboxylic acid reactive group, 2 amino reactive groups, or 2 carboxylic acid reactive groups. The linker has, for example, 4 to 20, 4 to 12, 5 to 10 carbon atoms. The linker may be ADH.

In one of the embodiments of the invention, MenA; Menc; or MenA and MenC are conjugated to a carrier protein (eg, tetanus toxoid) via a linker.

In one embodiment, at least one N. meningitidis saccharide is conjugated to a carrier protein via a linker using CDAP and EDAC. So, for example, MenA; Menc; or MenA and MenC are conjugated to the protein via a linker (eg, two hydrazino groups at the ends, such as ADH) using CDAP and EDAC as described above. For example, CDAP is used to conjugate a saccharide to a linker, and EDAC is used to conjugate a linker to a protein. Optionally, conjugation via a linker gives a saccharide:carrier protein ratio of 1:0.5 to 1:6; 1:1 to 1:5 or 1:2 to 1:4, for MenA; Menc; or MenA and MenC.

In one embodiment, the MenA capsular saccharide, if present at least partially in O-acetylated form, for example, has at least 50%, 60%, 70%, 80%, 90%, 95%, or 98% repeating units, is O-acetylated in at least one position. O-acetylation, for example, is achieved at least at the O-3 position for at least 50%, 60%, 70%, 80%, 90%, 95%, or 98% of the repeat units.

In one embodiment, the MenC capsular saccharide, if present at least partially in O-acetylated form, e.g., has at least 30%, 40%, 50%, 60%, 70%, 80%, 90% , 95% or 98% of (α2→9)-linked NeuNAc repeat units, is O-acetylated at least one or two positions. O-acetylation, for example, is achieved at the O-7 and/or O-8 positions for at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 98% of the repeat units .

In one embodiment, the MenW capsular saccharide, if present at least partially in O-acetylated form, e.g., has at least 30%, 40%, 50%, 60%, 70%, 80%, 90% , 95% or 98% of the repeating units, is O-acetylated in at least one or two positions. O-acetylation, for example, is achieved at the O-7 and/or O-9 positions for at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 98% of the repeat units .

In one embodiment, the MenY capsular saccharide, if present at least partially in O-acetylated form, e.g., has at least 20%, 30%, 40%, 50%, 60%, 70%, 80% , 90%, 95% or 98% of the repeating units is O-acetylated at least in one or two positions. O-acetylation is achieved at positions 7 and/or 9 for at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 98% of the repeat units.

The percentage of O-acetylation refers to the percentage of repeating units containing an O-acetyl group. This percentage can be determined in the saccharide before conjugation and/or after conjugation.

In one embodiment, the immunogenic composition, the saccharide present, or each N. meningitidis capsular saccharide present is conjugated to TT. In another embodiment of the invention, each N. meningitidis capsular saccharide is separately conjugated to a separate carrier protein. In another embodiment, each N. meningitidis capsular saccharide conjugate has a saccharide:carrier ratio of 1:5-5:1 or 1:1-1:4 (w/w). In another embodiment, at least one, two, or three N. meningitidis capsular saccharide conjugates are directly conjugated to a carrier protein. In another embodiment of the invention, Men W and/or MenY, MenW and/or MenC, MenY and/or MenC, or MenW and MenC and MenY are directly conjugated to a carrier protein. In another embodiment of the invention, at least one, two, or three N. meningitidis capsular saccharide conjugates are directly conjugated using a chemical CDAP technique. In another embodiment of the invention, the ratio of Men W and/or Y saccharide to carrier protein is from 1:0.5 to 1:2 (w/w). In another embodiment of the invention, the ratio of MenC saccharide to carrier protein is from 1:0.5 to 1:2 (w/w). In another embodiment, at least one, two, or three N. meningitidis capsular saccharides are conjugated to a carrier protein via a linker (which may be bifunctional, such as a linker having two reactive amino groups (such as ADH) or two reactive carboxyl groups). , or a reactive amino group at one end and a reactive carboxyl group at the other end). The linker may have from 4 to 12 carbon atoms. In another embodiment of the invention, this or each N. meningitidis capsular saccharide conjugated via a linker is conjugated to the linker using a chemical CDAP technique. In another embodiment of the invention, the carrier protein is conjugated to the linker using a chemical carbodiimide technique, for example using EDAC. In another embodiment of the invention, this or each N. meningitidis capsular saccharide is conjugated to a linker prior to conjugation of the carrier protein to the linker. In another embodiment of the invention, MenA is conjugated to a carrier protein via a linker (the ratio of MenA saccharide to carrier protein may be from 1:2 to 1:5 (w/w)). In another embodiment, MenC is conjugated to a carrier protein via a linker (the ratio of MenC saccharide to carrier protein may be 1:2 to 1:5 (w/w)).

Using native polysaccharide conjugates or small size polysaccharide conjugates, one or more of the following advantages can be achieved: 1) the conjugate is highly immunogenic and can be filtered through a 0.2 micron filter; 2) immune memory can be improved (as in Example 3); 3) the ratio of polysaccharide to protein in the conjugate can be changed so that the ratio of polysaccharide to protein (w/w) is increased (this may result in a reduced carrier suppression effect); 4) immunogenic conjugates prone to hydrolysis (such as MenA conjugates) can be stabilized using larger conjugated polysaccharides. The use of larger polysaccharides may result in more efficient cross-linking with the conjugate carrier and may reduce the release of free saccharide from the conjugate. Conjugate vaccines previously described in the literature tend to depolymerize the polysaccharides prior to conjugation, which improves conjugation. Meningococcal (or saccharide) conjugate vaccines that retain the larger saccharide may give a good immune response against meningococcal disease.

Thus, the immunogenic composition according to the invention may contain one or more saccharide conjugates, where the average size of each saccharide before conjugation exceeds 50 kDa, 75 kDa, 100 kDa, 110 kDa, 120 kDa or 130 kDa. In one embodiment of the invention, the conjugate after conjugation should be easily filtered through a 0.2 micron filter so that the sample yield after filtration is greater than 50%, 60%, 70%, 80%, 90%, or 95% compared to the sample yield before filtration. .

In particular, the immunogenic composition of the invention comprises N. meningitidis capsular saccharides of at least one, two, three, or four serogroups A, C, W, and Y conjugated to a carrier protein, wherein the average size (weight average molecular weight; Mw) at least one, two, three or four or each of the N. meningitidis saccharides is greater than 50 kDa, 60 kDa, 75 kDa, 100 kDa, 110 kDa, 120 kDa, or 130 kDa.

In a preferred embodiment, the average Mw of the MenA _AH -TT conjugate is at least 250 kDa, 260 kDa, 270 kDa, 280 kDa, or 290 kDa, most preferably about 300 kDa, and a maximum of 350 kDa or 330 kDa. In a preferred embodiment of the invention, the average Mw of the MenC _AH -TT conjugate is at least 150 kDa, 160 kDa, 170 kDa, 180 kDa or 190 kDa, most preferably about 200 kDa and a maximum of 250 kDa or 230 kDa. In a preferred embodiment of the invention, the average Mw of the MenW-TT conjugate is at least 240, 250 kDa, 260 kDa, or 270 kDa, most preferably about 280 kDa, and a maximum of 330 kDa or 310 kDa. In a preferred embodiment of the invention, the average Mw of the MenY-TT conjugate is at least 220 kDa, 230 kDa, 240 kDa, or 250 kDa, most preferably about 270 kDa, and a maximum of 320 kDa or 300 kDa.

The immunogenic composition may contain N. meningitidis capsular saccharides of at least one, two, three or four serogroups A, C, W and Y conjugated to a carrier protein, where at least one, two, three or four saccharides or each saccharide N. meningitidis is either a native saccharide or is sized 2, 3, 4, 5, 6, 7, 8, 9, or 10 times the weight average molecular weight of the native polysaccharide.

Used in the present invention, the term "native polysaccharide" means a saccharide that has not been subjected to processing in order to reduce the size of the saccharide. The size of the polysaccharide may decrease slightly during normal purification procedures. Such a saccharide is still native. And only after undergoing resizing procedures, such a polysaccharide is no longer considered as native.

As used herein, the term "2-fold size change" means that the saccharide has been subjected to a size reduction procedure, yet retains a size that is more than half the size of the native polysaccharide. ×3, ×4, etc. interpreted in the same way, that is, the saccharide was subjected to the procedure of reducing the size of the polysaccharide, but retained the size of more than a third, a quarter, etc. on the size of the native polysaccharide.

In one aspect of the invention, the immunogenic composition comprises N. meningitidis capsular saccharides of at least one, two, three, or four serogroups A, C, W, and Y conjugated to a carrier protein, wherein at least one, two, three, or four or each N. meningitidis saccharide is a native polysaccharide.

In one aspect of the invention, the immunogenic composition comprises N. meningitidis capsular saccharides of at least one, two, three, or four serogroups A, C, W, and Y conjugated to a carrier protein, wherein at least one, two, three, or four saccharides or each saccharide of N. meningitidis are resized by factors of 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

The immunogenic compositions of the invention include, but are not limited to, conjugates of: N. meningitidis serogroup C capsular saccharide (MenC); capsular saccharide serogroup A (MenA); capsular saccharide serogroup W135 (MenW); capsular saccharide serogroup Y (MenY); capsular saccharides of serogroups C and Y (MenCY); capsular saccharides of serogroups C and A (MenAC); capsular saccharides of serogroups C and W (MenCW); serogroup A and Y serogroup capsular saccharides (MenAY); capsular saccharides of serogroups A and W (MenAW); capsular saccharides of serogroups W and Y (Men WY); capsular saccharides of serogroups A, C and W (MenACW); capsular saccharides of serogroups A, C and Y (MenACY); capsular saccharides of serogroup A, W135 and Y (MenAWY); capsular saccharides of serogroup C, W135 and Y (MenCWY); or capsular saccharides of serogroups A, C, W135 and Y (MenACWY). This definition includes "one, two, three, or four", or "at least one of the saccharides" of serogroups A, C, W, and Y, or each N. meningitidis saccharide mentioned herein.

In one of the embodiments of the invention, the average size of at least one, two, three, four saccharides or each N. meningitidis saccharide is from 50 kDa to 1500 kDa, from 50 kDa to 500 kDa, from 50 kDa to 300 KDa, from 101 kDa to 1500 kDa, 101 kDa to 500 kDa, 101 kDa to 300 kDa, as determined by MALLS.

In one embodiment, the MenA saccharide, if present, has a molecular weight of 50-500 kDa, 50-100 kDa, 100-500 kDa, 55-90 kDa, 60-70 kDa or 70-80 kDa or 60-80 kDa.

In one embodiment, the MenC saccharide, if present, has a molecular weight of 100-200 kDa, 50-100 kDa, 100-150 kDa, 101-130 kDa, 150-210 kDa, or 180-210 kDa.

In one embodiment, the MenY saccharide, if present, has a molecular weight of 60-190 kDa, 70-180 kDa, 80-170 kDa, 90-160 kDa, 100-150 kDa, or 110-140 kDa, 50-100 kDa, 100-140 kDa, 140-170 kDa or 150-160 kDa.

In one embodiment, the MenW saccharide, if present, has a molecular weight of 60-190 kDa, 70-180 kDa, 80-170 kDa, 90-160 kDa, 100-150 kDa, 110-140 kDa, 50-100 kDa or 120-140 kDa.

Molecular weight or average molecular weight of the saccharide described here means the weight average molecular weight (Mw) of the saccharide, determined prior to conjugation and measured using MALLS.

The MALLS method is well known to those skilled in the art and is generally performed as described in Example 2. For MALLS analysis of meningococcal saccharides, a combination of two columns (TSKG6000 and 5000PWxl) can be used and these saccharides are eluted in water. Saccharides are detected with a light scattering detector (eg Wyatt Dawn DSP equipped with a 10 mW argon laser at 488 nm) and an interferometric refractometer (eg Wyatt Otilab DSP equipped with a P100 cell and a 498 nm red filter).

In one embodiment, the N. meningitidis saccharides are native polysaccharides or native polysaccharides that are reduced in size by a standard extraction process.

In one embodiment, the N. meningitidis saccharides are sized by mechanical digestion, such as microfluidization or sonication. Microfluidization or sonication have the advantage that they allow the size of larger native polysaccharides to be reduced to a level sufficient to obtain a filterable conjugate (eg, through a 0.2 micron filter). The change in size can be no more than 20, 10, 8, 6, 5, 4, 3, 2 or 1.5 times.

In one of the embodiments of the invention, the immunogenic composition contains N. meningitidis conjugates, obtained from a mixture of native polysaccharides and saccharides, the size of which is changed no more than 20 times. For example, MenC and/or MenA saccharides are native. Thus, for example, the MenY and/or MenW saccharides are sized no more than 20, 10, 8, 6, 5, 4, 3 or 2 times. Thus, for example, the immunogenic composition contains a conjugate derived from MenY and/or MenW and/or MenC and/or MenA, having a size changed by no more than 10 times, and/or subjected to microfluidization. For example, the immunogenic composition contains a conjugate derived from native MenA and/or MenC and/or MenW and/or MenY. For example, the immunogenic composition contains a conjugate derived from native MenC. Thus, for example, the immunogenic composition includes a conjugate derived from native MenC and MenA, having a size changed by no more than 10 times, and/or subjected to microfluidization. Thus, for example, the immunogenic composition includes a conjugate derived from native MenC and MenY, having a size changed by no more than 10 times and/or subjected to microfluidization.

In one embodiment of the invention, the saccharide polydispersity is 1-1.5, 1-1.3, 1-1.2, 1-1.1, or 1-1.05, and after conjugation with a carrier protein, the saccharide polydispersity is 1.0-2.5, 1.0-2.0, 1.0-1.5, 1.0-1.2, 1.5-2.5, 1.7-2.2 or 1 .5-2.0. All polydispersity measurements were performed using MALLS.

The saccharides are optionally 1.5, 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20 times the size of the polysaccharide isolated from bacteria.

In one embodiment, each N. meningitidis saccharide is either a native polysaccharide or a polysaccharide that differs by no more than 10-fold. In another embodiment of the invention, each N. meningitidis capsular saccharide is a native polysaccharide. In another embodiment, at least one, two, three, or four N. meningitidis capsular saccharides have been sized by microfluidization. In another embodiment of the invention, each N. meningitidis capsular saccharide is sized no more than 10 times. In another embodiment of the invention, N. meningitidis conjugates are prepared from a mixture of native polysaccharides and saccharides that have been resized no more than 10 times. In another embodiment, the serogroup Y capsular saccharide is sized no more than 10-fold. In another embodiment of the invention, the capsular saccharides of serogroups A and C are native polysaccharides and the saccharides of serogroups W135 and Y are sized no more than 10-fold. In another embodiment of the invention, the average size of each N. meningitidis capsular saccharide is 50 kDa - 300 KDa or 50 kDa - 200 kDa. In another embodiment of the invention, the immunogenic composition comprises a MenA capsular saccharide having an average size greater than 50 kDa, 75 kDa, 100 kDa, or an average size of 50-100 kDa or 55-90 kDa or 60-80 kDa. In another embodiment of the invention, the immunogenic composition comprises a MenC capsular saccharide having an average size greater than 50 kDa, 75 kDa, 100 kDa, or 100-200 kDa, 100-150 kDa, 80-120 kDa, 90-110 kDa, 150-200 kDa , 120-240 kDa, 140-220 kDa, 160-200 kDa or 190-200 kDa. In another embodiment of the invention, the immunogenic composition comprises a MenY capsular saccharide having an average size greater than 50 kDa, 75 kDa, 100 kDa or 60-190 kDa or 70-180 kDa or 80-170 kDa or 90-160 kDa or 100-150 kDa , 110-145 kDa or 120-140 kDa. In another embodiment of the invention, the immunogenic composition comprises a MenW capsular saccharide having an average size greater than 50 kDa, 75 kDa, 100 kDa or 60-190 kDa or 70-180 kDa or 80-170 kDa or 90-160 kDa or 100-150 kDa , 140-180 kDa, 150-170 kDa or 110-140 kDa.

In one embodiment, the saccharide doses of each of the at least two, three, four conjugates or each N. meningitidis saccharide conjugate are, but not necessarily, the same or approximately the same.

In one embodiment, the immunogenic composition of the invention is adjusted to or buffered at or adjusted to pH 7.0-8.0, pH 7.2-7.6, or approximately or exactly pH 7.4.

The immunogenic composition or vaccines of the invention are optionally lyophilized in the presence of a stabilizing agent, for example a polyol such as sucrose or trehalose.

In the case of the N. meningitidis saccharide combinations discussed above, it may be preferable not to use any aluminum salt adjuvant, or not to use an adjuvant at all.

The active agent may be present in the pharmaceutical composition or in the vaccine according to the invention in various concentrations. Typically, the minimum concentration of a substance is the amount necessary to use it for the desired purpose, and the maximum concentration is the maximum amount that will remain in solution or be homogeneously suspended in the original mixture. Thus, for example, the minimum amount of therapeutic agent is, but not necessarily, the amount that provides one therapeutically effective dose. In the case of bioactive substances, the minimum concentration is the amount necessary to achieve biological activity after dilution, and the maximum concentration is the amount at which a homogeneous suspension cannot be maintained.

In another embodiment of the invention, the composition comprises a conjugate of Neisseria meningitidis serogroup X capsular polysaccharide and a carrier molecule. The group X capsular polysaccharide structure consists of N-acetylglucosamine-4-phosphate residues linked to each other via α-4-phosphodiester bonds without O-acetyl groups. The carrier molecule can be a diphtheria or tetanus toxoid, CRM 197, or protein D. In a preferred embodiment, as shown in the Examples, the composition does not include a N. meningitidis serogroup X capsular polysaccharide conjugate.

Stability

The terms "stable" and "stability" refer to the ability of an antigen to remain immunogenic for a certain period of time. Stability can be assessed by activity over a period of time. The terms "stable" and "stability" also refer to the physical, chemical and conformational stability of the immunogenic composition. Protein composition instability can be caused by chemical degradation or aggregation of protein molecules to form higher order polymers; dissociation of heterodimers into monomers; deglycosylation; glycosylation modification; or any other structural modification that reduces at least one biological activity of the protein composition included in the present invention. Stability can be assessed by well-known methods, including measurement of light scattering in a sample, apparent light attenuation (absorptivity or optical density), size (for example, using size exclusion chromatography), in vitro or in vivo biological activity and / or properties using differential scanning calorimetry (DSC). Other stability assessment methods are known to those skilled in the art and may also be used in accordance with the present invention.

In some embodiments of the invention, the antigen in the stable composition according to the invention may retain, compared with the reference standard, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96% , 97%, 98%, 99%, or 100% activity for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, 12 months, 18 months, 24 months, 30 months , 36 months, 42 months, 48 months, 54 months or 60 months. In some embodiments of the invention, the antigen in the stable composition according to the invention may retain, compared with the reference standard, at least 50% activity for at least 1 year, 2 years, 3 years, 4 years or 5 years. The terms "stable" and "stability" also refer to the ability of an antigen to retain epitopes or immunoreactivity over a period of time. Thus, for example, the antigen of a stable composition according to the invention can retain, compared with a reference standard, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% , 98%, 99%, or 100% epitopes or immunoreactivity for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, 12 months, 18 months, 24 months, 30 months , 36 months, 42 months, 48 months, 54 months or 60 months. In some embodiments of the invention, the stability is measured depending on environmental conditions. Non-limiting examples of environmental conditions are light, temperature, freeze/thaw cycles, agitation, and pH. The person skilled in the art can determine the presence of antigenic epitopes or immunoreactivity by the methods described here or other known methods. In some embodiments of the invention, the stability of the antigen is measured from the moment of its receipt. In some embodiments of the invention, the stability of the antigen is measured since the change in storage conditions. Non-limiting examples of changing storage conditions are from frozen to chilled, from frozen to room temperature, from chilled to room temperature, from chilled to frozen, from room temperature to frozen, from room temperature to chilled, transfer from light into darkness or start mixing.

In one embodiment of the invention, the terms "stable" and "stability" refer to the ability of an antigen to bind to aluminum. Thus, for example, a stable composition according to the invention comprises at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% a protein that binds to aluminum (e.g., aluminum phosphate) in the composition compared to a reference standard for at least 1 hour, 6 hours, 12 hours, 18 hours, 24 hours, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, 12 months, 18 months, 24 months, 30 months, 36 months, 42 months, 48 months, 54 months or 60 months. See, for example, Example 13. In a preferred embodiment, at least 90%, more preferably at least 95%, and most preferably at least 99% of the total rLP2086 subfamily A polypeptide (e.g., a polypeptide that includes the amino acid sequence shown in SEQ ID NO: 1) binds to aluminum in the composition. In a preferred embodiment of the invention, at least 90%, more preferably at least 95%, and most preferably at least 99% of the entire rLP2086 subfamily B polypeptide (e.g., a polypeptide that includes the amino acid sequence shown in SEQ ID NO : 2) binds to aluminum in the composition.

Determination of bonding with aluminum . The composition containing aluminum and at least one protein antigen was centrifuged so as to precipitate aluminum. Centrifugation of proteins adsorbed on aluminum is known to those skilled in the art. See, for example, Egan et al., Vaccine, Vol. 27(24): 3175-3180 (2009). The aluminum bound protein precipitated while the non-aluminum bound protein remained in the supernatant. The total protein in the supernatant and in the pellet was determined using a Lowry assay. The percentage of bound protein was calculated by dividing the amount of total protein in the supernatant by the amount of total protein added to the composition and multiplying the result by 100%. Similarly, the percentage of unbound protein was calculated by dividing the amount of total protein in the supernatant by the amount of total protein added to the composition and multiplying the result by 100%. For formulations containing both subfamily A and subfamily B antigens, the concentrations of individual A and B subfamily proteins in the supernatant were determined by ion exchange chromatography. Separation and elution of subfamily A and B proteins was performed on a strong anion column with a high salt eluent. Both A and B subfamily proteins were detected and quantified using a series of fluorescence detectors at excitation wavelength=280 nm and emission wavelength=310 nm. Subfamily A and subfamily B proteins were eluted at different retention times and quantified by plotting a standard curve over the data compared to the reference protein rLP2086. The percentage of unbound protein was calculated by dividing the amount of total protein in the supernatant by the amount of total protein added to the composition and multiplying the result by 100%. The percent bound protein was calculated by subtracting the percent unbound protein from 100%.

POLYSORBATE-80

Polysorbate 80 (PS-80) is a non-ionic surfactant. Prospective stability studies using an in vitro activity assay based on a monoclonal antibody demonstrated the stability of the B subfamily protein at higher molar ratios of PS-80 to the rLP2086 MnB protein in the final composition. Additional experiments with various ratios of PS-80 indicated that the optimal molar ratio of PS-80 to the MnB rLP2086 protein is approximately 2.8 ± 1.4, indicating retention of activity.

The concentration of PS-80 in the composition depends on the molar ratio of PS-80 to polypeptide. In one of the embodiments of the invention, the composition has a molar ratio of PS-80 to the first polypeptide and to the second polypeptide of 2.8±1.4. In one embodiment, the composition has a molar ratio of PS-80 to the first polypeptide and to the second polypeptide of 2.8±1.1. In one embodiment, the composition has a PS-80 to polypeptide molar ratio of at least 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2 .6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2 or 3.3. In one embodiment, the composition has a molar ratio of PS-80 to polypeptide of at most 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3 , 3.2, 3.1, 3.0 or 2.9. Any minimum value can be combined with any maximum value described here to obtain the appropriate interval. Preferably, the composition has a PS-80 to polypeptide molar ratio of 2.8.

The molar ratio of PS-80 to polypeptide is determined by calculating from the measured PS-80 concentration and the measured total polypeptide concentration, both in moles. For example, the molar ratio of PS-80 to protein is determined by calculating the ratio of the measured concentration of PS-80 (for example, using reverse-phase high pressure liquid chromatography (RP-HPLC)) to the measured concentration of total protein (for example, using ion exchange chromatography -high pressure liquid chromatography (HPLC-HPLC)) in the final drug substance, where both values are expressed in moles.

RP-HPLC is performed to quantify the concentration of polysorbate 80 in vaccine compositions. The detergent concentration is determined after saponification of the fatty acid group, and Polysorbate 80 is converted to free oleic acid by alkaline hydrolysis at 40°C. The sample was separated by RP-HPLC on a C18 column and quantified with a UV detector at 200 nm.

The first and second polypeptides are separated by anion exchange HPLC. Subfamily A and B rLP2086 (fHBP) proteins were eluted with different retention times and were quantified by standard curve plotting data versus data for the reference protein rLP2086.

The term "molar ratio" and description of an immunogenic composition comprising fHBP and PS-80 is also disclosed in WO2012025873 and US Patent Application Publication 2013/0171194, which are incorporated herein by reference in their entirety.

As used herein, the term "molar ratio" means the ratio of the number of moles of two different elements in a composition. In some embodiments, the mole ratio is the ratio of moles of detergent to moles of polypeptide. In some embodiments of the invention, the molar ratio is the ratio of moles of PS-80 to moles of protein. In one embodiment of the invention, based on the concentrations of protein and polysorbate 80, the molar ratio can be calculated using the following equation:

Molar ratio=% PS-80×216

mg/ml protein

In one of the embodiments of the invention, the composition includes a molar ratio of PS-80 to protein MnB rLP2086 from 1.4 to 4.2, indicating retention of activity. In one of the embodiments of the invention, the composition has a molar ratio of at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7 or 2.8. In one of the embodiments of the invention, the composition has a molar ratio of at most 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.7, 3.6, 3.5, 3, 4, 3.3, 3.2, 3.1, 3.0, 2.9 or 2.8. Any minimum value can be combined with any maximum value described here to obtain the appropriate interval.

In one embodiment, the composition comprises about 0.0015, 0.0017, 0.0019, 0.0021, 0.0023, 0.0025, 0.0027, 0.0029, 0.0031, 0.0033, 0.0035, 0.0037, 0.0039, 0.0041, 0.0043, 0.0045, 0.0047, 0.0049, 0.0051 mg/mL PS-80. Preferably, the composition comprises about 0.0035 mg/ml PS-80.

In another embodiment of the invention, the composition comprises at least 10 μg, 11 μg, 12 μg, 13 μg, 14 μg, 15 μg, 16 μg, 17 μg, 18 μg, 19 μg, 20 μg, 21 μg, 22 μg, 23 mcg, 24 mcg, or 25 mcg PS-80. In another embodiment, the composition comprises a maximum of 30 μg, 29 μg, 28 μg, 27 μg, 26 μg, 25 μg, 24 μg, 23 μg, 22 μg, 21 μg, 20 μg, 19 μg, or 18 μg PS-80 . Any minimum value can be combined with any maximum value described here to obtain the appropriate interval. In a preferred embodiment of the invention, the composition comprises at least 10 μg and a maximum of 20 μg of PS-80. In the most preferred embodiment of the invention, the composition comprises approximately 18 μg of PS-80.

In another embodiment of the invention, the composition comprises PS-80 at a concentration ranging from 0.0005% to 1%. For example, the concentration of PS-80 in the composition may be at least 0.0005%, 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0. 06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0, 7%, 0.8%, 0.9%, 1% or 1.1% PS-80. In one of the embodiments of the invention, the concentration of PS-80 in the composition can be a maximum of 2.0%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4% , 1.3%, 1.2%, 1.1%, 1.0%, 0.9%, 0.8%, or 0.7% PS-80. In a preferred embodiment of the invention, the composition comprises approximately 0.07% PS-80. Any minimum value can be combined with any maximum value described here to obtain the appropriate interval.

The present inventors have surprisingly found that although a composition comprising a combination of a first composition and a second composition may have a different molar ratio of polysorbate-80 to rLP2086 MnB polypeptides compared to the molar ratio of polysorbate-80 to rLP2086 MnB polypeptides in the first composition, however, an additional surfactant for the combined composition is surprisingly not necessary to maintain the solubility and stability of the rLP2086 MnB polypeptides in the combined composition. Accordingly, in one embodiment of the invention, the kit does not contain more than 0.02 mg of polysorbate-80.

Aluminum

The composition includes aluminum in the form of aluminum phosphate. AlPO ₄ is added as a stabilizer to improve processing properties and improve stability. A process for producing aluminum phosphate is described in US Patent Application Publication 2009/0016946, which is incorporated herein by reference in its entirety. In one embodiment of the invention, the composition also does not include a polyvalent non-aluminum cation. In one of the embodiments of the invention, the composition also does not include Al(OH) ₃ or Al(SO ₄ ) ₃ .

In one embodiment, the composition comprises at least 50 µg, 60 µg, 70 µg, 80 µg, 90 µg, 100 µg, 110 µg, 120 µg, 130 µg, 140 µg, 150 µg, 160 µg, 170 µg , 180 mcg, 190 mcg, 200 mcg, 210 mcg, 220 mcg, 230 mcg, 240 mcg or 250 mcg aluminum. In one embodiment, the composition comprises a maximum of 500 µg, 490 µg, 480 µg, 470 µg, 460 µg, 450 µg, 440 µg, 430 µg, 420 µg, 410 µg, 400 µg, 390 µg, 380 µg, 370 mcg, 360 mcg, 350 mcg, 340 mcg, 330 mcg, 320 mcg, 310 mcg, 300 mcg, 290 mcg, 280 mcg, 270 mcg, 260 mcg or 250 mcg aluminum. Any minimum value can be combined with any maximum value described here to obtain the appropriate interval. In the most preferred embodiment of the invention, the composition includes 250 μg of aluminum.

In one of the embodiments of the invention, the composition comprises at least 0.005 mg/ml, 0.01 mg/ml, 0.02 mg/ml, 0.03 mg/ml, 0.04 mg/ml, 0.05 mg/ml ml, 0.06 mg/ml, 0.07 mg/ml, 0.08 mg/ml, 0.09 mg/ml, 0.10 mg/ml, 0.2 mg/ml, 0.3 mg/ml , 0.4 mg/ml or 0.5 mg/ml aluminum phosphate. In one of the embodiments of the invention, the composition includes a maximum of 2.0 mg/ml, 1.9 mg/ml, 1.8 mg/ml, 1.7 mg/ml, 1.6 mg/ml, 1.5 mg/ml ml, 1.4 mg/ml, 1.3 mg/ml, 1.2 mg/ml, 1.1 mg/ml, 1.0 mg/ml, 0.9 mg/ml, 0.8 mg/ml , or 0.7 mg/ml PS-80. In a preferred embodiment of the invention, the composition comprises approximately 0.07 mg/ml PS-80. Any minimum value can be combined with any maximum value described here to obtain the appropriate interval. In a preferred embodiment of the invention, the composition comprises 0.5 mg/ml aluminum phosphate. In the most preferred embodiment of the invention, the composition comprises 0.5 mg aluminum/ml as aluminum phosphate (AlPO ₄ ). This concentration retains the binding (at least 90% binding or higher) of the A and B subfamily proteins to aluminum.

The present inventors have surprisingly found that although a composition comprising a combination of a first composition and a second composition can alter the percentage of aluminum-bound MnB rLP2086 polypeptides compared to the percentage of aluminum-bound MnB rLP2086 polypeptides in the first composition, however, the combination of the first and the second formulation unexpectedly retained binding of at least 90% of all rLP2086 MnB polypeptides to aluminum. Accordingly, in one embodiment of the invention, the percentage of all rLP2086 MnB polypeptides relative to aluminum in the combined composition is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. Preferably, the percentage of all rLP2086 MnB polypeptides relative to aluminum in the combined composition is at least 90%, more preferably at least 95%, and most preferably at least 100%.

In another embodiment of the invention, the concentration of aluminum-bound polypeptides in the immunogenic composition did not decrease after 24 hours compared to the concentration of aluminum-bound polypeptides in the liquid composition prior to reconstitution of the lyophilized composition. In another embodiment of the invention, the concentration of the MenA _AH -TT conjugate in the immunogenic composition did not decrease after 24 hours compared to the concentration of the MenA _AH -TT conjugate in the lyophilized composition. In one of the embodiments of the invention, the concentration was reduced by a maximum of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% after 24 hours compared to the corresponding concentration in the liquid formulation before dilution.

In another embodiment of the invention, the concentration of the MenC _AH -TT conjugate in the immunogenic composition did not decrease after 24 hours compared to the concentration of the MenC _AH -TT conjugate in the lyophilized composition. In another embodiment of the invention, the concentration of the MenW-TT conjugate in the immunogenic composition did not decrease after 24 hours compared to the concentration of the MenW-TT conjugate in the lyophilized composition. In another embodiment of the invention, the concentration of the MenY-TT conjugate in the immunogenic composition did not decrease after 24 hours compared to the concentration of the MenY-TT conjugate in the lyophilized composition. In one of the embodiments of the invention, the concentration was reduced by a maximum of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% after 24 hours compared to the corresponding concentration in the lyophilized formulation prior to dilution.

Fillers

In one of the embodiments of the invention, the composition includes histidine. In one embodiment, the composition comprises at least 650 µg, 660 µg, 670 µg, 680 µg, 690 µg, 700 µg, 710 µg, 720 µg, 730 µg, 740 µg, 750 µg, 760 µg, 770 µg , 780 mcg, 790 mcg, 800 mcg, 810 mcg, 820 mcg, 830 mcg, 840 mcg or 850 mcg histidine. In one of the embodiments of the invention, the composition includes a maximum of 1560 μg, 1500 μg, 1400 μg, 1300 μg, 1200 μg, 1100 μg, 1000 μg, 950 μg, 900 μg, 890 μg, 880 μg, 870 μg, 860 μg, 850 mcg, 840 mcg, 830 mcg, 820 mcg, 810 mcg, 800 mcg, 790 mcg or 780 mcg histidine. Any minimum value can be combined with any maximum value described here to obtain the appropriate interval. Preferably, the composition includes 780 µg of histidine.

In one of the embodiments of the invention, the composition includes Tris buffer, phosphate buffer or succinate buffer. In a preferred embodiment of the invention, the composition does not include Tris buffer. In a preferred embodiment of the invention, the composition does not include a phosphate buffer. In one preferred embodiment of the invention, the composition does not include a succinate buffer. In a preferred embodiment of the invention, the composition comprises a histidine buffer.

In one of the embodiments of the invention, the composition includes sodium chloride. The concentration of sodium chloride in the MenABCWY composition can vary between 160.5 and 161.1 mM.

In one of the embodiments of the invention, the pH of the composition is 5.5-7.5. In a preferred embodiment of the invention, the pH of the composition is 5.8-7.0, and most preferably, pH 5.8-6.0. In one of the embodiments of the invention, the pH of the composition is a maximum of 6.1. In one embodiment of the invention, the pH of the composition is 5.8.

Sets

Another aspect of the invention is a kit for administering a dose of a composition for generating bactericidal antibodies against Neisseria meningitidis in a mammal.

In one aspect of the invention, the kit includes a first composition containing the first polypeptide described above and the second polypeptide described above. In a preferred embodiment, the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1. In another preferred embodiment, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2. The kit also includes a second composition containing a MenA conjugate _AH -TT, MenC conjugate _AH -TT, MenW-TT conjugate and MenY-TT conjugate. In one of the embodiments of the invention, the set includes at least two containers, where the first container contains the first composition, and the second container contains the second composition.

In one of the embodiments of the invention, the kit includes a liquid first composition and a lyophilized second composition. Preferably, the kit includes a liquid divalent MnB rLP2086 composition and a lyophilized MenACWY-TT composition.

The present inventors have surprisingly found that although the composition comprising the combination of the first composition and the second composition may have a different molar ratio of polysorbate-80 to the MnB rLP2086 polypeptides in the first composition, however, it was surprising that the additional surfactant for the combined composition is not necessary to maintain the solubility and stability of the rLP2086 MnB polypeptides in the combined composition. Accordingly, in one embodiment of the invention, the kit does not contain more than 0.02 mg of polysorbate-80.

In one embodiment, the kit also does not include any of the following commercially available immunogenic compositions: MENACTRA(R), MENVEO(R), ADACEL(R), HAVRIX(R), GARDASIL(R), REPEVAX, or any combination thereof . For example, the kit also preferably does not include a meningococcal polysaccharide A, C, Y and W-135 (MCV4) conjugate composition wherein the carrier protein is a diphtheria toxoid. In one embodiment, the kit also preferably does not include a meningococcal polysaccharide A, C, Y and W-135 (MCV4) conjugate composition wherein the carrier protein is CRM ₁₉₇ . In one embodiment of the invention, the kit also does not include the NIMENRIX vaccine, where NIMENRIX contains a diluent consisting of sodium chloride and water.

Bactericidal activity

The incidence of MnB is approximately 1 in 100,000, which means that a very large number of individuals (400,000 to over 6 million) may be needed to confirm a statistically significant efficacy estimate. Thus, the assay for serum bactericidal activity using human complement (hSBA), which is a surrogate marker of vaccine protection and efficacy, is used to assess immunogenicity in clinical trials.

Pfizer, from 2000 to 2006, collected a large set of MnB strains (N=at least 1263) including isolates that cause IMZ. MnB isolates have been systematically collected by the US Centers for Disease Control and Prevention (CDC) and Health Protection Laboratories and Reference Laboratories in Europe.

In one embodiment of the invention, the immune response induced by administration of the composition to a human is determined using a human complement serum bactericidal assay (hSBA) against four N. meningitidis serogroup B (MnB) strains. The MnB strains used in hSBA were selected from a pool of strains. The strain pool is a collection of systematically collected clinically relevant strains of N. meningitidis .

The high level of hSBA response to all tested strains, and in particular strains expressing 2086 lipoprotein variants with sequences homologous to the first polypeptide and the second polypeptide, suggests that such a composition is a broad-spectrum protective vaccine that is sufficient to impart a high seroprotective effect. activity against strains of N. meningitidis expressing rLP2086 (FHBP) of at least serogroup B, including additional serogroups such as serogroup X.

Subfamily A strains

In one embodiment, the hSBA strain is a N. meningitidis strain expressing LP2086 subfamily A protein (fHBP). In one embodiment, the hSBA strain is an LP2086 subfamily A (fHBP) strain that expresses a 2086 lipoprotein variant heterologous to an A05-expressing N. meningitidis strain. Thus, for example, in one embodiment of the invention, the hSBA strain is an LP2086 subfamily A (fHBP) strain that expresses a 2086 lipoprotein variant heterologous to the M98250771 strain.

In one embodiment, the hSBA strain is a N. meningitidis strain expressing fHBP A10. In one embodiment, the hSBA strain is a N. meningitidis strain expressing LP2086 (fHBP) A22. In one embodiment, the hSBA strain is a N. meningitidis strain expressing LP2086 (fHBP) A56. In another embodiment of the invention, the hSBA strains are strains LP2086 (fHBP) A22 and LP2086 (fHBP) A56. In another embodiment, the hSBA strain is a N. meningitidis strain expressing LP2086 A04. In one embodiment, the hSBA strain is a N. meningitidis strain expressing LP2086 A05. In one embodiment, the hSBA strain is a N. meningitidis strain expressing LP2086 A12. In one embodiment, the hSBA strain is a N. meningitidis strain expressing LP2086 A22. In one embodiment, the hSBA strain is a N. meningitidis strain expressing LP2086 A12. In one embodiment, the hSBA strain is a N. meningitidis strain expressing LP2086 A04. In one embodiment, the hSBA strain is a N. meningitidis strain expressing LP2086 A19. In one embodiment, the hSBA strain is a N. meningitidis strain expressing LP2086 A07. In another embodiment of the invention, the hSBA strain includes any of the strains expressing A22, A12, A19, A05 and A07. In one of the embodiments of the invention, the hSBA strains include any of the strains expressing A06, A15 and A29.

In one embodiment, the immune response is bactericidal against a N. meningitidis fHPB subfamily A strain of serogroup B that is heterologous to an A05-expressing N. meningitidis strain. In one embodiment, the immune response is directed against N. meningitidis serogroup B strain A22. In one embodiment, the immune response is directed against N. meningitidis serogroup B strain A56. In one embodiment, the immune response is directed against against strain A06 of N. meningitidis serogroup B. In one embodiment, the immune response is directed against strain A15 of N. meningitidis serogroup B. In one embodiment, the immune response is directed against strain A29 of N. meningitidis serogroup B. In one embodiment, the immune response is directed against N. meningitidis serogroup B strain A62. In one embodiment, the immune response is bactericidal against a strain of N. meningitidis serogroup B subfamily A that is heterologous to the N strain. meningitidis M98250771.

In one embodiment, the immune response is bactericidal against a N. meningitidis subfamily A strain of serogroup B that expresses a factor H binding protein and includes an amino acid sequence that is at least 80%, 81%, 82% 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the first polypeptide. In another embodiment, the immune response is bactericidal against a N. meningitidis subfamily A strain of serogroup B that expresses a factor H binding protein and includes an amino acid sequence that is at least 80%, 81%, 82%, 83 %, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to factor H binding protein expressed by N. meningitidis strain M98250771. In a preferred embodiment of the invention, the immune response is bactericidal against a N. meningitidis subfamily A strain of serogroup B that expresses a factor H binding protein and comprises an amino acid sequence that is at least 80%, and more preferably at least 84% identical to the factor H binding protein expressed by the N. meningitidis strain M98250771.

In another embodiment, the immune response is bactericidal against a N. meningitidis subfamily A strain of serogroup B that expresses a factor H binding protein and includes an amino acid sequence that is at most 81%, 82%, 83%, 84% 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the first polypeptide . In another embodiment, the immune response is bactericidal against a N. meningitidis subfamily A strain of serogroup B that expresses a factor H binding protein and includes an amino acid sequence that is at most 81%, 82%, 83%, 84% 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% protein identical, binding to factor H and expressed by the strain N. meningitidis M98250771. In a preferred embodiment of the invention, the immune response is bactericidal against a N. meningitidis subfamily A strain of serogroup B that expresses a factor H binding protein and comprises an amino acid sequence that is at most 85%, and more preferably at most 99% identical. factor H binding protein expressed by N. meningitidis strain M98250771. Any minimum value can be combined with any maximum value described here to obtain the appropriate interval.

In one embodiment, the immune response elicited by the composition is bactericidal not only against an N. meningitidis fHPB subfamily A strain of serogroup B, but also against an N. meningitidis strain expressing an fHBP subfamily A polypeptide where such serogroup is not is serogroup B. Thus, for example, in one of the preferred embodiments of the invention, the immune response generated by the composition has a bactericidal effect against a strain of subfamily A N. meningitidis serogroup B and against a strain of N. meningitidis serogroup C, which express the polypeptide of the subfamily A fHBP, heterologous fHBP A05. Thus, for example, in one embodiment of the invention, the immune response is directed against a strain of N. meningitidis serogroup C expressing fHBP A10. In another embodiment of the invention, the immune response is directed against a strain of N. meningitidis serogroup W expressing fHBP A19. In one embodiment, the immune response is bactericidal against a strain of N. meningitidis that expresses an fHBP subfamily A polypeptide, wherein said strain is heterologous to N. meningitidis strain M98250771.

Subfamily B strains

In one embodiment, the hSBA strain is a subfamily B LP2086 (fHBP) strain. In one embodiment, the hSBA strain is an LP2086 (fHBP) subfamily B strain expressing a 2086 lipoprotein variant that is heterologous to a B01-expressing N. meningitidis strain. Thus, for example, in one embodiment of the invention, the hSBA strain is an LP2086 (fHBP) subfamily B strain expressing a 2086 lipoprotein variant that is heterologous to the CDC1127 strain. In a preferred embodiment, the hSBA strain is an LP2086 (fHBP) subfamily B strain expressing a 2086 lipoprotein variant that is heterologous to the CDC1573 strain.

In one embodiment, the immune response is bactericidal against a N. meningitidis serogroup B fHPB subfamily B strain that is heterologous to a B01-expressing N. meningitidis strain. In one embodiment, the immune response is directed against the B24 strain of N. meningitidis serogroup B. In one embodiment, the immune response is directed against the B44 strain of N. meningitidis serogroup B. In one embodiment, the immune response is directed against serogroup B N. meningitidis strain B16. In one embodiment, the immune response is directed against N. meningitidis serogroup B strain B03. In one embodiment, the immune response is directed against N. meningitidis serogroup B strain B09. In one embodiment, the immune response is directed against N. meningitidis serogroup B strain B15. In one embodiment, the immune response is directed against N. meningitidis serogroup B strain B153. In one embodiment, the immune response is tank tericidal activity against N. meningitidis serogroup B fHPB subfamily strain, which is heterologous to N. meningitidis strain CDC1573.

In one embodiment, the immune response is bactericidal against a strain of subfamily B of N. meningitidis serogroup B that expresses a protein that binds to factor H and includes an amino acid sequence that is at least 80%, 81%, 82% 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the second polypeptide. In another embodiment, the immune response is bactericidal against a subfamily B strain of N. meningitidis serogroup B that expresses a factor H binding protein and includes an amino acid sequence that is at least 80%, 81%, 82%, 83 %, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to factor H binding protein expressed by N. meningitidis CDC1573. In a preferred embodiment, the immune response is bactericidal against a N. meningitidis serogroup B subfamily B strain that expresses a factor H binding protein and includes an amino acid sequence that is at least 80%, and more preferably at least 87% identical to the factor H binding protein expressed by N. meningitidis CDC1573. In another preferred embodiment of the invention, the immune response is bactericidal against a strain of subfamily B of N. meningitidis serogroup B that expresses a factor H binding protein and includes an amino acid sequence that is 100% identical to the factor H binding protein expressed by the strain. N. meningitidis CDC1573.

In one embodiment, the immune response is bactericidal against a subfamily B strain of N. meningitidis serogroup B that expresses a protein that binds to factor H and includes an amino acid sequence that is at most 81%, 82%, 83%, 84% , 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the second polypeptide. In another embodiment, the immune response is bactericidal against a N. meningitidis subfamily A strain of serogroup B that expresses a factor H binding protein and includes an amino acid sequence that is at most 81%, 82%, 83%, 84% 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% protein identical, binding to factor H and expressed by N. meningitidis CDC1573. In a preferred embodiment of the invention, the immune response is bactericidal against a strain of subfamily B of N. meningitidis serogroup B that expresses a protein that binds to factor H and includes an amino acid sequence that is at most 88%, and more preferably at least 99% % is identical to the protein that binds to factor H and is expressed by the strain N. meningitidis CDC1573. Any minimum value can be combined with any maximum value described here to obtain the appropriate interval.

In one embodiment, the hSBA strain is B24 LP2086 (fHBP) strain. In another embodiment of the invention, the hSBA strain is the B44 LP2086 (fHBP) strain. In another embodiment of the invention, the hSBA strains include strains B24 LP2086 (fHBP) and B44 LP2086 (fHBP). In one embodiment, the hSBA strains include A22 LP2086 (fHBP), A56 LP2086 (fHBP), B24 LP2086 (fHBP) and B44 LP2086 (fHBP). In one of the embodiments of the invention, the hSBA strain includes B15. In one embodiment, the hSBA strain includes B153. In another embodiment of the invention, the hSBA strain is the B16 LP2086 strain. In one embodiment, the hSBA strain is the B03 LP2086 strain. In one embodiment, the hSBA strain is the B09 LP2086 strain. In another embodiment of the invention, the hSBA strains include B24, B16, B44, B03 and B09, or any combination thereof. In another embodiment of the invention, the hSBA strains include B24, B16, B44, A22, B03, B09, A12, A19, A05 and A07, or any combination thereof. In another embodiment, the hSBA strains include A06, A07, A12, A15, A19, A29, B03, B09, B15 and B16, or any combination thereof.

In one embodiment, the method induces an immune response against N. meningitidis fHPB subfamily A strain of serogroup B and against N. meningitidis fHPB subfamily B strain of serogroup B. Preferably, the immune response is bactericidal against N. meningitidis fHPB subfamily A strain serogroup B and against a strain of subfamily B fHPB N. meningitidis serogroup B.

In one embodiment, the immune response elicited by the composition is bactericidal not only against an fHPB subfamily B strain of N. meningitidis serogroup B, but also against a N. meningitidis strain of fHBP subfamily B polypeptide where such serogroup is not is serogroup B. Thus, for example, in one of the preferred embodiments of the invention, the immune response generated by the composition has a bactericidal effect against a strain of subfamily B of N. meningitidis serogroup B and against a strain of N. meningitidis serogroup Y, which express the polypeptide of the subfamily In fHBP, heterologous fHBP B01. Thus, for example, in one embodiment of the invention, the immune response is directed against a N. meningitidis serogroup A strain expressing B16 fHBP. In another embodiment of the invention, the immune response is directed against a N. meningitidis serogroup Y strain expressing B47 fHBP. In one embodiment, the immune response is directed against a N. meningitidis serogroup X strain expressing B49 fHBP. In one embodiment, the immune response is bactericidal against a strain of N. meningitidis that expresses an fHBP subfamily B polypeptide, wherein said strain is heterologous to a strain of N. meningitidis serogroup B CDC1573.

Credits

In one of the embodiments of the invention, the composition induces an increase in bactericidal titer in humans compared to bactericidal titer in humans prior to administration of the dose of the composition, where the assessment is carried out under identical conditions in hSBA. In one embodiment of the invention, the increase in bactericidal titer is compared to the bactericidal titer in a human prior to administration of the first dose of the composition, if such an assessment is carried out under identical conditions in hSBA. In one embodiment of the invention, an increase in titer is observed after administration of the second dose of the composition compared to the bactericidal titer in humans prior to administration of the second dose of the composition, if such an assessment is carried out under identical conditions in hSBA. In another embodiment of the invention, an increase in titer is observed after the administration of the third dose of the composition compared to the bactericidal titer in humans before the administration of the third dose of the composition, if such an assessment is carried out under identical conditions in hSBA.

In one embodiment, the composition induces a bactericidal titer in a post-dose human, wherein the bactericidal titer is at least 1-fold greater than the bactericidal titer in a pre-dose human when such assessment is performed under identical conditions in hSBA. So, for example, the bactericidal titer can be at least 1.01 times, 1.1 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 fold, 11 fold, 12 fold, 13 fold, 14 fold, 15 fold, or 16 fold the post-dose human titer of the composition compared to the pre-dose human bactericidal titer when such assessment is performed under identical hSBA conditions.

In one embodiment, the term “responder” refers to a human in which the composition induces a post-dose bactericidal titer, and where the bactericidal titer is at least 1-fold greater than the bactericidal titer in the pre-dose human. In a preferred embodiment of the invention, the respondent has an increase in hSBA titer of at least ≥ 4 times compared to the human bactericidal titer prior to dosing. Such a respondent may be referred to as a respondent having a protective titer. In some embodiments, the protective titer is greater than 1:4.

In one embodiment, the hSBA titer is inversely proportional to the highest dilution of the serum sample that produces a measurable effect. Thus, for example, in one embodiment of the invention, the hSBA titer is inversely proportional to the highest 2-fold dilution of the test serum, which gives at least a 50% reduction in MnB bacteria (50% bacterial survival) compared to the c.f.u. T30 (i.e., number of bacteria surviving after incubation in assay wells containing all assay components except test serum; 100% bacterial survival).

In one embodiment, the composition induces a bactericidal titer in a human after the first dose that is at least 2-fold greater than the bactericidal titer in a human before the first dose (e.g., greater than the bactericidal titer in a human in the absence of the first dose), if such the assessment is carried out under identical conditions in hSBA. In one embodiment of the invention, the composition induces a bactericidal titer in a human that is at least 4 times the bactericidal titer in a human prior to the first dose if such assessment is carried out under identical conditions in a human serum bactericidal assay that uses human complement (hSBA). In one embodiment of the invention, the composition induces a bactericidal titer in a human that is at least 8 times the bactericidal titer in a human prior to the first dose if such assessment is carried out under identical conditions in a human serum bactericidal assay that uses human complement (hSBA).

In a preferred embodiment of the invention, the human serum complement is derived from a human with low natural bactericidal activity against a given test hSBA strain. Low natural bactericidal activity means, for example, that the bactericidal titer is effective against a given test strain in hSBA at a dilution of at least less than 1:4. In one embodiment, the human complement is from a human whose hSBA titer is effective against the strain tested in hSBA at a dilution of at least less than 1:4, such as 1:2 if the composition was not administered to a human.

The human may have an hSBA titer of less than 1:4 prior to administration of the composition, such as rLP2086 divalent formulation, or the individual may have an hSBA titer of ≧1:4 prior to administration of the composition. Accordingly, in preferred embodiments and examples, administration of at least one dose of the composition to a human will result in at least a 4-fold increase in hSBA titer compared to the human titer prior to administration. In some embodiments, administration of at least one dose of the composition to a human will result in an increase in hSBA titer of at least more than 1:4, e.g. hSBA titer ≥1:8, hSBA titer ≥1:16, and hSBA titer ≥1:32. The Examples presented here describe the evaluation of the ratio of the number of individuals in which the hSBA titer was ≥1:8 and/or ≥1:16 when the bivalent formulation of rLP2086 was administered to a human. In some embodiments of the invention, a 4-fold increase in titer in humans after administration of the composition compared to the titer before administration of the composition indicates that such protection is associated with administration of the composition. In some embodiments of the invention, such a preferred assessment of hSBA titers greater than 1:4 indicates that a protective effect, ie, a bactericidal immune response, induced in the human is associated with the composition.

In one embodiment, the human has an hSBA titer equal to or greater than the lower limit of hSBA quantification (LLOQ) after administration of the first dose of the composition. In another embodiment of the invention, the human has an hSBA titer equal to or greater than the hSBA LLOQ after administration of the second dose of the composition. In another embodiment of the invention, the human has an hSBA titer equal to or greater than the hSBA LLOQ after administration of the third dose of the composition.

Methods and Introduction

In one of its aspects, the present invention relates to a method for inducing an immune response against N. meningitidis in a human. In another aspect, the present invention relates to a method for vaccinating a human. In one embodiment, the method comprises administering to a human at least one dose of the composition described above. In a preferred embodiment of the invention, the method comprises administering to a human a maximum of one dose of the composition described above. In another embodiment of the invention, the method includes administering to a human at least a first dose and a second dose of the composition described above.

In one embodiment, the second dose is administered at least 20, 30, 50, 60, 100, 120, 160, 170, or 180 days after the first dose, and at most 250, 210, 200, or 190 days after the first dose. doses. Any minimum value can be combined with any maximum value described here to obtain the appropriate interval.

In another embodiment of the invention, the second dose is administered approximately 30 days after the first dose. In another embodiment of the invention, the second dose is administered approximately 60 days after the first dose, such as a 0.2 month immunization schedule. In another embodiment of the invention, the second dose is administered approximately 180 days after the first dose, such as a 0.6 month immunization schedule. In another embodiment of the invention, the second dose is administered approximately 120 days after the first dose, for example, according to the immunization schedule of 2, 6 months.

In one embodiment of the invention, the method comprises administering two doses and a maximum of two doses of the composition to a human. In one embodiment, two doses are administered over a period of approximately 6 months after the first dose. In one of the embodiments of the invention, the method does not include additional administration of a booster dose to a person. As used herein, the term "booster dose" refers to additional administration of the composition to a human. It may be preferable to administer a maximum of two doses of the composition to a human. Such advantages are, for example, facilitating a person's full compliance with an administration regimen and reducing the material cost of such an administration regimen.

In one embodiment, a first dose and a second dose are administered to a human over a period of approximately 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170 , 180, 190 or 200 days, and a maximum of 400, 390, 380, 370, 365, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210 or 200 days after the first dose. Any minimum value can be combined with any maximum value described here to obtain the appropriate interval. Preferably, the first and second doses are administered at least 4 weeks apart, eg, ≥8 weeks apart, ≥2 months apart, ≥3 months apart, ≥6 months apart, and the like.

In one embodiment of the invention, the first dose and the second dose are administered to a human over a period of approximately 30 days. In another embodiment of the invention, the first dose and the second dose are administered to a human over a period of approximately 60 days. In another embodiment of the invention, the first dose and the second dose are administered to a human for about 180 days.

Preferably, the first dose is given substantially at the same time (for example, at the same medical consultation or doctor's visit, or 24 hours after the first dose of meningococcal vaccine) with another vaccine, such as hepatitis B virus vaccine, diphtheria vaccine , tetanus vaccine, pertussis vaccine (cellular or preferably non-cellular), Haemophilus influenzae type b vaccine, Streptococcus pneumoniae vaccine and/or polio vaccine (preferably inactivated poliovirus vaccine). Each of these optionally co-administered vaccines may be a monovalent vaccine or part of a combination vaccine (eg, part of a DTP vaccine).

Preferably, the second dose is given substantially at the same time (for example, at the same medical consultation or doctor's visit or 24 hours after the first dose of meningococcal vaccine) with the other vaccine, for example, simultaneously with the hepatitis B virus vaccine. , diphtheria vaccine, tetanus vaccine, pertussis vaccine (cellular or non-cellular), Haemophilus influenzae type b vaccine, Streptococcus pneumoniae vaccine, polio vaccine (preferably inactivated poliovirus vaccine), influenza vaccine, chickenpox vaccine, vaccine against measles, mumps and/or rubella vaccines. Each of these optionally co-administered vaccines may be a monovalent vaccine or part of a combination vaccine (eg, part of an MMR vaccine).

Preferably, the third dose is given substantially at the same time (for example, at the same medical consultation or doctor's visit, or 24 hours after the third dose of meningococcal vaccine) with another vaccine, for example, simultaneously with the hepatitis B virus vaccine. , diphtheria vaccine, tetanus vaccine, pertussis vaccine (cellular or non-cellular), Haemophilus influenzae type b vaccine, Streptococcus pneumoniae vaccine, polio vaccine (preferably inactivated poliovirus vaccine), influenza vaccine, chickenpox vaccine, vaccine against measles, mumps and/or rubella vaccines. Each of these optionally co-administered vaccines may be a monovalent vaccine or part of a combination vaccine (eg, part of an MMR vaccine).

Three doses

In one embodiment, the three dose schedule of the composition induces a bactericidal titer in humans against multiple strains expressing LP2086 (fHBP) heterologous to the first and/or second polypeptide at a higher percentage than the two dose schedule.

In one embodiment, the method comprises administering three doses of the composition to a human. In another embodiment of the invention, the method comprises administering a maximum of three doses of the composition to a human. In one embodiment of the invention, three doses are administered approximately 6 months after the first dose. In one embodiment, the method comprises administering a booster dose to a human after the third dose. In another embodiment of the invention, the method does not include administering a booster dose to the human after the third dose. In another embodiment of the invention, the method also does not include administering a quarter dose or booster dose of the composition to a human. In another embodiment of the invention, a maximum of three doses are administered to a human over a period of approximately 6 months.

In a representative embodiment of the invention, the second dose is administered approximately 30 days after the first dose, and the third dose is administered approximately 150 days after the second dose, for example, in a 0, 1, 6 month immunization schedule. In another representative embodiment of the invention, the second dose is administered approximately 60 days after the first dose, and the third dose is administered approximately 120 days after the second dose, for example, in a 0, 2, 6 month immunization schedule.

In one embodiment, the first dose, second dose, and third dose are administered to a human for approximately 150, 160, 170, or 180 days, and a maximum of 240, 210,200, or 190 days. Any minimum value can be combined with any maximum value described here to obtain the appropriate interval. Preferably, the first dose, second dose, and third dose are administered to a human over a period of approximately 180 days or 6 months. Thus, for example, a second dose may be administered to a human approximately 60 days after the first dose, and a third dose may be administered to a human approximately 120 days after the second dose. Accordingly, a representative administration schedule includes administering a dose to a human for approximately 0, 2, and 6 months.

As described above, multiple doses of an immunogenic composition may be administered to a human, and the number of days between administration of each dose may vary. The advantage of such a method is, for example, the ease with which such administration regimens can be followed by a person.

In one embodiment, the method comprises administering a maximum of three doses of the same immunogenic composition to a human. Thus, for example, in a preferred embodiment of the invention, the method does not include administering a first dose of a first composition to a human, administering a second dose of a second composition to a human, and administering a third dose of a third composition to a human, wherein the first, second, and third compositions are not identical. In another embodiment of the invention, the method comprises administering a maximum of four doses of the same immunogenic composition to a human.

Examples

The following examples illustrate embodiments of the invention. Unless otherwise noted, the following Examples consider a bivalent composition of MnB rLP2086 at a dose of 120 μg of bivalent rLP2086, where a preferred representative formulation includes: 60 μg of the first lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 per dose 0, 5 ml; 60 μg of a second lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 per 0.5 ml dose; the molar ratio of polysorbate-80 to the first polypeptide of 2.8; the molar ratio of polysorbate-80 to the second polypeptide of 2.8; 0.5 mg Al ³⁺ /ml composition; 10 mM histidine and 150 mM sodium chloride.

More specifically, the experimental bivalent recombinant rLP2086 vaccine administered at a dose of 120 μg of bivalent rLP2086 comprises: (a) 60 μg of a first lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1; (b) 60 μg of a second lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2; (c) 18 μg polysorbate-80; (d) 250 µg aluminum; (e) 780 µg histidine, and (f) 4380 µg sodium chloride. Each dose is 0.5 ml.

Unless otherwise noted, the following Examples refer to MenACWY-TT, which is a preferred representative tetravalent meningococcal polysaccharide conjugated composition and which includes Neisseria meningitidis A, C, W-135, and Y capsular polysaccharides, each of which associated with tetanus toxoid as a carrier protein. Neisseria meningitidis serogroups A and C polysaccharides are conjugated with adipic acid dihydrazide (AH) spacer and indirectly conjugated with tetanus toxoid, while W-135 and Y polysaccharides are conjugated directly with tetanus toxoid. This composition does not contain any preservatives or adjuvants.

More specifically, the MenACWY-TT lyophilized formulation described in the Examples below comprises 5 micrograms of Neisseria meningitidis serogroup A polysaccharide conjugated to a tetanus toxoid carrier protein; 5 micrograms of Neisseria meningitidis serogroup C polysaccharide conjugated to a tetanus toxoid carrier protein; 5 micrograms of Neisseria meningitidis serogroup W-135 polysaccharide conjugated to a tetanus toxoid carrier protein; 5 micrograms of Neisseria meningitidis serogroup Y polysaccharide conjugated to a tetanus toxoid carrier protein; 28 mg sucrose; 97 micrograms of trometamol per dose (0.5 ml).

Example 1: Composition MenABCWY

The final MenABCWY formulation is prepared by diluting the lyophilized MenACWY-TT drug product (described in Example 2 below) in a vial with 0.67 milliliters of rLP2086 divalent MnB drug product (described in Example 3 below) to produce a 0.5 ml dose of MenABCWY vaccine for intramuscular injection . All components used to prepare the MenABCWY vaccine and their functions are described in Table 1 below.

Table 1. Composition of the MenABCWY vaccine Ingredients Quantity/dose MnB rLP2086 subfamily A (SEQ ID NO: 1) 60 mcg MnB rLP2086 subfamily B (SEQ ID NO: 2) 60 mcg MenA _AH -TT conjugate
(mean ratio TT/polysaccharide: ~3) 5 mcg MenA
~7.5 µg TT MenC _AH -TT conjugate
(mean ratio TT/polysaccharide: ~3) 5 mcg MenC
~7.5 µg TT MenW-TT conjugate
(mean ratio TT/polysaccharide: ~1.5) 5 mcg MenW
~3.75 µg TT MenY-TT conjugate
(mean ratio TT/polysaccharide: ~1.3) 5 mcg MenY
~3.25 µg TT Tris-HCl 97 mcg Sodium chloride ^a 4.69-4.71 mg sucrose 28 mg L-Histidine 0.78 mg Polysorbate 80 (PS80) 0.02 mg aluminum phosphate 0.25 mg aluminum Water for injections Ven. count up to 0.5 ml ^a The concentration of sodium chloride in the MenABCWY vaccine can vary between 160.5 and 161.1 mM based on the batch composition of the NIMENRIX clinical and commercial drug product (DP).

Example 2: Description and composition of the divalent drug product MnB rLP2086

The rLP2086 MnB bivalent drug product is a sterile liquid composition consisting of rLP2086 subfamily A and B proteins formulated at 120 µg/mL/subfamily in 10 mM histidine buffer, 150 mM sodium chloride (NaCl) at pH 6.0 s 0. 5 mg/ml aluminum as aluminum phosphate (AlPO4). Polysorbate 80 (PS-80) is added to the drug substance to obtain the desired PS-80:protein molar ratio. Therefore, PS-80 is not added during the preparation of the drug product, but it is present in the final drug product in the same ratio. This medicinal product is filled into 1 ml syringes. One dose of the vaccine is 0.5 ml and contains no preservatives.

Table 2 Composition of the bivalent drug product MnB rLP2086 Ingredients Quantity/dose MnB rLP2086 subfamily A (SEQ ID NO: 1) 120 µg/ml MnB rLP2086 subfamily B (SEQ ID NO: 2) 120 µg/ml Sodium chloride L-Histidine aluminum phosphate 0.50 mg aluminum phosphate/ml Water for injections Ven. count up to 1 ml ^a Polysorbate 80 (PS-80) is part of the drug substance. PS-80 functions as a surfactant in the drug product.
^b Equivalent to 0.25 mg aluminum per dose

Effect of Polysorbate 80 concentration

Polysorbate 80 (PS-80) is a non-ionic surfactant. It is used to stabilize and solubilize the rLP2086 MnB A and B subfamily proteins in the formulation by preventing aggregation and adsorption that can be caused by temperature changes, filtration, tubing, container/closed system contact, and mixing. Stability studies using an in vitro monoclonal antibody activity assay demonstrated instability of the B subfamily protein at higher molar ratios of PS-80 to rLP2086 MnB protein in the final composition. Experiments with different molar ratios of PS-80 to protein demonstrated that the optimal molar ratio of PS-80 to MnB rLP2086 protein is approximately 1.4-4.2, indicating retention of activity.

Example 3 Description and Composition of MenACWY-TT

The MenACWY-TT drug product consists of purified Neisseria meningitidis serogroups A, C, W, and Y polysaccharides, each conjugated to tetanus toxoid (TT) in relation to polysaccharide ~3, ~3, ~1.5, and ~1.3 , respectively.

The MenACWY-TT drug product is presented as a lyophilized powder supplied in a suitable 3 ml glass vial for lyophilization with a bromobutyl rubber stopper and aluminum screw caps. All components used to prepare the MenACWY-TT drug product and their functions are shown in Table 3.

Table 3. Composition of the drug product MenACWY-TT Ingredients Quantity/dose MenA _AH -TT conjugate
(mean ratio TT/polysaccharide: ~3) 5 mcg MenA
~15 µg TT MenC _AH -TT conjugate
(mean ratio TT/polysaccharide: ~3) 5 mcg MenC
~15 µg TT MenW-TT conjugate
(mean ratio TT/polysaccharide: ~1.5) 5 mcg MenW
~7.5 µg TT MenY-TT conjugate
(mean ratio TT/polysaccharide: ~1.3) 5 mcg MenY
~6.5 µg TT Tris-HCl 97 mcg sucrose 28 mg Sodium chloride ^a 306.0-325.0 mg ^a The lyophilized filter cake also contains sodium chloride resulting from the presence of salt in each of all purified TT conjugates. The concentration of sodium chloride varies between 10.5-11.1 mm depending on the composition of clinical and commercially available batches.

Example 4: Getting the MenABCWY Composition

The final MenABCWY formulation was prepared in the clinic by diluting the lyophilized MenACWY-TT drug product in a vial with 0.67 milliliters of divalent MnB rLP2086. The resulting MenABCWY composition (liquid vaccine drug product) contains rLP2086 subfamily A and B proteins at 120 μg/ml/subfamily, purified Neisseria meningitis type A, C, W, and Y polysaccharides at 10 μg/ml/type, conjugated with tetanus toxoid in ratios ~3, ~3, ~1.5 and ~3, respectively, in 10 mM histidine and 1.6 mM Tris buffer containing 160.5-161.1 mM sodium chloride, 0.5 mg/ml aluminum as aluminum phosphate (AlPO4), 0.035 mg/ml polysorbate 80 and 56 mg/ml sucrose at pH 6.05 for intramuscular injection.

The MenABCWY vaccine is prepared by mixing two drug products, MenACWY-TT and divalent MnB rLP2086. Buffering components and excipients were selected depending on the individual method of preparation of each component and have been shown to provide the necessary stability profile for longer shelf life of the product.

Dose verification studies have been conducted to demonstrate that the MenACWY-TT drug product and the bivalent MnB drug product rLP2086 are compatible when mixed to administer the MenABCWY vaccine, and that all drug products and dosing solutions are compatible with the administered components, and that dosing solutions are stable in the presence of the administered components for a period of time sufficient to prepare doses and conduct administration schemes. The stability of the MenABCWY vaccine prepared by reconstituting the MenACWY-TT drug product with 0.67 ml of the bivalent MnB drug product rLP2086 over a period of time at ambient temperature and under light conditions was confirmed in dilution vials and dosing syringes.

The MenABCWY dosing solution samples were tested by stability assessment methods such as RP-HPLC for antigen binding and purity, bioplex activity assay, ELISA, and ICP-MS according to predefined acceptance criteria. The results of this study showed acceptable stability of the MenABCWY vaccine for 24 hours at room temperature and under light conditions. These data are presented and described below in Examples 5-15.

Example 5: Evaluation of the MenABCWY Vaccine

A study was conducted to evaluate acceptable physical compatibility and short-term stability when the lyophilized MenACWY-TT formulation was reconstituted with the divalent formulation of MnB rLP2086. The lyophilized MenACWY-TT composition and the liquid MnB rLP2086 bivalent composition were combined and stored for 24 hours under uncontrolled conditions at room temperature, close to actual storage conditions. It has been demonstrated that the lyophilized MenACWY-TT formulation can be reconstituted with liquid MnB rLP2086 divalent formulation with gentle manual agitation and the combined pH and osmolality are within typical injection ranges. All key features of the conjugates and proteins were similar to the control features under uncontrolled conditions at room temperature for 24 hours.

Physical compatibility was assessed by determining the pH, appearance, ease of dilution and osmolality of the combined drug product. Antigen stability was assessed by calculating the concentration, purity and relative in vitro antigenicity (IVRA) of the rLP2086 subfamily A and subfamily B proteins, and calculating the concentrations of conjugated meningococcal polysaccharides A, C, Y and W-135 by ELISA.

Examples 5-15 demonstrate that the combination of a lyophilized MenACWY-TT formulation and a liquid divalent MnB rLP2086 formulation, ie a MenABCWY formulation, is compatible and stable for at least 24 hours at room temperature.

ELISA for determination of concentrations of meningococcal polysaccharides A, C, Y and W-135 in MenABCWY formulation - Development of ELISA for meningococcal A, C, Y and W-135 and pAb screening for detection

Six antibodies were selected for screening for use in ELISA assays. Each of four groups of ten rabbits were immunized with Men A, C, Y, or W-135 polysaccharide-TT conjugates, where the rabbits were bled after antibody administration. Each rabbit was individually screened with Men A, C, Y or W-135 polysaccharides conjugated to CRM ₁₉₇ carrier protein for binding and specificity. Rabbit sera were screened for a positive binding signal, which is equivalent to an optical density three times the background optical density. In addition, rabbit sera were screened for low non-specific binding, which can be consistent with any optical density data for a combination of sera without antigen, second or detecting antibodies, exceeding the optical density data for control, as well as low cross-reactivity, which is any optical density data. densities for heterolygic serotypes that exceed background optical density. Groups of rabbits were selected that met the screening criteria. A standard curve was built for a specific data interval for the CRM conjugates, and this curve confirmed the dilution of the lyophilized MenACWY-TT formulation. A standard curve was built for a specific data interval for the CRM conjugates, and this curve confirmed the dilution of the lyophilized MenACWY-TT formulation.

The feasibility of quantifying conjugates A, C, Y and W in the combined drug product (MenABCWY formulations) was evaluated. It was determined that one divalent formulation of MnB rLP2086 was not detected in this assay. In addition, if aluminum phosphate was solubilized in MenABCWY composition samples, then full conjugate yields were obtained. Therefore, it was determined that the divalent composition of MnB rLP2086 does not affect the quantification of MenACWY-TT conjugates in ELISA.

Example 6 Evaluation of Applicability of Methods for Assessing Bivalent Composition MnB rLP2086 in the Presence of Composition MenACWY-TT

IOC-HPLC was evaluated for its applicability to determine the activity of subfamily A and B proteins in divalent MnB rLP2086 in the presence of the MenACWY-TT formulation. Results were obtained for total protein and bound protein in the divalent MnB formulation rLP2086 in the presence and absence of the MenACWY-TT formulation. The overlapping chromatograms are shown in Fig. one.

Table 4 Obazets Subfamily Bound protein, % Bivalent composition MnB rLP2086 without composition MenACWY-TT Protein A (SEQ ID NO: 1) 107 Protein B (SEQ ID NO: 2) 104 Bivalent composition MnB rLP2086 together with composition MenACWY-TT Protein A (SEQ ID NO: 1) 108 Protein B (SEQ ID NO: 2) 103

Example 7 Evaluation of Purity and Peak Ratio of Bivalent MnB rLP2086 Composition in the Presence of MenACWY-TT Composition

RP-HPLC was evaluated for its utility in determining the purity of the divalent MnB composition rLP2086 in the presence of the MenACWY-TT composition. Purity results were compared for the divalent MnB formulation rLP2086 in the presence of the MenACWY-TT formulation and in the absence of the MenACWY-TT formulation. The overlapping chromatograms are shown in Fig. 2 . An example of impurity peak integration is shown as an inset in FIG. 2. The results of the evaluation showed that the presence of the MenACWY-TT composition did not affect the purity evaluation of the divalent MnB composition rLP2086 using the RP-HPLC method.

Example 8: IVRA evaluation for bivalent MnB formulation rLP2086 in the presence of MenACWY-TT formulation

The IVRA method was evaluated for its applicability to determine the in vitro relative antigenicity of the MnB rLP2086 bivalent composition for subfamily A (SEQ ID NO: 1) and subfamily B (SEQ ID NO: 2) proteins in the presence of the MenACWY-TT composition.

IVRA results were compared for subfamily A and subfamily B proteins in the bivalent MnB formulation rLP2086 in the presence and absence of the MenACWY-TT formulation. The results of the acceptability assessment showed that in the case of assay variability, such results are comparable, and that the presence of the MenACWY-TT composition does not affect the determination of relative in vitro antigenicity.

Example 9 Dilution of MenACWY-TT Composition with Bivalent Composition of MnB rLP2086 in Vessels

Drug products of the MenACWY-TT formulation and rLP2086 bivalent MnB formulation were prepared using the MenACWY-TT formulation in vials diluted with the drug product of the rLP2086 bivalent MnB formulation. Vessel contents of MenACWY-TT formulation diluted with either saline or MenACWY-TT formulation taken as a placebo matrix were used as controls, depending on the method.

Table 5 Product Component Composition Composition MenACWY-TT Neisseria meningitidis
Group A polysaccharide
Group C polysaccharide
Polysaccharide group W-135
Group Y polysaccharide
Tetanus toxoid carrier protein
sucrose
Tromethanol 10 µg/ml
10 µg/ml
10 µg/ml
10 µg/ml
88 mcg/ml Duvalent composition MnB rLP2086 Subfamily A protein rLP2086 (SEQ ID NO: 1)
Subfamily B protein rLP2086 (SEQ ID NO: 2)
AlPO4
Histidine
NaCl 120 µg/ml
120 µg/ml
0.5 mg/ml
pH 6.0 Bivalent formulation placebo MnB rLP2086 AlPO4
Histidine
NaCl 0.5 mg/ml
pH 6.01 General bivalent composition MnB rLP2086 DP Subfamily A protein rLP2086 (SEQ ID NO: 1)
Subfamily B protein rLP2086 (SEQ ID NO: 2)
AlPO4
Histidine
NaCl 120 µg/ml
120 µg/ml
0.5 mg/ml
pH 6.0

Determination of the Dilution Volume of MenACWY-TT Composition with Saline

A package of the commercially available NIMENRIX® product contains a vial containing a lyophilized MenACWY-TT formulation and a syringe containing 0.9% saline for reconstitution. To restore the final concentration of NIMENRIX® in a commercially available vaccine after dilution with the divalent formulation of MnB rLP2086, the amount of saline injected using a syringe from a commercially available product can be determined. The same volume of the divalent formulation of MnB rLP2086 can then be used in all dilution studies.

Dilution of the MenACWY-TT composition with the divalent composition of MnB rLP2086 in vessels

The divalent composition of MnB rLP2086The was collected in a 10 ml glass vial. Approximately 800 μl of the solution was withdrawn into a 1 ml syringe. The adjusted content of the syringe was injected into the vial containing the MenACWY-TT composition. The vessel was shaken to dissolve the contents.

The pH and appearance of the MenABCWY composition were determined in duplicate samples. Osmomolality was measured in triplicate for MenACWY-TT formulation diluted with saline and MenACWY-TT formulation diluted with rLP2086 bivalent MnB formulation.

Example 10: SEC-MALLS to evaluate the stability of meningococcal polysaccharides A, C, Y and W-135 in a DP matrix.

Meningococcal polysaccharides A, C, Y and W-135 were used as surrogates to determine if any instability of conjugated meningococcal polysaccharides A, C, Y and W-135 could be expected in the combined drug product (MenABCWY formulations).

Treatment of meningococcal polysaccharides A, C, Y and W-135

Reagent Preparation (“Complete Buffer Matrix of MenABCWY Composition”)

2.24 g of sucrose and 7.8 mg of Tris (Tromethamine) were added to 20 ml of 2× buffer matrix bivalent formulation of MnB rLP2086 with MnB rLP2086 proteins (20 mM histidine, pH 6.0, 300 mM NaCl, 0.07 mg/ml PS-80, 1 mg/ml (8 mM) AlPO ₄ , rLP2086 subfamily A (SEQ ID NO: 1) and subfamily B (SEQ ID NO: 2) proteins, 240 μg/ml each).

Sample preparation

Each meningococcal polysaccharide was diluted 1:1 with MenABCWY Composition Complete Buffer Matrix and incubated for 0, 6, and 24 hours at 5°C, 25°C, and 37°C. After incubation, the sample suspension was centrifuged for 1 minute at 14,000 rpm. The supernatant was analyzed using SEC-MALLS.

Example 11 Stability of MenABCWY Composition - Assessment of pH, Appearance and Osmolality of Combination of Bivalent MnB rLP2086 Composition and MenACWY-TT Composition

The pH and appearance of the combination of the bivalent MnB rLP2086 formulation and the MenACWY-TT formulation, ie the MenABCWY formulation, were assessed immediately after dilution and again 24 hours later.

All results were as expected (Table 6).

Table 6 Appearance and pH of the MenABCWY composition # sample Sample Time watch Appearance pH one Composition MenABCWY, Rep1 0 Homogeneous white suspension 5.8 2 Composition MenABCWY, Rep2 0 Homogeneous white suspension 5.8 3 Composition MenABCWY, Rep 1 24 Homogeneous white suspension 5.8 four Composition MenABCWY, Rep 2 24 Homogeneous white suspension 5.8 5 Composition of MenACWY-TT with saline 0 Transparent, colorless 6.3 6 Composition of MenACWY-TT with saline 24 Transparent, colorless 6.4

The mean osmolality of the MenACWY-TT formulation diluted with rLP2086 bivalent MnB formulation was 3% of the mean osmolality of the MenACWY-TT formulation diluted with saline.

Table 7 Vessel Breeding agent Data per 1 mOsm Data at 2 mOsm Data at 3 mOsm Average mOsm Composition MenACWY-TT Saline 471 473 478 474 Composition MenACWY-TT Bivalent composition MnB rLP2086 487 487 489 488

Example 12 Conjugate Concentrations meningococcal polysaccharides A, C, Y and W-135 in the combined drug product

The concentration of meningococcal conjugates A, C, Y and W-135-TT in the MenABCWY formulation was assessed immediately and again after 24 hours. The concentrations of the four conjugates were stable over 24 hours (Table 8).

Table 8 Short term stability results of MnA, C, Y, and W conjugates in MenABCWY formulation assessed by ELISA Serotype Composition MenACWY-TT + bivalent composition MnB rLP2086 Composition MenACWY-TT+saline solution Initial, µg/ml After 24 hours, mcg/ml Stability ratio Initial, µg/ml After 24 hours, mcg/ml Stability ratio A 6.7 6.8 1.0 6.4 6.7 1.1 C 6.9 6.6 1.0 6.5 6.7 1.0 Y 8.1 8.7 1.1 9.6 9.8 1.0 W 8.5 8.8 1.0 8.8 9.0 1.0

Example 13 Stability Evaluation of Bivalent MnB rLP2086 Proteins in MenABCWY Composition

Concentrations of total and associated protein of subfamily A (SEQ ID NO: 1) and subfamily B of rLP2086 (SEQ ID NO: 2) in the combined drug product.

Samples of the MenABCWY composition were analyzed by IOC-HPLC to determine protein concentrations. As shown in Table 9, the total protein, associated (aluminum) protein and % bound bivalent proteins subfamily A (SEQ ID NO: 1) and subfamily B (SEQ ID NO: 2) MnB rLP2086 (aluminum associated) did not change during 24 hours, indicating that rLP2086 subfamily A and subfamily B proteins were stable for 24 hours.

Table 9 Stability of Total and Bound Protein Time watch Subfamily Total protein, mcg/ml Bound protein, µg/ml Bound protein, % 0 A 83 85 102 B 88 87 99 24 A 87 88 101 B 92 91 99

Example 14 Purity and Peak Ratio of rLP2086 Protein in MenABCWY Combined Formulation

Samples of the MenABCWY composition were analyzed by RP-HPLC to determine the purity and peak ratio of the rLP2086 proteins. See fig. 3. The peak at 11.9 minutes was excluded from the purity score.

IVRA of rLP2086 subfamily A and subfamily B proteins in combination drug product

The IVRA of the MenABCWY composition samples was evaluated 24 hours after mixing. The relative antigenicity of subfamily A (SEQ ID NO: 1) and subfamily B (SEQ ID NO: 2) rLP2086 proteins in the MenABCWY formulation was determined to be stable for 24 hours.

Example 15 Stability of Meningococcal Polysaccharides A, C, Y, and W-135 in Complete MenABCWYA Composition Buffer Matrix Determined by SEC-MALS

Stability of meningococcal PS A in complete MenABCWYA composition buffer matrix determined by SEC-MALS after incubation for 6 and 24 hours at various temperatures

Meningococcal polysaccharides A, C, W and Y were mixed with the complete MenABCWYA composition buffer matrix and evaluated for stability by SEC-MALS after incubation at 5°C, 25°C and 37°C for 24 hours. All four polysaccharides were stable for 24 hours at 5°C and 25°C. Some degree of degradation was observed at 37° C. for meningococci A and Y. The degree of degradation could not be determined for meningococcal Y polysaccharides due to the formation of high molecular weight aggregates under all conditions tested except for baseline.

Table 10 Sample Incubation time, hours Incubation temperature Mw (kDa) ∆Mw(%) Meningococcal PS A 0 NA 169 N/A 24 5°C 171 one 25°C 157 -7 37°C 126 -26 Meningococcal PS C 0 NA 213 N/A 24 5°C 216 one 25°C 215 one 37°C 220 3 Meningococcal PS Y* 0 NA 294 N/A 24 5°C 734 149 25°C 756 157 37°C 696 136 Meningococcal PS W-135 0 NA 205 N/A 24 5°C 230 12 25°C 211 3 37°C 219 7

Example 16 Evaluation of hSBA activity induced by bivalent MnB formulation rLP2086 with cross-protective effect against MenACWXY

The bivalent composition of TRUMENBA MnB rLP2086 contains two lipidized factor H binding proteins (fHBP), one from each subfamily, and has a protective effect against Neisseria meningitides serogroup B. It was found that fHBP is expressed at different levels in serogroups A, C, W, Y, and X, suggesting that the divalent formulation of MnB rLP2086 may provide protection against these serogroups. The authors took the serum used in study B1971015 to test the concept that the divalent TRUMENBA MnB rLP2086 formulation could confer protection against serogroups A, C, W, Y, and X. Strains were collected from around the world (including strains of MnW taken after the recent outbreak in the UK and MnX, which has just appeared in Africa). For comparison, a method for selecting a candidate strain for development of hSBA and immune response in a subset of individuals immunized with a bivalent MnB formulation rLP2086 and in individuals immunized with a quadrivalent meningococcal capsular polysaccharide (MCV4) conjugate vaccine is described.

Sera used for assessing immunogenicity

A subset of sera from Study B1971015, Phase 2, which is a blinded, randomized, actively controlled study in which two groups of individuals received either MENACTRA (a conjugate vaccine based on meningococcal polysaccharide A, C, Y, and W-135 [MCV4]) and ADACEL (tetanus toxoid vaccine, attenuated diphtheria toxoid vaccine, non-cellular pertussis vaccine [Tdap]) or bivalent rLP2086 (TRUMENBA [meningococcal serogroup B vaccine] approved for use in the United States) were used to assess immunogenicity in hSBA using MnACWYX strains selected as described below.

Baseline seropositivity score

Baseline seropositivity in hSBA on microcolonies was assessed using non-immune or pre-vaccination sera taken from adolescents in study B1971015. For these purposes, seropositivity was defined as hSBA titer ≥1:8, which was predicted in the LLOQ analysis. To design an assay, strains must have low baseline seropositivity (ie, hSBA titer ratio ≥ 1:8 using baseline serum approximately <40%).

Sera used to determine immunogenicity

A subset of sera from Study B1971015, Phase 2, which is a blinded, randomized, actively controlled study in which two groups of individuals received either MENACTRA (a conjugate vaccine based on meningococcal polysaccharide A, C, Y, and W-135 [MCV4]) and ADACEL (tetanus toxoid vaccine, attenuated diphtheria toxoid vaccine, non-cellular pertussis vaccine [Tdap]) or bivalent rLP2086 (TRUMENBA [meningococcal serogroup B vaccine] approved for use in the United States) were evaluated for immunogenicity in hSBA using MnACWYX strains selected as described below.

Results and discussion

Serological group A

A pool of strains for SBA serogroup A was obtained, which included 17 strains from South Africa, 4 strains from the USA and one strain from the Netherlands. 73 percent of serogroup A strains in this collection express B16 and 23% express B22. The predominant clonal complexes in this collection of serogroup A strains are ST-1 and ST-5. Two strains expressing B16 and one strain expressing B22 with fHBP expression levels above 1100 MFI were selected for further testing. 36 parties C' were used to screen the relationship T30/T0 C'. PMB1546 was excluded because relevant human serum complement sources that did not non-specifically kill the strain could not be identified according to the C' transit rate (Table 11). PMB3143 was excluded due to high baseline seropositivity rates of 52% using non-immune adolescent sera. PMB3257 met the selection criteria: appropriate complement sources were identified; the low baseline seropositivity rates assessed using non-immune adolescent sera were, for example, 0%, and hSBAs were technically feasible.

Table 11. Candidate serogroup A strains tested Strain fHBP variant fHBP expression ^a epidemiological marker Country of origin of the strain Passing speed C' Compatible hSBA Baseline seropositivity score PMB3257 B16 1725 Complex ST-1/
subgroup I/II South Africa 33% Yes ^b 0% ^c PMB3143 B16 1657 Complex ST-1/
subgroup I/II USA fourteen% Yes 52% ^d PMB1546 B22 2657 Complex ST-5/
subgroup III South Africa 0% ND ^e ND a. average fluorescence intensity determined in the MEASURE assay b. the strain formed certain colonies and was destroyed using an indicator serum in hSBA c. adolescents, pre-vaccination serum d. high level of non-immune response e. did not The selected serogroup A strain is shown in bold .

Serological group C

A strain pool was obtained for SBA serogroup C, which included 49 strains from the USA and 10 strains from the Netherlands. The fHBP sequence is more heterogeneous in serogroup C. The strain pool contains at least 17%, namely 19% of MenC strains that are strains expressing fHBP A10, and 10% of strains expressing A15. The predominant clonal complexes in this collection of serogroup C strains are the ST-11/ET-37 and ST-103 complex. For further testing, one strain expressing A10 and three strains expressing A15 with fHBP expression levels above 1100 MFI were selected. 36 parties C' were used to screen the relationship T30/T0 C'. PMB5180 and PMB5196 expressing A15 for fHBP were excluded because corresponding human serum complement sources that did not non-specifically kill the strain could not be identified according to the C' transit rate (Table 12). For PMB5043, the original C' screening test failed the SBA analysis due to the T30/T0 ratio. PMB5208 met the selection criteria: appropriate complement sources were identified; the low baseline seropositivity rates assessed using non-immune adolescent sera was 17% and hSBA was technically feasible.

Table 12. Candidate strains tested for serogroup C Strain fHBP variant fHBP expression ^a epidemiological marker Country of origin of the strain Passing speed C' Compatible hSBA Baseline seropositivity score PMB5208 A10 1563 Complex ST11/ET-37 USA 56% ^b 17% ^d PMB5180 A15 3237 ST-103 USA 0% ND ^e ND PMB5196 A15 3353 ST-103 USA 3% ND ND PMB5043 A15 2803 ST-103 USA eight% No ^c ND a. average fluorescence intensity determined in the MEASURE assay b. the strain formed certain colonies and was destroyed using an indicator serum in hSBA c. there were no corresponding complement sources available, which does not allow determination of the T30/T0 ratio in SBA d. adolescents, pre-vaccination serum e. did not The selected strain of serogroup C is shown in bold .

Serological group W

A pool of strains for SBA serogroup W was obtained, which included 14 strains from the USA, 9 strains from the Netherlands and 6 strains from the UK after a recent outbreak. The fHBP sequence is more homogeneous in this collection of serogroup W strains. 45% of the strains in this pool express the fHBP A19 variant and 45% the fHBP A10 variant. The predominant clonal complex for fHBP A19 variant strains in the W serogroup pool is ST-22, and the predominant clonal complex for fHBP A10 variant strains is the ST-11/ET-37 complex. From the US strains, one strain expressing A19 and one strain expressing A10 were selected. For further testing, two strains were selected from the UK after the outbreak, expressing A10, where the expression levels of fHBP exceeded 1100 MFI. 36 parties C' were used to screen the relationship T30/T0 C'. All four strains passed this initial screening test C'. Based on the level of non-immune response, PMB5524 and PMB5163 were excluded because the seropositivity rate for non-immune serum in adolescents was 74% and 48%, respectively. PMB5248 and PMB5523 met the selection criteria: appropriate complement sources were identified; the low baseline seropositivity rates assessed using non-immune adolescent sera were, for example, 26% and 28%, respectively, and hSBA was technically feasible (Table 13).

Table 13. Candidate strains tested for serogroup W Strain fHBP variant fHBP expression ^a epidemiological marker Country of origin of the strain Passing speed C' Compatible hSBA Baseline seropositivity score PMB5248 A19 2684 Complex ST-22 USA fourteen% Yes ^b 26% ^c PMB5524 A10 1197 Complex ST-11/ET-37 Great Britain 34% Yes 74% ^d PMB5163 A10 1565 Complex ST-11/Complex ET-37 USA 6% Yes 48% ^d PMB5523 A10 1796 Complex ST-11 / complex ET-37 Great Britain 32% Yes 28% a. average fluorescence intensity determined in the MEASURE assay b. the strain formed certain colonies and was destroyed using an indicator serum in hSBA c. adolescents, pre-vaccination serum d. high level of non-immune response The selected strain of serogroup W is shown in bold .

Serological group X

A pool of strains for SBA serogroup X was obtained, which included 8 strains from Africa and one strain from the Netherlands. Five strains from Africa expressed the fHBP B49 variant, three strains had a new type of fHBP, and one strain from the Netherlands expressed fHBP B09. The expression of fHBP for all nine strains exceeded 1100 MFI. Three strains from Africa after the outbreak and one strain from the Netherlands were selected for further testing. 36 parties C' were used to screen the relationship T30/T0 C'. All four strains passed this initial screening test C'. Based on the level of non-immune response, PMB5467 was excluded because the seropositivity rate for non-immune serum in adolescents was 68%. PMB5537, PMB5540 and PMB5539 met the selection criteria: appropriate complement sources were identified; the low baseline seropositivity rates assessed using non-immune adolescent sera were, for example, 0%, and hSBAs were technically feasible (Table 14). PMB5540 was selected for evaluation.

Table 14. Candidate strains tested for serogroup X Strain fHBP variant fHBP expression ^a epidemiological marker Country of origin of the strain Passing speed C' Compatible hSBA Baseline seropositivity score PMB5467 B09 1795 Complex ST-1157 Netherlands 6% yes ^b 68% ^d PMB5537 B239 4896 Complex ST-181 Burkina Faso eight% yes ^b 4% ^c PMB5540 B49 8612 Complex ST-181 Burkina Faso 53% yes ^b 0% PMB5539 B49 13706 Must be identified Uganda 83% yes ^b 0% a. average fluorescence intensity determined in the MEASURE assay b. the strain formed certain colonies and was destroyed using an indicator serum in hSBA c. there were no corresponding complement sources available, which does not allow determination of the T30/T0 ratio in SBA With. adolescents, pre-vaccination serum d. high level of non-immune response The selected strain of serogroup X is shown in bold .

Serological group Y

A pool of strains for SBA serogroup Y was obtained, which included 87 strains from the USA and 30 strains from the Netherlands. The fHBP sequence is more homogeneous in this collection of serogroup Y strains. 66% of serogroup Y strains in this pool express the A15 fHBP variant. The predominant clonal complex for strains expressing the fHBP A15 variant in this collection of serogroup Y strains is ST-23/cluster A3. And only 15% of serogroup Y strains in this collection express fHBP above 1100 MFI. Three strains of the A15 variant group with fHBP expression levels above 1100 MFI were selected for additional testing. 36 parties C' were used to screen the relationship T30/T0 C'. PMB5122, PMB5053, and PMB5050 were excluded because the corresponding human serum complement sources that did not non-specifically kill the strain could not be identified according to the C' transit rate (Table 15). PMB5187 with fHBP B47 were selected for T30/T0 C' screening and sources of C' could be identified that could not non-specifically kill the strain (Table 15). Even though PMB5187 did not express the predominant fHBP variant for serogroup Y, however, this strain met other selection criteria: appropriate complement sources were identified; the low baseline seropositivity rates estimated using non-immune adolescent sera were, for example, 4% and hSBA was technically feasible.

Table 15. Candidate serogroup Y tested strains: Strain fHBP variant fHBP expression ^a epidemiological marker Country of origin of the strain Passing speed C' Compatible hSBA Baseline seropositivity score PMB5122 A15 1011 ST-23/Cluster A3 USA 0% ^NDd ND PMB5053 A15 1608 ST-23/Cluster A3 USA 0% ND ND PMB5050 A15 1811 ST-23/Cluster A3 USA 0% ND ND PMB5187 B47 5063 ST-23/ Cluster A3 USA 64% Yes ^b four% ^c a. average fluorescence intensity determined in the MEASURE assay b. the strain formed certain colonies and was destroyed using an indicator serum in hSBA c. adolescents, pre-vaccination serum d. did not The selected strain of serogroup Y is shown in bold .

MnACYWX strains were selected for hSBA development based on the predominant fHBP variants for the respective serogroup, fHBP expression above 1100 MFI, and the feasibility of testing (eg, human complement identification and baseline seropositivity). The characteristics of the six selected MnACYWX strains, as well as the median fHBP expression for each group of variants, are summarized in Table 16.

Table 16. Characteristics of the tested strains MnACYWX Strain Serological group fHBP variant fHBP expression clonal complex Country of origin of the strain PMB3257 A B16 1725 Complex ST-1 South Africa PMB5208 C A10 1563 Complex CC11/ET-37 USA PMB5248 W A19 2684 CC-22 USA PMB5523 W A10 1796 Complex CC-11/ET-37 Great Britain. PMB5187 Y B47 5063 CC-23/Cluster A3 USA PMB5540 X B49 8612 Complex ST-181 Burkina Faso

Immunogenicity assay

MnACYWX test strains were used in hSBAs to assess the immune response elicited by subgroups of healthy adolescents aged 10 to <13 years enrolled in the Phase 2 Collaborative Study B1971015. The hSBA responses produced by TRUMENBA were compared with the hSBA responses produced by MENACTRA (meningococcal polysaccharides A, C, Y and W-135 conjugate vaccine [MCV4]) for six selected strains (Table 17).

High levels of bactericidal humoral responses were observed in a large number of vaccinated individuals with hSBA titer ≥1:8, which is a more stringent criterion than the acceptable correlate of protection (i.e., hSBA titer ≥1:4) for MenCWYX strains, with the exception of MenA in 28% of individuals immunized with TRUMENBA compared to 97% of individuals immunized with MCV4. TRUMENBA response peaked one month after dose 2 for two strains of serogroup W (PMB5248 100%, PMB5523, 97%) and one month after dose 3 for strains of serogroups C, Y, X with response rates of 83% -100%. MCV4 responses peaked one month after dose 1 with no response to MenX. Antibodies produced by TRUMENBA showed a certain potential for protection against MenX, which was not achieved in the case of MCV4. TRUMENBA response rates (ie, % ≥1:8) after 3 doses against MnC, W, or Y strains were comparable to MCV4 response levels after 1 dose. Immunogenicity data obtained for MnACYWX strains tested in hSBA supported the concept of a protective effect against non-B serogroups.

Table 17: Percentage of SBA titers ≥1:8 for MnACWYX strains Vaccine group Group 2: MCV4, month 0 Group 3: Trumenba, months 0, 2, 6 Strain Serological group Sample time N n %(GMT) N n %(GMT) PMB3257 A Month 0 thirty one 3% (2) thirty one 3% (2) Month 1 thirty 29 97% (95) thirty four 13% (3) Month 3 thirty 22 73% (31) thirty 6 20% (4) Month 7 thirty fourteen 47% (11) 29 eight 28% (5) PMB5208 C Month 0 thirty 6 20% (3) thirty 7 23% (4) Month 1 thirty 27 90% (119) thirty 16 53% (8) Month 3 thirty 28 93% (88) thirty 21 70% (12) Month 7 thirty 27 90% (38) thirty 28 93% (29) PMB5248 W Month 0 thirty 12 40% (5) thirty eight 27% (4) Month 1 thirty 29 97% (88) thirty 22 73% (18) Month 3 29 28 97% (58) thirty thirty 100% (47) Month 7 29 26 90% (41) thirty thirty 100% (77) PMB5523 W Month 0 29 16 55% (8) thirty 13 43% (7) Month 1 thirty 29 97% (60) thirty twenty 67% (15) Month 3 thirty 29 97% (58) thirty 29 97% (21) Month 7 thirty 29 97% (44) thirty thirty 100% (42) PMB5187 X Month 0 thirty four 13% (3) thirty 3 10% (3) Month 1 thirty 29 97% (79) thirty fourteen 47% (7) Month 3 thirty 28 93% (58) thirty 28 93% (31) Month 7 thirty 27 90% (36) thirty thirty 100% (58) PMB5540 Y Month 0 thirty 0 0% (2) thirty 2 7% (2) Month 1 thirty 0 0% (2) thirty 5 17% (3) Month 3 thirty 0 0% (2) thirty 16 53% (7) Month 7 thirty 0 0% (2) thirty 25 83% (20)

Conclusions: MnACYWX strains were selected for hSBA development based on the predominant variants for the respective serogroup, fHBP expression above the fHBP MnB expression threshold, and the feasibility of testing (eg, human complement identification and baseline seropositivity). Using sera from individuals immunized with Trumenba in hSBA using MnACYWX strains, a proof of concept was obtained that antibodies produced by Trumenba may be protective against non-B serogroups. titers ≥1:8) after 3 doses were comparable to the responses produced by MCV4 after 1 dose for strains of serogroups C, W, and Y, but were lower for strains of serogroup A. In addition, hSBA titers, produced by divalent rLP2086 and ≥1:8 in a large number of individuals indicate a protective effect against MenX, but this effect was not observed in the case of MCV4.

Example 17: Trumenba Generates Bactericidal Antibodies Against Non-Serogroup B Meningococci

Introduction. Neisseria meningitidis (Men) is the leading cause of bacterial meningitis and sepsis in children, adolescents and young adults. There are 5 major disease causing meningococcal serogroups A, B, C, Y and W, and a sixth serogroup X found in Africa. Quadrivalent meningococcal conjugate vaccines (MCV4) are used to protect against disease caused by meningococcal serogroups A, C, Y and W in various regions of the world. The recently validated TRUMENBA® vaccine (MenB-FHbp, divalent rLP2086, Pfizer Inc, Collegeville, PA) provides protection against serogroup B disease and consists of 2 recombinant lipoproteins, 1 from each of 2 phylogenetic subfamily proteins that bind to factor H (FHbp). The gene encoding FHbp has been found to be present in almost all invasive strains of Men, regardless of serogroup classification. Preclinical studies have demonstrated that MenB-FHbp can generate a protective effect against other disease-causing serogroups. In preliminary studies, the Men B BEXSERO® vaccine (MenB-4C; Novartis Inc, Cambridge, MA), which includes a single FHbp variant antigen, was found to be able to mount a bactericidal immune response against MenX and MenW strains. The aim of this study is to extend the observations from exploratory assays towards studying the production of antibodies in response to MenB-FHbp of bactericidal activity against strains of MenA, C, W, Y and X ( Fig. 7 ) in adolescents, and thereby the production of a protective action against all serogroups that cause meningococcal disease.

Experimental review. Of the candidate strains ( Fig. 8 ), strains were selected that:

- were not susceptible to the destruction of only the human complement;

- were destroyed, as shown by hSBA using a serum that has been shown to produce bactericidal antibodies against FHbp expressed by MenB strains;

- have low baseline titers in the case of pre-vaccination sera (25 pre-vaccination sera; see Fig. 9 ).

For selected strains ( Table 18 , Fig. 10 ), exploratory hSBA assays were developed. See fig. 9 for serum tested in hSBA. Response rate=percentage of individuals with hSBA titers ≥1:8 greater than the established protection correlate (≥1:4) ( FIGS. 11-16 ).

Table 18. Characteristics of selected test strains MenA, MenC, MenY, MenW and MenX Strain Serological group fHBP variant fHBP expression (MFI) Median fHBP expression (MFI) for serogroup clonal complex Country of origin of the strain PMB3257 A B16 1725 1192 ST-1 complex / subgroup I/II South Africa PMB5208 C A10 1563 1563 Complex ST-11/Complex ET-37 USA PMB5248 W A19 2684 460 Complex ST-22 USA PMB5523 W A10 1796 460 ST-11 complex / ET-37 complex England/Wales PMB5187 Y B47 5063 290 Complex ST-23 / Cluster A3 USA PMB5540 X B49 8612 7555 Complex ST-181 Burkina Faso

As shown in FIG. 9 , 25 sera from Group A at month 0 were used to study the primary immune response. Sera taken from 30 individuals in Group A at months 0 and 1 and from Group B at months 0, 1, 3 and 7 were used for hSBA testing. The response rate endpoint is the percentage of serum with titers > 1:8.

Fig. ten- Distribution expression levels of surface FHbp (MFI) determined in experiments by flow cytometry using mAb MN 994-11 that reacts with FHbp. FHbp surface expression for each of the strains of a particular serogroup is shown as black dots, and FHbp surface expression levels for selected test strains of each serogroup are shown with a colored asterisk.

Fig. 11 - hSBA response rate (percentage of individuals with hSBA titers ≥1:8) for MenA PMB3257 (B16). Response rates and 95% confidence intervals are shown for sera collected before immunization (month 0) and 1 month after administration of 1, 2, and 3 doses of MenBFHbp. The geometric mean of the obtained titers (GMT) were 2, 3, 4, and 5, respectively. The response rates for individuals in the positive control group were 3% before vaccination and 97% one month after MCV4 administration. GMT for the positive control group were 2 and 95, respectively.

Fig. 12 - hSBA response rate (percentage of individuals with hSBA titers ≥1:8) for MenC PMB5208 (A10). Response rates and 95% confidence intervals are shown for sera collected before immunization (month 0) and 1 month after administration of 1, 2, and 3 doses of MenBFHbp. The resulting GMTs were 4, 8, 12 and 29, respectively. The response rates for individuals in the positive control group were 20% before vaccination and 90% 1 month after MCV4 administration. GMT for the positive control group were 3 and 119, respectively.

Fig. 13 - hSBA response rate (percentage of individuals with hSBA titers ≥1:8) for MenW PMB5248 (A19). Response rates and 95% confidence intervals are shown for sera collected before immunization (month 0) and 1 month after administration of 1, 2, and 3 doses of MenBFHbp. The resulting GMTs were 4, 18, 47 and 77, respectively. The response rates for individuals in the positive control group were 40% before vaccination and 97% one month after MCV4 administration. GMT for the positive control group were 5 and 88, respectively.

Fig. fourteen- hSBA response rate (percentage of individuals with hSBA titers ≥1:8) for MenW PMB5523 (A10). Response rates and 95% confidence intervals are shown for sera collected before immunization (month 0) and 1 month after administration of 1, 2, and 3 doses of MenBFHbp. The resulting GMTs were 7, 15, 21 and 42, respectively. The response rates for individuals in the positive control group were 55% before vaccination and 97% one month after MCV4 administration. GMT for the positive control group were 8 and 60, respectively.

Fig. 15 - hSBA response rate (percentage of individuals with hSBA titers ≥1:8) for MenY PMB5187 (B47). Response rates and 95% confidence intervals are shown for sera collected before immunization (month 0) and 1 month after administration of 1, 2, and 3 doses of MenBFHbp. The resulting GMTs were 3, 7, 31 and 58, respectively. The response rates for individuals in the positive control group were 13% before vaccination and 97% one month after MCV4 administration. GMT for the positive control group were 3 and 79, respectively.

Fig. 16 - hSBA response rate (percentage of individuals with hSBA titers ≥1:8) for MenX PMB5540 (B49). Response rates and 95% confidence intervals are shown for sera collected before immunization (month 0) and 1 month after administration of 1, 2, and 3 doses of MenBFHbp. The resulting GMTs were 2, 3, 7 and 20, respectively. The response rates for individuals in the positive control group were 0% before vaccination and 0% one month after MCV4 administration. GMT for the positive control group were 2 and 2, respectively.

Conclusions. The proportion of responses elicited by MenB-FHbp was maximal at 1 month post-dose 2 (Month 3) for the MenW strain, PMB5248 (100%), and 1 month post-dose 3 (Month 7) for the MenC, MenY, and MenX strains , as well as for the second strain MenW (response rates in the range of 83%-100%).

The response rates measured with hSBA for the tested MenA strain were significantly lower and peaked at 28% after 3 doses of MenB-FHbp.

The identified correlate of protection against meningococcal disease is an hSBA titer ≥1:4. The ability of MenB-FHbp to produce hSBA titers of at least 1:8 supports the concept that MenB-FHbp may have a protective effect against disease caused by non-B meningococcal serogroups, including MenX, but this effect is not conferred by MCV4.

Example 18: Phase 2 randomized, blinded, controlled trial to characterize the immunogenicity, safety, and tolerability of Neisseria meningitidis serogroup B recombinant lipoprotein 2086 bivalent vaccine (bivalent rLP2086, i.e., the currently used TRUMENBA® vaccine) in healthy aged individuals ≥24 months to <10 years (B1971017-syn).

Study protocol: This study is a Phase 2, randomised, controlled, multicentre, blinded, multicentre study designed to evaluate the immunogenicity, safety, and tolerability of bivalent rLP2086 at a dose of 120 mcg administered to healthy individuals ≥24 months to <10 years of age on a monthly schedule 0, 2 and 6 (Table 19). A program was developed in which approximately 400 individuals were randomly assigned to 1 of 2 groups in a 3:1 ratio. Group 1 received bivalent rLP2086 at month 0 (Visit 1) followed by vaccinations at months 2 and 6. Group 2 received a licensed childhood hepatitis A virus (HAV) vaccine at month 0 (Visit 1) and month 6 and saline injection at month 2 to ensure blind research. Follow-up visits were made 1 month after each vaccination and 6 months after the third vaccination to collect safety data and/or draw a blood sample. Individuals participated in the study for up to 13 months.

Table 19 Study Protocol Vaccination 1 Follow-up after vaccination 1 Vaccination 2 Blood sampling after vaccination 2 Vaccination 3 Blood sampling after vaccination 3 Observation and blood sampling for month 12 Visit one 2 3 four 5 6 7 Approximate month 0 one 2 3 6 7 12 Group 1
(300 individuals) Bivalent rLP2086 Bivalent rLP2086 Bivalent rLP2086 Group 2
(100 individuals HAV vaccine Saline HAV vaccine Blood sampling
(all individuals) 5-10 ml 5-10 ml 5-10 ml 5-10 ml HAV=hepatitis A virus

Administered Vaccines: Group 1 individuals were administered bivalent rLP2086 by intramuscular injection into the upper deltoid muscle of the arm for months 0, 2 and 6. Individuals of Group 2 were administered the HAV vaccine/saline/HAV vaccine in the upper deltoid muscle of the arm for months 0, 2 and 6, respectively.

Immunogenicity assessments : To facilitate immunogenicity analysis, approximately 5-10 ml of blood (depending on age) was taken from individuals immediately before the first vaccination, 1 month after the 2nd vaccination, and 1 and 6 months after the third vaccination. To assess immune response to bivalent rLP2086, functional antibodies were analyzed in hSBA for validity using the 4 primary test MnB strains selected using an unbiased algorithm and adjusted for epidemiological prevalence based on the adjusted baseline for the pool of MnB strains in the Pfizer serum bactericidal assay (S.B.A.). In hSBA, human serum antibodies were evaluated that initiate complement-dependent destruction of the target meningococcal strain. Four (4) MnB primary test strains, PMB80 (A22), PMB2001 (A56), PMB2948 (B24), and PMB2707 (B44), each expressing a variant factor H-binding protein (fHBP) heterologous to vaccine antigen components, were used in hSBA to determine immunogenicity endpoints in this study. These analyzes used sera obtained from all individuals prior to the first experimental vaccination, 1 month after the second experimental vaccination, and 1 and 6 months after the third experimental vaccination. For primary analyses, 2 primary test strains (PMB80 [A22] and PMB2948 [B24]) were analyzed at each time of blood sampling for one half of the individuals (in both groups), and the other 2 primary test strains (PMB2001 [A56] and PMB2707 [B44]) were analyzed at each point of blood sampling for the other half of the individuals.

Primary immunogenicity endpoints are: number of individuals aged ≥24 months to <4 years (at baseline) with hSBA titer ≥lower limit(s) of quantification (LLOQ) for each of the 4 primary MnB strains tested through 1 month after the third vaccination with bivalent rLP2086; and the number of individuals aged ≥4 years to <10 years (at baseline) with hSBA titer ≥LLOQ for each of the 4 primary MnB strains tested 1 month after the third vaccination with divalent rLP2086.

The secondary endpoints of immunogenicity are:

- In healthy subjects aged ≥24 months to <10 years at baseline:

- Number of individuals with hSBA titer≥LLOQ for each of the 4 primary MnB strains tested 1 month after the third vaccination with divalent rLP2086.

- In healthy individuals aged ≥24 months to <4 years at baseline, in healthy individuals aged ≥4 years to <10 years at baseline, and in the pooled age stratified group:

- Number of individuals with hSBA titer≥LLOQ for each of the 4 primary MnB strains tested 1 month after the second vaccination and 1 and 6 months after the third vaccination with divalent rLP2086.

- Number of individuals achieving hSBA titers ≥1:4, ≥1:8, ≥1:16, ≥1:32, ≥1:64 and ≥1:128 for each of the 4 primary test strains at baseline, 1 month after the second vaccination and 1 and 6 months after the third vaccination with divalent rLP2086.

- GMT in hSBA for each of the 4 primary test strains at baseline, 1 month after the second vaccination and 1 and 6 months after the third vaccination with divalent rLP2086.

Secondary immunogenicity endpoints were summarized for the immunogenicity group and for the mITT group.

The following experimental immunogenicity endpoints were used to describe responses in healthy individuals aged ≥24 months to <4 years at baseline, healthy individuals aged ≥4 years to <10 years at baseline, and in a pooled age stratified group:

- Number of individuals with hSBA titers ≥1:4, ≥1:8, ≥1:16, ≥1:32, ≥1:64 and ≥1:128 at each appropriate time of blood sampling.

- GMT in hSBA for each of the 4 primary strains at each appropriate time of blood sampling.

- Number of individuals achieving at least a 4-fold increase in hSBA titer from baseline 1 month after the third vaccination with divalent rLP2086 for each of the 4 primary test strains:

- For individuals whose baseline hSBA titer was below the limit of detection (LOD) or hSBA titer <1:4, a 4-fold response was observed, defined as hSBA titer ≥1:16 or LLOQ (however high this titer is was).

- For individuals with baseline hSBA titer ≥LOD (ie, hSBA titer ≥1:4) and < LLOQ, a 4-fold response was observed, defined as hSBA titer ≥ 4 times LLOQ.

- For individuals with baseline hSBA titer≥LLOQ, a 4-fold response was observed, defined as hSBA titer ≥4 times baseline titer.

results

Individuals: A total of 400 individuals aged ≥24 months to <10 years were randomly included in this study. Of all randomly selected individuals, 294 individuals were included in Group 1 (bivalent rLP2086) and 106 individuals were included in Group 2 (HAV/saline). Each stratified age group included 200 randomly selected individuals ranging in age from ≥24 months to <4 years and from ≥4 years to <10 years.

Of 400 randomly selected individuals, 390 (97.5%) individuals completed the entire vaccination phase (within 1 month of the last experimental vaccination) of this study. A total of 387 (96.8%) individuals completed a 6-month follow-up by telephone contact. It is considered that only those individuals who completed the vaccination phase and completed the 6-month telephone follow-up were considered to have completed the entire survey. Overall, only 375 (93.8%) subjects completed all study procedures and this completion was the same for each age stratified group. A total of 371 (92.8%) individuals were included in the immunogenicity evaluation group, and 29 (7.3%) individuals were excluded from this group. All 400 randomly selected individuals were included in the mITT group.

Immunogenicity results : The main objective of this study is to characterize the immune response in individuals to bivalent rLP2086, as determined by hSBA, against 4 primary MnB strains tested, 2 of which expressed the LP2086 subfamily A protein and 2 expressed the LP2086 subfamily B protein, as was determined 1 month after the third vaccination in healthy individuals aged ≥24 months to <4 years and ≥4 years to <10 years. The second objective is to describe immune responses for patients in a pooled cohort stratified by age (≥24 months to <10 years). The primary goal endpoints are the number of individuals in each age stratified group who achieved hSBA titers ≥LLOQ for each of the 4 primary MnB strains 1 month after the third vaccination.

A sustained immune response was observed in children aged ≥24 months to <10 years 1 month after the third dose of bivalent rLP2086, as evidenced by the number of individuals with hSBA titer ≥LLOQ (1:8 for A56, B24 and B44; 1:16 for A22) for each of the 4 primary MnB strains tested ranging from 80.0% to 100.0% for individuals aged ≥24 months to <4 years and from 78.3% to 100.0% for individuals aged ≥4 years to <10 years after 3 doses. The number of individuals in this pooled age stratified group with hSBA titer≥LLOQ for each of the 4 primary MnB strains tested 1 month after the third vaccination ranged from 79.1% to 100.0%. These data were also confirmed by high GMT (ranging from 19.1 to 191) and the number of individuals who achieved hSBA titers ≥1:4 (81.5% - 100%) or ≥1:16 (75.4% - 100%) against each of the 4 primary tested strains of MnB after administration of 3 doses of divalent rLP2086 compared with baseline levels for two age stratified groups. In addition, the number of individuals in the pooled age stratified group whose hSBA titer was ≥4 compared to baseline 1 month after the third vaccination for each of the 4 primary MnB strains tested ranged from 76.9% to 93.5% .

The second objective of the study is to characterize immune responses 1 month after the second dose of bivalent rLP2086, as determined by ≥LLOQ responses, determined hSBA titers and GMT hSBA for 2 age stratified groups and in the pooled age stratified group. For the pooled age stratified group, the number of individuals with hSBA titer≥LLOQ ranged from 48.5% to 100.0%, and no significant differences were observed between the younger and older stratified groups. These data were further confirmed for the pooled age stratified group with increasing GMT (ranging from 11.1 to 96.6) and for the number of individuals who achieved hSBA titer ≥1:4 (57.7% - 100%) or ≥1:16 (43.1% - 100%) after 2 doses of divalent rLP2086 compared to baseline against each of the 4 primary MnB strains tested. GMT were similar for the 2 age stratified groups. In addition, the number of individuals in the pooled age stratified group whose hSBA titer was ≥4 compared to baseline 1 month after the second vaccination for each of the 4 primary MnB strains tested ranged from 42.3% to 91.0% .

Immunopersistence was also assessed 6 months after the third dose of bivalent rLP2086, where the number of individuals with hSBA titer ≥LLOQ decreased from 79.1%-100% 1 month after the third vaccination to 10.4%-82.4% by 6- th month after the third vaccination for the pooled age stratified group. No differences were observed between the 2 age stratified groups except for A22, where there were more older children with a titer ≥LLOQ than younger children (46%, 95% with CI=33.4, 59.1 and 19% , 95% with CI=10.2, 30.9). However, pre-vaccination baseline ≥LLOQ titers were higher for A22 in the age stratified group (13.6% and 4.4%). A similar trend was also observed for the pooled age stratified group in individuals with a protective hSBA titer ≥1:4 ranging from 13.3%-84.0%, and a GMT ranging from 5.1-31.3 6 months after the third vaccination.

Overall, bivalent rLP2086 given at 3 doses on a schedule for months 0, 2, and 6 elicited a robust immune response in infants and children ≥24 months to <10 years of age with protective antibody titers achieved as showed hSBA in a large number of individuals after the third dose. No significant clinical differences were observed between young children, i.e., aged ≥24 months to <4 years, and children aged ≥4 years to <10 years. Humoral responses decreased 6 months after the third dose, but remained higher than baseline levels before vaccination.

Conclusion(s ): In conclusion, bivalent rLP2086 given to infants and children ≥24 months to <10 years of age on a 3-dose schedule for months 0, 2, and 6 elicits a robust immune response in most children after second and third doses, with protective antibody titers achieved after the third dose as determined by hSBA. hSBA titers declined 6 months after the 3 dose series. The vaccine administered in this study was safe and well tolerated with an acceptable safety profile in infants and children ≥24 months to <10 years of age.

Example 19: Phase 2 randomized, blinded, controlled trial to characterize the immunogenicity, safety and tolerability of Neisseria meningitidis serogroup B recombinant lipoprotein 2086 bivalent vaccine (bivalent rLP2086, i.e. the currently used TRUMENBA® vaccine) in healthy aged individuals ≥24 months to <10 years (B1971017-CSR).

The original composition of divalent rLP2086 (which did not include polysorbate-80) was evaluated in Phase 1 studies in adults, adolescents, children and young children with satisfactory safety, tolerability and immunogenicity profiles demonstrated for these groups. The parent formulation has been shown to have an acceptable safety profile at doses up to 200 μg, and is immunogenic as determined by hSBA. In children aged 18 to 36 months, the parent formulation was tested at doses of 20 µg, 60 µg and 200 µg (study 6108A1-502-AU). In study 6108A1-502-AU, the rate of local reactions in each dose-matched group was generally higher than in the hepatitis A vaccine (HAV)/placebo group, but in most cases, mild reactions or reactions were observed. moderate severity. Cases of fever tended to increase with increasing dose. The number of individuals who experienced any increase in temperature after any dose was 36.4% in the 20 µg dose group, 39.1% in the 60 µg dose group, and 54.5% in the 200 µg dose group. The frequency of other systemic side effects in each vaccine group was generally comparable to that for HAV/placebo. Most individuals showed an hSBA response to MnB strains after the third vaccination.

Compared to the original composition, the method of preparation of the drug substance and the composition of the drug product were modified upwards (including the addition of polysorbate-80) to increase the scale of production and ensure long-term stability of the final vaccine composition. The safety data for adults and adolescents participating in studies using the final formulation of bivalent rLP2086 were consistent with the safety data for the parent formulation. Local reactions and systemic effects were generally mild to moderate for all age groups. Severe side effects were relatively rare. In addition, observational adverse event (AE) data from the 6108A1 1002-AU, 6108A1 2001 and B1971033 immunopersistence studies conducted 6 to 48 months after the third dose of bivalent rLP2086 did not raise any safety concerns. Severe adverse events (SAEs) were rare and generally not associated with the experimental vaccine. However, there have been several cases of withdrawals from the research program due to AE.

Two (2) Phase 2 studies (B1971017 and B1971035) were conducted to evaluate the immunogenicity and safety of the vaccine in children (12 months to <10 years of age) administered the final vaccine composition. Study B1971035 is ongoing and was designed to evaluate the safety, tolerability, and immunogenicity of 2 different doses (60 mcg and 120 mcg) in healthy children aged 12 months to <24 months. This study (B1971017) assessed the immunogenicity, safety and tolerability of bivalent rLP2086 at a dose of 120 μg (final formulation) administered to healthy individuals ≥24 months to <10 years of age on a schedule for months 0, 2 and 6. In accordance with program, approximately 400 individuals were randomly assigned to 1 of 2 groups in a 3:1 ratio. Group 1 received bivalent rLP2086 at month 0 (Visit 1) followed by vaccinations at months 2 and 6. Group 2 received HAV vaccine at month 0 (Visit 1) and at month 6 and saline injection at month 2. Randomization was stratified to ensure inclusion of an equal number of individuals in an age stratified group that included individuals aged ≥24 months to <4 years and ≥4 years to <10 years. This study (B1971017) is a phase 2, randomized, controlled, multicenter, blinded study in which, according to the design, approximately 400 individuals were randomly assigned to 1 of 2 groups in a 3:1 ratio. Group 1 received bivalent rLP2086 at month 0 (Visit 1) followed by vaccinations at months 2 and 6. Group 2 received a licensed childhood HAV vaccine at month 0 (Visit 1) and month 6 and saline injection at month 2 to enable study blind method. Randomization was stratified to ensure that an equal number of individuals were included in the age stratified group, which included individuals aged ≥24 months to <4 years and ≥4 years to <10 years. Approximately 200 individuals aged ≥24 months to <4 years and approximately 200 individuals aged ≥4 years to <10 years were included in this study according to the program.

This study assessed the immunogenicity, safety, and tolerability of bivalent rLP2086 at a dose of 120 μg administered to healthy individuals aged ≥24 months to <10 years on a schedule for months 0, 2, and 6. Follow-up visits were made 1 month after each vaccination and 6 months after the third vaccination for safety data and/or blood sampling. Individuals participated in the study up to 13 months.

Bivalent rLP2086 (containing 60 μg of each purified rLP2086 subfamily A and subfamily B protein adsorbed to aluminum in a sterile buffered isotonic suspension) was prepared at a dose of 0.5 ml for injection.

The licensed pediatric HAV vaccine was formulated as a 0.5 ml injection.

For Group 1, 59.31% of individuals aged ≥24 months to <4 years and 57.05% of individuals aged ≥4 years to <10 years received concomitant treatment. For Group 2, 58.18% of individuals aged ≥24 months to <4 years and 52.94% of individuals aged ≥4 years to <10 years received concomitant treatment. The most common concomitant treatment given during the study included the administration of ibuprofen, paracetamol, and amoxicillin.

Serum bactericidal activity assay using divalent rLP2086 - Primary test strains

To evaluate the immune response to bivalent rLP2086, functional antibodies were analyzed in hSBA for validity using the 4 primary MnB test strains selected using an unbiased algorithm and adjusted for epidemiological prevalence based on the adjusted baseline for the pool of MnB strains in the Pfizer serum bactericidal assay (S.B.A.). In hSBA, human serum antibodies were evaluated that initiate complement-dependent destruction of the target meningococcal strain. Four (4) MnB primary test strains, PMB80 (A22), PMB2001 (A56), PMB2948 (B24), and PMB2707 (B44), each expressing an fHBP variant heterologous to vaccine antigen components, were used in hSBA to determine immunogenicity endpoints in this study. These analyzes used sera obtained from all individuals prior to the first experimental vaccination, 1 month after the second experimental vaccination, and 1 and 6 months after the third experimental vaccination.

For primary analyses, 2 primary test strains (PMB80 [A22] and PMB2948 [B24]) were analyzed at each time of blood sampling for one half of the individuals (in both groups), and the other 2 primary test strains (PMB2001 [A56] and PMB2707 [B44]) were analyzed at each point of blood sampling for the other half of the individuals.

Immunogenicity assay

There are no hypotheses for testing for immunogenicity testing. The evaluation method was applied to achieve the first, second and experimental goals.

The number of individuals in each group that reached the hSBA titer ≥ lower limit of quantification (LLOQ) at 1 month after the second and third vaccinations and 6 months after the third vaccination was calculated for each test strain, together with a two-sided 95% exact confidence interval ( CI) for each age stratified group and pooled age stratified group.

Primary endpoints of immunogenicity

The primary endpoints of immunogenicity are:

- number of individuals aged ≥24 months to <4 years (at baseline) with hSBA titer ≥LLOQ for each of the 4 primary MnB strains tested 1 month after the third vaccination with bivalent rLP2086.

- Number of individuals aged ≥4 years to <10 years (at baseline) with hSBA titer ≥LLOQ for each of the 4 primary MnB strains tested 1 month after the third vaccination with bivalent rLP2086.

Secondary endpoints of immunogenicity

The secondary endpoints of immunogenicity are:

- In healthy subjects aged ≥24 months to <10 years at baseline:

- Number of individuals with hSBA titer≥LLOQ for each of the 4 primary MnB strains tested 1 month after the third vaccination with divalent rLP2086.

- In healthy individuals aged ≥24 months to <4 years at baseline, in healthy individuals aged ≥4 years to <10 years at baseline, and in the pooled age stratified group:

- Number of individuals with hSBA titer≥LLOQ for each of the 4 primary MnB strains tested 1 month after the second vaccination and 1 and 6 months after the third vaccination with divalent rLP2086.

- Number of individuals achieving hSBA titers ≥1:4, ≥1:8, ≥1:16, ≥1:32, ≥1:64 and ≥1:128 for each of the 4 primary test strains at baseline, 1 month after the second vaccination and 1 and 6 months after the third vaccination with divalent rLP2086.

- GMT in hSBA for each of the 4 primary test strains at baseline, 1 month after the second vaccination and 1 and 6 months after the third vaccination with divalent rLP2086.

Experimental Immunogenicity Endpoints

To achieve experimental endpoints, testing was not performed on all 4 primary MnB strains tested. Instead, 50% of individuals were tested using strains PMB2001 (A56) and PMB2707 (B44), but not PMB80 (A22) or PMB2948 (B24). The remaining 50% of individuals were tested using strains PMB80 (A22) or PMB2948 (B24), but not PMB2001 (A56) or PMB2707 (B44). All experimental endpoints defined below can be used to obtain hSBA results in all individuals who received divalent rLP2086 and can be tested for the appropriate strain at the indicated time(s).

The following experimental endpoints were used to describe responses in healthy individuals aged ≥24 months to <4 years at baseline, healthy individuals aged ≥4 years to <10 years at baseline, and in a pooled age stratified group:

- Number of individuals with hSBA titers ≥1:4, ≥1:8, ≥1:16, ≥1:32, ≥1:64 and ≥1:128 at each appropriate time of blood sampling.

- GMT in hSBA for each of the 4 primary test strains at each appropriate time of blood sampling.

- Number of individuals achieving at least a 4-fold increase in hSBA titer from baseline 1 month after the third vaccination with bivalent rLP2086 for each of the 4 primary test strains:

- For individuals whose baseline hSBA titer is below the limit of detection (LOD) or hSBA titer <1:4, a 4-fold response is defined as hSBA titer ≥1:16 or LLOQ (however high that titer is).

- For individuals with a baseline hSBA titer≥LOD (ie, hSBA titer≥1:4) and < LLOQ, a 4-fold response is defined as an hSBA titer ≥4 times LLOQ.

- For individuals with a baseline hSBA titer≥LLOQ, a 4-fold response is defined as an hSBA titer ≥4 times baseline titer.

Analysis Methods

Primary immunogenicity analysis includes an estimate of the number of individuals in each group that achieved hSBA titer ≥LLOQ at 1 month after the third vaccination for each test strain, together with a two-sided 95% exact confidence interval, CI, for each age stratified group and pooled age stratified groups.

All binary endpoints (including primary endpoints) were classified according to a two-tailed 95% CI using an exact method. The GMT of hSBA results was also systematized by 95% CI.

LLOQ is 1:16 for PMB80 (A22), 1:8 for PMB2001 (A56), 1:8 for PMB2707 (B44) and 1:8 for PMB2948 (B24).

To calculate GMT, hSBA results below LLOQ were reported as 0.5 × LLOQ for the primary analysis.

Primary Endpoint Analysis

Primary analyzes to achieve primary goals were performed for groups assessed for immunogenicity. The number of individuals in each group that achieved hSBA titer≥LLOQ 1 month after the third vaccination was calculated for each strain tested along with a two-sided 95% exact CI. To meet the 2 primary goals, these data are presented for 2 age stratified groups: ≥24 months to <4 years of age and ≥4 years to <10 years of age.

All binary endpoints (including primary endpoints) were classified according to a two-tailed 95% CI using an exact method. The GMT of hSBA results was also systematized by 95% CI.

To confirm the correct interpretation of the primary analyzes, an identical analysis was performed for the mITT group.

Analysis of secondary and experimental endpoints

The purpose of the following assays is to evaluate secondary and experimental immunogenicity:

- The number of individuals achieving hSBA titer≥LLOQ for each of the 4 primary strains at 1 month after the second vaccination and 6 months after the third vaccination was analyzed for two age stratified groups separately and in combination in groups assessed for immunogenicity and in mITT groups.

- Number of individuals achieving hSBA titers ≥1:4, ≥1:8, ≥1:16, ≥1:32, ≥1:64 and ≥1:128 for each of the 4 primary strains at baseline, through 1 month after the second vaccination and 1 and 6 months after the third vaccination, were analyzed for two age stratified groups separately and in combination in groups assessed for immunogenicity and in groups mITT.

- GMT hSBA for each of the 4 primary test strains at baseline 1 month after the second vaccination and 1 and 6 months after the third vaccination, analyzed for 2 age stratified groups separately and in combination in groups assessed for immunogenicity and in groups mITT.

Experimental endpoints were systematized for 2 age stratified groups separately and in combination at each appropriate time point for the immunogenicity group and the mITT group.

Inverse Cumulative Distribution Curves

Empirical reverse cumulative distribution curves (RCDCs) were graphically analyzed for each of the 4 primary strains at each sampling point for the immunogenicity group.

Assessment of immunogenicity

Analyzed groups

The group assessed for immunogenicity is the primary immunogenicity test group. The mITT group was used as an additional group for immunogenicity analysis.

A total of 371 (92.8%) individuals were included in the group assessed for immunogenicity, and 29 (7.3%) individuals were excluded from this group. Individuals may be excluded from immunogenicity evaluation groups for more than 1 reason. Only 21 (5.3%) individuals were excluded from the group assessed for immunogenicity, because they had a baseline blood sample taken before the first dose of vaccine or after the third vaccination, 15 (3.8%) individuals did not receive significant and confirmatory test results at any of the visits, 11 (2.8%) individuals were not or could not be included in the study before vaccination or 1 month after the visit for the third vaccination, 11 (2.8%) individuals did not receive the vaccine because they randomly made all vaccination visits and 4 (1.0%) individuals had significant protocol deviations identified by the attending physician. Overall, the 2 treatment groups and the 2 age stratified groups were comparable in terms of the percentage of individuals that were excluded from the immunogenicity group.

All 400 randomly selected individuals were included in the mITT group.

Immunogenicity Test Results

The results of analyzes for primary immunogenicity endpoints, secondary immunogenicity endpoints, and experimental immunogenicity endpoints are presented in the sections below.

Primary and secondary endpoints

Number of individuals achieving hSBA titer≥LLOQ

Primary immunogenicity endpoints are the number of individuals aged ≥24 months to <4 years (at baseline), and aged ≥4 years to <10 years (at baseline), with hSBA titer ≥LLOQ for each of 4 primary MnB test strains 1 month after the third vaccination with divalent rLP2086. Secondary endpoints are the number of all individuals in the pooled age stratified group (baseline) with hSBA titer ≥LLOQ for each of the 4 primary MnB strains tested 1 month after the third vaccination with bivalent rLP2086, together with the number of individuals in single and pooled age stratified groups with hSBA titre≥LLOQ for each of the 4 primary MnB strains tested 1 month after the second vaccination with bivalent rLP2086.

The number of individuals in each age stratified group with hSBA titer≥LLOQ for each of the 4 primary MnB strains tested is shown in Table 20 for the immunogenicity group.

The number of individuals aged ≥24 months to <4 years and aged ≥4 years to <10 years in Group 1 with hSBA titer ≥LLOQ at baseline is 4.4% and 13.6%, respectively, for PMB80 (A22); 1.5% and 15.4%, respectively, for PMB2001 (A56); 3.0% and 7.5%, respectively, for PMB2948 (B24); and 0.0%, for both age stratified PMB2707 (B44). Overall, the number of individuals in the pooled age stratified group with hSBA titer ≥LLOQ at baseline is 9.0% for PMB80 (A22), 8.3% for PMB2001 (A56), 5.2% for PMB2948 (B24), and 0.0% for PMB2707 (B44) in Group 1.

The number of individuals aged ≥24 months to <4 years and ≥4 years to <10 years in Group 1 with hSBA titer ≥LLOQ 1 month after the second vaccination is 59.4% and 78.8%, respectively, for PMB80(A22); 100.0% for both age stratified groups for PMB2001 (A56); 49.2% and 65.1%, respectively, for PMB2948 (B24); and 57.1% and 40.3%, respectively, for PMB2707 (B44).

Overall, the number of individuals in the pooled age stratified group with hSBA titer ≥LLOQ 1 month after second vaccination is 69.2% for PMB80 (A22), 100.0% for PMB2001 (A56), 57.0% for PMB2948 ( B24), and 48.5% for PMB2707 (B44) in Group 1.

The number of individuals aged ≥24 months to <4 years and ≥4 years to <10 years in Group 1 with hSBA titer ≥LLOQ 1 month after the third vaccination is 83.8% and 91.0%, respectively, for PMB80 (A22); 100.0% for both age stratified groups for PMB2001 (A56); 85.7% and 92.1%, respectively, for PMB2948 (B24); and 80.0% and 78.3%, respectively, for PMB2707 (B44).

Overall, the number of individuals in the pooled age stratified group with hSBA titer ≥LLOQ 1 month after the third vaccination is 87.4% for PMB80 (A22), 100.0% for PMB2001 (A56), 88.9% for PMB2948 ( B24), and 79.1% for PMB2707 (B44) in Group 1. In general terms, the number of individuals in Group 2 with hSBA titer ≥LLOQ did not change over a period of time compared to baseline. The number of individuals in the pooled age stratified group with hSBA titer≥LLOQ at any given time ranges from 4.4% to 8.5% for PMB80 (A22); 14.9% to 20.9% for PMB2001 (A56); 0.0% to 8.9% for PMB2948 (B24); and 0.0% at each time for PMB2707 (B44).

The results for the mITT group were similar to those for the immunogenicity group.

An estimate of the number of individuals in subgroups with hSBA titer≥LLOQ for each of the 4 primary tested MnB strains is presented for the group assessed for immunogenicity by sex, race and country. There were no clinically significant differences in the subgroup analyses.

Immunosistence: Number of individuals achieving hSBA titer ≥LLOQ 6 months after the third vaccination

Number of individuals aged ≥24 months to <4 years, ≥4 years to <10 years, and in a pooled age stratified group (≥24 months to <10 years), with hSBA titer ≥LLOQ for each of the 4 primary of MnB strains tested 6 months after the third vaccination with bivalent rLP2086 represents a secondary immunogenicity endpoint. The number of individuals with hSBA titer≥LLOQ for each of the 4 primary MnB strains tested in each age stratified group is shown in Table 20 for the immunogenicity group.

In general terms, there was a decrease in the number of individuals with hSBA titer≥LLOQ for each of the 4 primary MnB strains tested, observed in Group 1 individuals in both age stratified groups 6 months after the third vaccination.

For individuals aged ≥24 months to <4 years, starting 1 month after the third vaccination and up to 6 months after the third vaccination, the number of individuals with hSBA titer ≥LLOQ decreased from 83.8% to 19.0%, respectively, for PMB80 (A22); from 100.0% to 80.3%, respectively, for PMB2001 (A56); from 85.7% to 9.2%, respectively, for PMB2948 (B24); and from 80.0% to 12.1%, respectively, for PMB2707 (B44).

For individuals aged ≥4 years to <10 years, from 1 month after the third vaccination to 6 months after the third vaccination, the number of individuals with hSBA titer ≥LLOQ decreased from 91.0% to 46.0%, respectively, for PMB80 (A22); from 100.0% to 84.3%, respectively, for PMB2001 (A56); from 92.1% to 21.9%, respectively for PMB2948 (B24); and from 78.3% to 8.7%, respectively, for PMB2707 (B44).

Overall, for the pooled age stratified group, from 1 month after the third vaccination to 6 months after the third vaccination, the number of individuals with hSBA titer ≥LLOQ decreased from 87.4% to 32.5% for PMB80 (A22); from 100.0% to 82.4% for PMB2001 (A56); from 88.9% to 15.5% for PMB2948 (B24); and from 79.1% to 10.4% for PMB2707 (B44).

In general, the number of Group 2 individuals with hSBA titer≥LLOQ did not change over a period of time compared to baseline.

Table 20. Individuals with hSBA Titer ≥ LLOQ for Primary Strains - Group Assessed for Immunogenicity Vaccine group (randomized) Group 1 Group 2 rLP2086 HAV/saline solution Taking a sample of a strain (variant) at a given point in time for an age stratified group N ^a n ^b (%) (95% CI) ^c N ^a n ^b (%) (95% CI) ^c PMB80(A22) Before vaccination 1 ≥24 months to <10 years 134 12 (9.0) (4.7, 15.1) 47 3 (6.4) (1.3, 17.5) ≥24 months to <4 years 68 3 (4.4) (0.9, 12.4) 26 one (3.8) (0.1, 19.6) ≥4 years to <10 years 66 9 (13.6) (6.4, 24.3) 21 2 (9.5) (1.2, 30.4) 1 month after vaccination 2 ≥24 months to <10 years 130 90 (69.2) (60.5, 77.0) 45 2 (4.4) (0.5, 15.1) ≥24 months to <4 years 64 38 (59.4) (46.4, 71.5) 24 0 (0.0) (0.0, 14.2) ≥4 years to <10 years 66 52 (78.8) (67.0, 87.9) 21 2 (9.5) (1.2, 30.4) 1 month after vaccination 3 ≥24 months to <10 years 135 118 (87.4) (80.6, 92.5) 45 3 (6.7) (1.4, 18.3) ≥24 months to <4 years 68 57 (83.8) (72.9, 91.6) 25 one (4.0) (0.1, 20.4) ≥4 years to <10 years 67 61 (91.0) (81.5, 96.6) twenty 2 (10.0) (1.2, 31.7) 6 months after vaccination 3 ≥24 months to <10 years 126 41 (32.5) (24.5, 41.5) 47 four (8.5) (2.4, 20.4) ≥24 months to <4 years 63 12 (19.0) (10.2, 30.9) 26 2 (7.7) (0.9, 25.1) ≥4 years to <10 years 63 29 (46.0) (33.4, 59.1) 21 2 (9.5) (1.2, 30.4) PMB2001(A56) Before vaccination 1 ≥24 months to <10 years 132 eleven (8.3) (4.2, 14.4) 47 7 (14.9) (6.2, 28.3) ≥24 months to <4 years 67 one (1.5) (0.0, 8.0) 24 2 (8.3) (1.0, 27.0) ≥4 years to <10 years 65 ten (15.4) (7.6, 26.5) 23 5 (21.7) (7.5, 43.7) 1 month after vaccination 2 ≥24 months to <10 years 133 133 (100.0) (97.3, 100.0) 43 7 (16.3) (6.8, 30.7) ≥24 months to <4 years 66 66 (100.0) (94.6, 100.0) 21 2 (9.5) (1.2, 30.4) ≥4 years to <10 years 67 67 (100.0) (94.6, 100.0) 22 5 (22.7) (7.8, 45.4) 1 month after vaccination 3 ≥24 months to <10 years 139 139 (100.0) (97.4, 100.0) 43 9 (20.9) (10.0, 36.0) ≥24 months to <4 years 68 68 (100.0) (94.7, 100.0) 24 one (4.2) (0.1, 21.1) ≥4 years to <10 years 71 71 (100.0) (94.9, 100.0) 19 eight (42.1) (20.3, 66.5) 6 months after vaccination 3 ≥24 months to <10 years 131 108 (82.4) (74.8, 88.5) 46 9 (19.6) (9.4, 33.9) ≥24 months to <4 years 61 49 (80.3) (68.2, 89.4) 24 four (16.7) (4.7, 37.4) ≥4 years to <10 years 70 59 (84.3) (73.6, 91.9) 22 5 (22.7) (7.8, 45.4) PMB2948 (B24) Before vaccination 1 ≥24 months to <10 years 134 7 (5.2) (2.1, 10.5) 47 2 (4.3) (0.5, 14.5) ≥24 months to <4 years 67 2 (3.0) (0.4, 10.4) 26 one (3.8) (0.1, 19.6) ≥4 years to <10 years 67 5 (7.5) (2.5, 16.6) 21 one (4.8) (0.1, 23.8) 1 month after vaccination 2 ≥24 months to <10 years 128 73 (57.0) (48.0, 65.7) 45 four (8.9) (2.5, 21.2) ≥24 months to <4 years 65 32 (49.2) (36.6, 61.9) 24 2 (8.3) (1.0, 27.0) ≥4 years to <10 years 63 41 (65.1) (52.0, 76.7) 21 2 (9.5) (1.2, 30.4) 1 month after vaccination 3 ≥24 months to <10 years 126 112 (88.9) (82.1, 93.8) 46 2 (4.3) (0.5, 14.8) ≥24 months to <4 years 63 54 (85.7) (74.6, 93.3) 26 2 (7.7) (0.9, 25.1) ≥4 years to <10 years 63 58 (92.1) (82.4, 97.4) twenty 0 (0.0) (0.0, 16.8) 6 months after vaccination 3 ≥24 months to <10 years 129 twenty (15.5) (9.7, 22.9) 47 0 (0.0) (0.0, 7.5) ≥24 months to <4 years 65 6 (9.2) (3.5, 19.0) 26 0 (0.0) (0.0, 13.2) ≥4 years to <10 years 64 fourteen (21.9) (12.5, 34.0) 21 0 (0.0) (0.0, 16.1) PMB2707 (B44) Before vaccination 1 ≥24 months to <10 years 138 0 (0.0) (0.0, 2.6) fifty 0 (0.0) (0.0, 7.1) ≥24 months to <4 years 67 0 (0.0) (0.0, 5.4) 26 0 (0.0) (0.0, 13.2) ≥4 years to <10 years 71 0 (0.0) (0.0, 5.1) 24 0 (0.0) (0.0, 14.2) 1 month after vaccination 2 ≥24 months to <10 years 130 63 (48.5) (39.6, 57.4) fifty 0 (0.0) (0.0, 7.1) ≥24 months to <4 years 63 36 (57.1) (44.0, 69.5) 26 0 (0.0) (0.0, 13.2) ≥4 years to <10 years 67 27 (40.3) (28.5, 53.0) 24 0 (0.0) (0.0, 14.2) 1 month after vaccination 3 ≥24 months to <10 years 134 106 (79.1) (71.2, 85.6) fifty 0 (0.0) (0.0, 7.1) ≥24 months to <4 years 65 52 (80.0) (68.2, 88.9) 26 0 (0.0) (0.0, 13.2) ≥4 years to <10 years 69 54 (78.3) (66.7, 87.3) 24 0 (0.0) (0.0, 14.2) 6 months after vaccination 3 ≥24 months to <10 years 135 fourteen (10.4) (5.8, 16.8) 49 0 (0.0) (0.0, 7.3) ≥24 months to <4 years 66 eight (12.1) (5.4, 22.5) 26 0 (0.0) (0.0, 13.2) ≥4 years to <10 years 69 6 (8.7) (3.3, 18.0) 23 0 (0.0) (0.0, 14.8) Abbreviation: hSBA=serum bactericidal activity using human complement; LLOQ=lower limit of quantification
Note: LLOQ=1:16 for A22; 1:8 for A56, B24, and B44
a. N=number of individuals with confirmed and determined hSBA titers for a given strain.
b. n=number of individuals with an observed hSBA titer ≥ LLOQ for a given strain at a given time.
c. Exact 2-tailed CI based on the observed number of individuals using the method of Klopper and Pearson.
Program ID: study B1971017/CP IMM_LLOQ.SAS. Database reporting series creation date: 01JUN2017. Passing time ID: June 16, 2017 11:22. File ID: T_2_2_IMM_LLOQ_EVL.HTM.

GMT hSBA. The secondary endpoint is hSBA GMT for each of the 4 primary test strains at baseline, 1 month after the second vaccination, and 1 month after the third vaccination with bivalent rLP2086. Table 21 shows the hSBA GMTs for the 4 primary MnB strains in the group assessed for immunogenicity.

For Group 1 individuals aged ≥24 months to <4 years and ≥4 years to <10 years, GMT hSBA at baseline was 8.3 and 9.1, respectively, for PMB80 (A22); 4.1 and 5.8, respectively, for PMB2001 (A56); 4.3 and 4.6, respectively, for PMB2948 (B24); and 4.0 in both age stratified groups for PMB2707 (B44).

For Group 1 individuals aged ≥24 months to <10 years, GMT hSBA at baseline was 8.7 for PMB80 (A22), 4.9 for PMB2001 (A56), 4.5 for PMB2948 (B24) and 4. 0 for PMB2707 (B44).

For Group 1 individuals aged ≥24 months to <4 years and ≥4 years to <10 years, the hSBA GMT at 1 month post-vaccination 2 was 17.4 and 23.1, respectively, for PMB80 (A22); 103.8 and 90.0, respectively, for PMB2001 (A56); 9.1 and 13.7, respectively, for PMB2948 (B24); and 17.1 and 8.2, respectively, for PMB2707 (B44).

For Group 1 individuals aged ≥24 months to <10 years, GMT hSBA 1 month post-vaccination 2 was 20.1 for PMB80 (A22), 96.6 for PMB2001 (A56); 11.1 for PMB2948 (B24) and 11.7 for PMB2707 (B44).

For Group 1 individuals aged ≥24 months to <4 years and ≥4 years to <10 years, the hSBA GMT at 1 month post-vaccination 3 was 33.7 and 38.2, respectively, for PMB80 (A22); 175.6 and 191.0, respectively, for PMB2001 (A56); 19.1 and 26.8, respectively, for PMB2948 (B24); and 43.6 and 36.5, respectively, for PMB2707 (B44).

For Group 1 individuals aged ≥24 months to <10 years, GMT hSBA 1 month post-vaccination 3 was 35.8 for PMB80 (A22), 183.3 for PMB2001 (A56); 22.6 for PMB2948 (B24), and 39.8 for PMB2707 (B44).

In general terms, GMT hSBA for individuals in Group 2 did not change over a period of time compared to baseline. For the pooled age stratified group, hSBA GMT at any given time ranged from 8.6 to 8.9 for PMB80 (A22); 5.6 to 6.0 for PMB2001 (A56); 4.0 to 4.8 for PMB2948 (B24); and 4.0 at each time for PMB2707 (B44).

Table 21. GMT hSBA for Primary Strains - Group Assessed for Immunogenicity Vaccine group (randomized) Group 1 Group 2 rLP2086 HAV/saline solution Taking a sample of a strain (variant) at a given point in time for an age stratified group N ^a GMT ^b (95% CI) ^c N ^a GMT ^b (95% CI) ^c PMB80(A22) Before vaccination 1 ≥24 months to <10 years 134 8.7 (8.3, 9.1) 47 8.9 (7.8, 10.1) ≥24 months to <4 years 68 8.3 (7.9, 8.8) 26 8.2 (7.8, 8.7) ≥4 years to <10 years 66 9.1 (8.3, 9.9) 21 9.8 (7.2, 13.2) 1 month after vaccination 2 ≥24 months to <10 years 130 20.1 (17.4, 23.2) 45 8.6 (7.7, 9.7) ≥24 months to <4 years 64 17.4 (14.2, 21.4) 24 8.0 (NE, NE) ≥4 years to <10 years 66 23.1 (18.9, 28.3) 21 9.4 (7.4, 12.0) 1 month after vaccination 3 ≥24 months to <10 years 135 35.8 (30.5, 42.2) 45 8.8 (7.8, 9.8) ≥24 months to <4 years 68 33.7 (26.4, 42.9) 25 8.7 (7.3, 10.3) ≥4 years to <10 years 67 38.2 (30.6, 47.6) twenty 8.9 (7.6, 10.4) 6 months after vaccination 3 ≥24 months to <10 years 126 12.4 (10.9, 14.2) 47 8.7 (7.9, 9.7) ≥24 months to <4 years 63 10.9 (9.0, 13.1) 26 8.4 (7.8, 9.1) ≥4 years to <10 years 63 14.2 (11.8, 17.0) 21 9.1 (7.4, 11.3) PMB2001(A56) Before vaccination 1 ≥24 months to <10 years 132 4.9 (4.3, 5.5) 47 5.6 (4.4, 7.2) ≥24 months to <4 years 67 4.1 (3.9, 4.3) 24 4.9 (3.7, 6.6) ≥4 years to <10 years 65 5.8 (4.6, 7.3) 23 6.5 (4.3, 9.8) 1 month after vaccination 2 ≥24 months to <10 years 133 96.6 (83.0, 112.5) 43 5.8 (4.4, 7.6) ≥24 months to <4 years 66 103.8 (84.2, 127.9) 21 5.0 (3.6, 7.1) ≥4 years to <10 years 67 90.0 (71.9, 112.7) 22 6.6 (4.2, 10.5) 1 month after vaccination 3 ≥24 months to <10 years 139 183.3 (156.7, 214.4) 43 6.0 (4.6, 7.7) ≥24 months to <4 years 68 175.6 (139.1, 221.6) 24 4.5 (3.5, 5.7) ≥4 years to <10 years 71 191.0 (153.9, 237.1) 19 8.6 (5.4, 13.8) 6 months after vaccination 3 ≥24 months to <10 years 131 31.3 (25.3, 38.7) 46 6.0 (4.6, 7.8) ≥24 months to <4 years 61 27.0 (19.7, 36.9) 24 6.0 (4.0, 8.9) ≥4 years to <10 years 70 35.7 (26.6, 47.8) 22 6.0 (4.2, 8.7) PMB2948 (B24) Before vaccination 1 ≥24 months to <10 years 134 4.5 (4.1, 4.9) 47 4.4 (3.9, 4.9) ≥24 months to <4 years 67 4.3 (3.8, 4.9) 26 4.3 (3.7, 5.1) ≥4 years to <10 years 67 4.6 (4.0, 5.2) 21 4.4 (3.6, 5.4) 1 month after vaccination 2 ≥24 months to <10 years 128 11.1 (9.2, 13.5) 45 4.8 (4.0, 5.8) ≥24 months to <4 years 65 9.1 (7.0, 11.9) 24 4.8 (3.7, 6.2) ≥4 years to <10 years 63 13.7 (10.3, 18.2) 21 4.9 (3.6, 6.6) 1 month after vaccination 3 ≥24 months to <10 years 126 22.6 (19.1, 26.8) 46 4.3 (3.9, 4.8) ≥24 months to <4 years 63 19.1 (14.9, 24.5) 26 4.6 (3.8, 5.6) ≥4 years to <10 years 63 26.8 (21.3, 33.9) twenty 4.0 (NE, NE) 6 months after vaccination 3 ≥24 months to <10 years 129 5.6 (4.8, 6.5) 47 4.0 (NE, NE) ≥24 months to <4 years 65 5.1 (4.1, 6.3) 26 4.0 (NE, NE) ≥4 years to <10 years 64 6.2 (4.9, 7.7) 21 4.0 (NE, NE) PMB2707 (B44) Before vaccination 1 ≥24 months to <10 years 138 4.0 (NE, NE) fifty 4.0 (NE, NE) ≥24 months to <4 years 67 4.0 (NE, NE) 26 4.0 (NE, NE) ≥4 years to <10 years 71 4.0 (NE, NE) 24 4.0 (NE, NE) 1 month after vaccination 2 ≥24 months to <10 years 130 11.7 (9.3, 14.7) fifty 4.0 (NE, NE) ≥24 months to <4 years 63 17.1 (11.8, 24.8) 26 4.0 (NE, NE) ≥4 years to <10 years 67 8.2 (6.3, 10.6) 24 4.0 (NE, NE) 1 month after vaccination 3 ≥24 months to <10 years 134 39.8 (30.6, 51.6) fifty 4.0 (NE, NE) ≥24 months to <4 years 65 43.6 (29.9, 63.6) 26 4.0 (NE, NE) ≥4 years to <10 years 69 36.5 (25.2, 52.7) 24 4.0 (NE, NE) 6 months after vaccination 3 ≥24 months to <10 years 135 5.1 (4.4, 5.9) 49 4.0 (NE, NE) ≥24 months to <4 years 66 5.2 (4.2, 6.4) 26 4.0 (NE, NE) ≥4 years to <10 years 69 5.0 (4.1, 6.2) 23 4.0 (NE, NE) Abbreviations: GMT=geometric mean titer; hSBA = serum bactericidal activity assay using human complement; LLOQ=lower limit of quantification; NE=not rated
Note: LLOQ=1:16 for A22; 1:8 for A56, B24 and B44. Titers below LLOQ mean 0.5 x LLOQ for analysis.
a. N=number of individuals with confirmed and determined hSBA titers for a given strain
b. GMT was calculated for all individuals with confirmed and determined hSBA titers at a given point in time.
c. CI stands for inverse transform of confidence levels based on Student's t-distribution for the mean logarithm of hSBA titers.

Subgroup hSBA GMT analyzes for each of the 4 primary MnB strains tested are presented for the gender, race and country immunogenicity assessment group and for the mITT group. There were no clinically significant differences in the subgroup analyses.

Immunopersistence: GMT hSBA

The secondary endpoint is hSBA GMT for each of the 4 primary test strains 6 months after the third vaccination with divalent rLP2086. Table 21 shows the hSBA GMTs for the 4 primary MnB strains for the group assessed for immunogenicity.

In general, a decrease in hSBA GMT was observed from 1 month after the third vaccination to 6 months after the third vaccination for each of the 4 primary test strains in individuals in Group 1 in both age stratified groups.

For Group 1 individuals aged ≥24 months to <4 years, from 1 month after the third vaccination to 6 months after the third vaccination, hSBA GMT decreased from 33.7 to 10.9 for PMB80 (A22), c 175 .6 to 27.0 for PMB2001(A56), c 19.1 to 5.1 for PMB2948 (B24), and c 43.6 to 5.2 for PMB2707 (B44).

For Group 1 individuals aged ≥4 years to <10 years, from 1 month after the third vaccination to 6 months after the third vaccination, hSBA GMT decreased from 38.2 to 14.2 for PMB80 (A22), c 191 .0 to 35.7 for PMB2001 (A56), c 26.8 to 6.2 for PMB2948 (B24), and c 36.5 to 5.0 for PMB2707 (B44).

For Group 1 individuals aged ≥24 months to <10 years, from 1 month after the third vaccination to 6 months after the third vaccination, hSBA GMT decreased from 35.8 to 12.4 for PMB80 (A22), c 183 .3 to 31.3 for PMB2001(A56), c 22.6 to 5.6 for PMB2948 (B24), and c 39.8 to 5.1 for PMB2707 (B44).

In general terms, for Group 2 individuals, GMT hSBA did not change over a period of time from baseline.

Defined hSBA titers

The secondary immunogenicity endpoint is the number of individuals aged ≥24 months to <4 years, ≥4 years to <10 years, and in the pooled age stratified group who achieved hSBA titers ≥1:4, ≥1:8, ≥1:16, ≥1:32, ≥1:64 and ≥1:128 for each of the 4 primary test strains at baseline, 1 month after the second vaccination, and 1 month after the third vaccination with bivalent rLP2086.

The number of individuals who achieved certain hSBA titers for the 4 primary MnB strains was evaluated for the immunogenicity evaluation group.

Individuals who achieved hSBA titers ≥1:4 and ≥1:16 are described below. hSBA titer ≥1:4 is widely recognized as a correlate of protection against IMZ; however, a more conservative hSBA titer of ≥1:16 is considered to be indicative of a 4-fold vaccine effect for pre-vaccination seronegative individuals.

The number of individuals aged ≥24 months to <4 years, and ≥4 years to <10 years, in Group 1 with hSBA titer ≥1:4 at baseline is 5.9% and 19.7%, respectively, for PMB80 (A22); 3.0% and 18.5%, respectively, for PMB2001 (A56); 4.5% and 9.0%, respectively, for PMB2948 (B24); and 0.0% and 1.4%, respectively, for PMB2707 (B44). The number of individuals aged ≥24 months to <4 years and ≥4 years to <10 years in Group 1 with hSBA titer ≥1:16 at baseline is 4.4% and 13.6%, respectively, for PMB80 (A22); 1.5% and 15.4%, respectively, for PMB2001 (A56); 3.0% and 6.0%, respectively, for PMB2948 (B24); and 0.0% for both age stratified groups for PMB2707 (B44).

The number of individuals in Group 1 with pooled age stratification with hSBA titer ≥1:4 and ≥1:16 at baseline is 12.7% and 9.0%, respectively, for PMB80 (A22); 10.6% and 8.3%, respectively, for PMB2001 (A56); 6.7% and 4.5%, respectively, for PMB2948 (B24); and 0.7% and 0.0%, respectively, for PMB2707 (B44).

The number of individuals aged ≥24 months to <4 years and ≥4 years to <10 years in Group 1 with hSBA titer ≥1:4 1 month after the second vaccination is 65.6% and 83.3%, respectively , for PMB80 (A22); 100.0% for both age stratified groups for PMB2001 (A56); 53.8% and 68.3%, respectively, for PMB2948 (B24); and 49.3% and 66.7%, respectively, for PMB2707 (B44). The number of individuals aged ≥24 months to <4 years and ≥4 years to <10 years, in Group 1 with hSBA titer ≥1:16 1 month after the second vaccination is 59.4% and 78.8%, respectively, for PMB80 (A22); 98.5% and 100.0%, respectively, for PMB2001 (A56); 43.1% and 58.7%, respectively, for PMB2948 (B24); and 31.3% and 55.6%, respectively, for PMB2707 (B44).

The number of individuals in Group 1 with pooled age stratification and hSBA titer ≥1:4 and ≥1:16 1 month after the second vaccination is 74.6% and 69.2%, respectively, for PMB80 (A22); 100.0% and 99.2%, respectively, for PMB2001 (A56); 60.9% and 50.8%, respectively, for PMB2948 (B24); and 57.7% and 43.1%, respectively, for PMB2707 (B44).

The number of individuals aged ≥24 months to <4 years, and ≥4 years to <10 years, in Group 1 with hSBA titer ≥1:4 at 1 month after the third vaccination is 86.8% and 98.5% , respectively, for PMB80 (A22); 100.0% for each age stratified group for PMB2001 (A56); 90.5% and 95.2% for PMB2948 (B24); and 81.5% and 82.6%, respectively, for PMB2707 (B44). The number of individuals aged ≥24 months to <4 years and ≥4 years to <10 years, in Group 1 with hSBA titer ≥1:16 1 month after the third vaccination is 83.8% and 91.0%, respectively, for PMB80 (A22); 100.0% for each age stratified group for PMB2001 (A56); 81.0% and 88.9%, respectively, for PMB2948 (B24); and 81.5% to 82.6% and 80.0% and 75.4%, respectively, for PMB2707 (B44).

The number of individuals in Group 1 with pooled age stratification and hSBA titer ≥1:4 and ≥1:16 at 1 month after the third vaccination is 92.6% and 87.4%, respectively, for PMB80 (A22); 100.0% and 100.0% respectively for PMB2001 (A56); 92.9% and 84.9%, respectively, for PMB2948 (B24); and 82.1% and 77.6%, respectively, for PMB2707 (B44).

In general terms, the number of Group 2 individuals who achieved certain hSBA titers did not change over a certain period of time compared to baseline.

The results for the mITT group were similar to those for the immunogenicity group.

Immunopersistence: Defined hSBA titers

The secondary immunogenicity endpoint is the number of individuals aged ≥24 months to <4 years, ≥4 years to <10 years, and in the pooled age stratified group who achieved hSBA titers ≥1:4, ≥1:8, ≥1:16, ≥1:32, ≥1:64, and ≥1:128 for each of the 4 primary test strains 6 months after the third vaccination with divalent rLP2086. The number of individuals who achieved certain hSBA titers for the 4 primary MnB strains was evaluated for the group assessed for immunogenicity.

In general, there was a decrease in the number of Group 1 individuals with two age stratifications who achieved certain hSBA titers over a period of time from 1 month after the third vaccination to 6 months after the third vaccination.

For Group 1 individuals aged ≥24 months to <4 years and ≥4 years to <10 years, from 1 month after the third vaccination to 6 months after the third vaccination, the number of individuals with hSBA titer ≥1:4 decreased from 86.8% to 25.4% and from 98.5% to 55.6%, respectively, for PMB80 (A22); from 100.0% to 82.0% and from 100.0% to 85.7%, respectively for PMB2001 (A56); from 90.5% to 13.8% and from 95.2% to 26.6%, respectively, for PMB2948 (B24); and from 81.5% to 13.6% and from 82.6% to 13.0%, respectively, for PMB2707 (B44).

For Group 1 individuals with a pooled age stratification over time from 1 month after the third vaccination to 6 months after the third vaccination, the number of individuals with hSBA titer ≥1:4 decreased from 92.6% to 40.5% for PMB80 (A22) ; from 100.0% to 84.0% for PMB2001 (A56); from 92.9% to 20.2% for PMB2948 (B24); and from 82.1% to 13.3% for PMB2707 (B44).

For Group 1 individuals aged ≥24 months to <4 years and ≥4 years to <10 years, from 1 month after the third vaccination to 6 months after the third vaccination, the number of individuals with hSBA titer ≥1:4 decreased from 83.8% to 19.0% and from 91.0% to 46.0%, respectively, for PMB80 (A22); from 100.0% to 77.0% and from 100.0% to 82.9%, respectively, for PMB2001 (A56); from 81.0% to 9.2% and from 88.9% to 20.3%, respectively, for PMB2948 (B24); and from 80.0% to 9.1% and from 75.4% to 7.2%, respectively, for PMB2707 (B44).

For Group 1 individuals with a pooled age stratification over time from 1 month after the third vaccination to 6 months after the third vaccination, the number of individuals with hSBA titer ≥1:16 decreased from 87.4% to 32.5% for PMB80 (A22) ; from 100.0% to 80.2% for PMB2001 (A56); from 84.9% to 14.7% for PMB2948 (B24); and from 77.6% to 8.1% for PMB2707 (B44).

In general terms, the number of Group 2 individuals who achieved certain hSBA titers did not change over a certain period of time compared to baseline.

Experimental Immunogenicity Endpoints

Analysis of some experimental immunogenicity endpoints was performed based on hSBA results for strains PMB2001 (A56) and PMB2707 (B44) in one half of the individuals and for strains PMB80 (A22) and PMB2948 (B24) in the other half of the individuals. The analyzed experimental endpoint is an hSBA titer that is ≥4 times baseline.

hSBA titer 4 times baseline

Table 22 shows the number of individuals with hSBA titers that are ≥4 times baseline for the 4 primary strains tested.

The number of individuals aged ≥24 months to <4 years and ≥4 years to <10 years, in Group 1 with an hSBA titer that is ≥4 times baseline titer 1 month after vaccination 2 is 56.3 % and 63.6%, respectively, for PMB80 (A22); 100.0% and 82.1%, respectively, for PMB2001 (A56); 43.1% and 54.0%, respectively, for PMB2948 (B24); and 54.0% and 31.3%, respectively, for PMB2707 (B44).

The number of individuals in Group 1 with pooled age stratification who have an hSBA titer ≥4 times baseline titer at 1 month post-vaccination 2 is 60.0% for PMB80 (A22), 91.0% for PMB2001 (A56) , 48.4% for PMB2948 (B24), and 42.3% for PMB2707 (B44).

The number of individuals aged ≥24 months to <4 years, and ≥4 years to <10 years, in Group 1 with an hSBA titer that is ≥4 times baseline titer at 1 month post-vaccination 3 is 79, 4% and 77.6%, respectively, for PMB80 (A22); 98.5% and 88.7%, respectively, for PMB2001 (A56); 77.8% and 82.5%, respectively, for PMB2948 (B24); and 78.5% and 75.4%, respectively, for PMB2707 (B44).

The number of individuals in Group 1 with pooled age stratification who have an hSBA titer ≥4 times baseline titer at 1 month post-vaccination 3 is 78.5% for PMB80 (A22), 93.5% for PMB2001 (A56) , 80.2% for PMB2948 (B24), and 76.9% for PMB2707 (B44).

Number of individuals aged ≥24 months to <4 years and ≥4 years to <10 years, in Group 1 with an hSBA titer that is ≥4 times baseline titer, up to 6 months after vaccination 3, is 19.0% and 36.5%, respectively, for PMB80 (A22); 75.4% and 64.3%, respectively, for PMB2001 (A56); 6.2% and 17.2%, respectively, for PMB2948 (B24); and 7.6% and 7.2%, respectively, for PMB2707 (B44).

The number of individuals in Group 1 with pooled age stratification who have an hSBA titer ≥4 times baseline titer at 6 months post-vaccination 3 is 27.8% for PMB80 (A22), 69.5% for PMB2001 (A56) , 11.6% for PMB2948 (B24), and 7.4% for PMB2707 (B44).

Similar results were obtained for the mITT group.

Table 22. Individuals with hSBA titer that is ≥4 times higher for primary strains - Group assessed for immunogenicity Vaccine group (randomized) Group 1 Group 2 rLP2086 HAV/saline solution Taking a sample of a strain (variant) at a given point in time for an age stratified group N ^a n ^b (%) (95% CI) ^c N ^a n ^b (%) (95% CI) ^c hSBA titer ≥4 times baseline ^d PMB80(A22) 1 month after vaccination 2 ≥24 months to <10 years 130 78 (60.0) (51.0, 68.5) 45 0 (0.0) (0.0, 7.9) ≥24 months to <4 years 64 36 (56.3) (43.3, 68.6) 24 0 (0.0) (0.0, 14.2) ≥4 years to <10 years 66 42 (63.6) (50.9, 75.1) 21 0 (0.0) (0.0, 16.1) 1 month after vaccination 3 ≥24 months to <10 years 135 106 (78.5) (70.6, 85.1) 45 one (2.2) (0.1, 11.8) ≥24 months to <4 years 68 54 (79.4) (67.9, 88.3) 25 one (4.0) (0.1, 20.4) ≥4 years to <10 years 67 52 (77.6) (65.8, 86.9) twenty 0 (0.0) (0.0, 16.8) 6 months after vaccination 3 ≥24 months to <10 years 126 35 (27.8) (20.2, 36.5) 47 2 (4.3) (0.5, 14.5) ≥24 months to <4 years 63 12 (19.0) (10.2, 30.9) 26 2 (7.7) (0.9, 25.1) ≥4 years to <10 years 63 23 (36.5) (24.7, 49.6) 21 0 (0.0) (0.0, 16.1) PMB2001(A56) 1 month after vaccination 2 ≥24 months to <10 years 133 121 (91.0) (84.8, 95.3) 43 four (9.3) (2.6, 22.1) ≥24 months to <4 years 66 66 (100.0) (94.6, 100.0) 21 one (4.8) (0.1, 23.8) ≥4 years to <10 years 67 55 (82.1) (70.8, 90.4) 22 3 (13.6) (2.9, 34.9) 1 month after vaccination 3 ≥24 months to <10 years 139 130 (93.5) (88.1, 97.0) 43 eight (18.6) (8.4, 33.4) ≥24 months to <4 years 68 67 (98.5) (92.1, 100.0) 24 one (4.2) (0.1, 21.1) ≥4 years to <10 years 71 63 (88.7) (79.0, 95.0) 19 7 (36.8) (16.3, 61.6) 6 months after vaccination 3 ≥24 months to <10 years 131 91 (69.5) (60.8, 77.2) 46 6 (13.0) (4.9, 26.3) ≥24 months to <4 years 61 46 (75.4) (62.7, 85.5) 24 3 (12.5) (2.7, 32.4) ≥4 years to <10 years 70 45 (64.3) (51.9, 75.4) 22 3 (13.6) (2.9, 34.9) PMB2948 (B24) 1 month after vaccination 2 ≥24 months to <10 years 128 62 (48.4) (39.5, 57.4) 45 3 (6.7) (1.4, 18.3) ≥24 months to <4 years 65 28 (43.1) (30.8, 56.0) 24 one (4.2) (0.1, 21.1) ≥4 years to <10 years 63 34 (54.0) (40.9, 66.6) 21 2 (9.5) (1.2, 30.4) 1 month after vaccination 3 ≥24 months to <10 years 126 101 (80.2) (72.1, 86.7) 46 2 (4.3) (0.5, 14.8) ≥24 months to <4 years 63 49 (77.8) (65.5, 87.3) 26 2 (7.7) (0.9, 25.1) ≥4 years to <10 years 63 52 (82.5) (70.9, 90.9) twenty 0 (0.0) (0.0, 16.8) 6 months after vaccination 3 ≥24 months to <10 years 129 fifteen (11.6) (6.7, 18.5) 47 0 (0.0) (0.0, 7.5) ≥24 months to <4 years 65 four (6.2) (1.7, 15.0) 26 0 (0.0) (0.0, 13.2) ≥4 years to <10 years 64 eleven (17.2) (8.9, 28.7) 21 0 (0.0) (0.0, 16.1) PMB2707 (B44) 1 month after vaccination 2 ≥24 months to <10 years 130 55 (42.3) (33.7, 51.3) fifty 0 (0.0) (0.0, 7.1) ≥24 months to <4 years 63 34 (54.0) (40.9, 66.6) 26 0 (0.0) (0.0, 13.2) ≥4 years to <10 years 67 21 (31.3) (20.6, 43.8) 24 0 (0.0) (0.0, 14.2) 1 month after vaccination 3 ≥24 months to <10 years 134 103 (76.9) (68.8, 83.7) fifty 0 (0.0) (0.0, 7.1) ≥24 months to <4 years 65 51 (78.5) (66.5, 87.7) 26 0 (0.0) (0.0, 13.2) ≥4 years to <10 years 69 52 (75.4) (63.5, 84.9) 24 0 (0.0) (0.0, 14.2) 6 months after vaccination 3 ≥24 months to <10 years 135 ten (7.4) (3.6, 13.2) 49 0 (0.0) (0.0, 7.3) ≥24 months to <4 years 66 5 (7.6) (2.5, 16.8) 26 0 (0.0) (0.0, 13.2) ≥4 years to <10 years 69 5 (7.2) (2.4, 16.1) 23 0 (0.0) (0.0, 14.8) Abbreviations: hSBA = serum bactericidal activity assay using human complement; LLOQ=lower limit of quantification; LOD=limit of detection
Note: LLOQ=1:16 for A22; 1:8 for A56, B24, and B44
. Note: A 4-fold increase is defined as follows: (1) For individuals with baseline hSBA titer below LOD (hSBA titer <1:4), response is defined as hSBA titer ≥1:16 or LLOQ (however high that titer was not). (2) For individuals with a baseline hSBA titer ≥LOD and <LLOQ, response is defined as an hSBA titer that is ≥4 times the LLOQ. (3) For individuals with a baseline hSBA titer ≥LLOQ, response is defined as an hSBA titer that is ≥4 times the baseline titer.
a. For an hSBA titer that is ≥4 times baseline titer, N=number of individuals with confirmed and determined hSBA titers for a given strain at a given time point and at baseline.
b. For an hSBA titer that is ≥4 times baseline titer, n=number of individuals with hSBA titer
≥4 times the base titer for this strain.
c. Exact two-tailed CI based on the observed number of individuals using the method of Klopper and Pearson
d. Baseline is defined as the time of blood sampling prior to vaccination 1.

Additional tests for immunogenicity

hSBA Data Exclusion Evaluation for MnB Primary Test Strains

Only validated and valid hSBA results were included in all immunogenicity assays.

hSBA results have been excluded from immunogenicity assays (or being considered for exclusion) for the following reasons:

- The individual refused to participate in the study.

- The individual did not have blood samples taken for analysis, but did not withdraw from the study.

- The amount of blood was insufficient for analysis. This was recorded as "insufficient quantity" to obtain the results of the analysis.

- The sample was analyzed, but the numerical amount of the titer could not be determined. This was recorded as "unspecified" for analysis results.

Inverse Cumulative Distribution Curves

RCDCs were estimated for the number of individuals with an hSBA response (≥LLOQ) for each of the 4 primary strains and at each point of blood sampling for the pooled age stratified group. The RCDC was also assessed for each of the 4 primary strains and at each time of blood sampling for individuals aged ≥24 months to <4 years. RCDC was also assessed for each of the 4 primary strains and at each time of blood sampling for individuals aged ≥4 years to <10 years.

The RCDC indicated that the majority of individuals in both age stratified groups had a significant hSBA response to each of their primary MnB strains tested 1 month after the second and third doses of bivalent rLP2086.

Conclusions on immunogenicity

The primary objective of this study is to characterize the immune response in individuals to bivalent rLP2086, as determined by hSBA, against 4 primary MnB strains tested, 2 of which expressed LP2086 subfamily A protein and 2 expressed LP2086 subfamily B protein, as determined 1 month after third vaccination in healthy individuals aged ≥24 months to <4 years and ≥4 years to <10 years. The second objective is to describe immune responses for patients in a pooled age stratified group (≥24 months to <10 years). The primary goal endpoints are the number of individuals in each age stratified group who achieved hSBA titers ≥LLOQ for each of the 4 primary MnB strains 1 month after the third vaccination.

A sustained immune response was observed in children aged ≥24 months to <10 years 1 month after the third dose of bivalent rLP2086, as evidenced by the number of individuals with hSBA titer ≥LLOQ (1:8 for A56, B24 and B44; 1:16 for A22) for each of the 4 primary MnB strains tested ranging from 80.0% to 100.0% for individuals aged ≥24 months to <4 years and from 78.3% to 100.0% for individuals aged ≥4 years to <10 years after 3 doses. The number of individuals in this pooled age stratified group with hSBA titer≥LLOQ for each of the 4 primary MnB strains tested 1 month after the third vaccination ranged from 79.1% to 100.0%. These data were also confirmed by high GMT (ranging from 19.1 to 191) and the number of individuals who achieved hSBA titers ≥1:4 (81.5% - 100%) or ≥1:16 (75.4% - 100%) against each of the 4 primary MnB strains tested after 3 doses of bivalent rLP2086 compared to baseline levels for both age stratified groups. In addition, the number of individuals in the pooled age stratified group whose hSBA titer was ≥4 compared to baseline 1 month after the third vaccination for each of the 4 primary MnB strains tested ranged from 76.9% to 93.5% .

The second objective of the study is to characterize immune responses 1 month after a second dose of bivalent rLP2086 as assessed by ≥LLOQ responses, defined hSBA titers and hSBA GMT for 2 age stratified groups and pooled age stratified group. For the pooled age stratified group, the number of individuals with hSBA titer ≥LLOQ ranged from 48.5% to 100.0%, and no significant differences were observed between the younger and older stratified groups. These data were further confirmed for the pooled age stratified group with increasing GMT (ranging from 11.1 to 96.6) and for the number of individuals who achieved hSBA titer ≥1:4 (57.7% - 100%) or ≥1:16 (43.1% - 100%) after 2 doses of divalent rLP2086 compared to baseline against each of the 4 primary MnB strains tested. GMT were similar for the 2 age stratified groups. In addition, the number of individuals in the pooled age stratified group whose hSBA titer was ≥4 compared to baseline 1 month after the second vaccination for each of the 4 primary MnB strains tested ranged from 42.3% to 91.0% .

Immunopersistence was also assessed 6 months after the third dose of bivalent rLP2086, where the number of individuals with hSBA titer ≥LLOQ decreased from 79.1%-100% 1 month after the third vaccination to 10.4%-82.4% at 6 months after the third vaccination for the combined age stratified group. No differences were observed between the 2 age stratified groups, with the exception of A22, where there were more older children with ≥LLOQ titer than younger children (46%, 95% with CI=33.4, 59.1 and 19 %, 95% with CI=10.2, 30.9). However, pre-vaccination baseline ≥LLOQ titers were higher for A22 in the age stratified group (13.6% and 4.4%). A similar trend was also observed for the pooled age stratified group in individuals with a protective hSBA titer ≥1:4, ranging from 13.3%-84.0% and GMT ranging from 5.1-31.3 6 months after the third vaccination.

Overall, bivalent rLP2086 given at 3 doses on a schedule for months 0, 2, and 6 elicited a robust immune response in infants and children ≥24 months to <10 years of age with protective antibody titers achieved as showed hSBA in a large number of individuals after the third dose. No significant clinical differences were observed between young children, i.e., aged ≥24 months to <4 years, and children aged ≥4 years to <10 years. Humoral responses decreased 6 months after the third dose, but remained higher than baseline levels before vaccination.

Discussion and general conclusions

Discussion of immunogenicity

The results of this Phase 2 immunogenicity study on a 3-dose schedule (months 0, 2, and 6) of bivalent rLP2086 administered to infants and children ≥24 months to <10 years of age are consistent with previous studies. studies involving adolescents and young adults. Immunogenic responses to vaccination with bivalent rLP2086 were assessed with hSBA for validity using 4 primary MnB test strains, each expressing fHBP variants heterologous to vaccine component antigens, by more stringent criteria than the generally accepted correlate of protection (hSBA titer ≥1: four). Based on hSBA titer ≥LLOQ for the 4 primary MnB strains tested at 1 month post-vaccination 3, infants and older children in this study had similar immune responses to adolescents (10 years to < 19 years old) in study B1971009, where the number of individuals with hSBA titer ≥LLOQ after the third vaccination (in the schedule of administration at 0, 2 and 6 months) ranged from 79.1% to 100% in this study and from 87 .1% to 99.5% in study B1971009. There were no significant differences in the number of individuals with hSBA titer ≥LLOQ for the 4 primary test strains at 1 month post-vaccination 3 in these 2 studies, although study B1971009 included a much larger number of adolescents with hSBA titer ≥ LLOQ before vaccination compared with titers in infants and older children participating in this study, and in particular for test strains A22 (33.2% and 9%, respectively) and A56 (27.5% and 8 .3%, respectively). Bivalent rLP2086 appears to be highly immunogenic in individuals in the ≥24 months to <10 years group and appears to have a protective effect against MnB infection similar to that expected in adolescents based on the hSBA correlate of protection.

For other targets, immune responses in this study for a pooled age stratified group (≥24 months to <10 years) 1 month after a second dose of bivalent rLP2086 were relatively robust for the 4 primary MnB strains tested, where the number of individuals which achieved hSBA-titer≥LLOQ ranged from 48.5% to 100%. With the exception of the A56 strain, these responses were lower than those observed in adolescents (10 to <19 years) of study B1971009 who received 2 doses of bivalent rLP2086 2 months apart, ranging from 64% to 99.1%. Immunopersistence was assessed in this study in both infants and older children by measuring hSBA titers 6 months post-vaccination month post-vaccination 3 to 10.4%-82.4% at 6 months post-vaccination 3. GMT and number of individuals with defined hSBA titers also decreased 6 months post-vaccination 3. Although the number of individuals achieving hSBA titer ≥ LLOQ at 6 months post-vaccination 3 in this study (10.4%-82.4%) was lower than the number of adolescents (11 to <19 years of age) participating in study 6108A1-2001 (36.7%- 89.4%), however, it was still higher than the number of individuals aged ≥24 months to <10 years with hSBA titer ≥LLOQ at baseline (0% - 9%). It has been well established that the degree of colonization of meningococci increases with age. Differences between individuals in the age group ≥24 months to <10 years and in the older age group may be partly due to the number of individuals with baseline titer ≥LLOQ, which was a maximum of 27.5% (A56) and 33.2% (A22) in study B1971009, and 7.4%-13.4% for subfamily A strains in study 6108A1-2001. In addition, a recent study showed that carriers of disease-associated serogroup B strains were more common among individuals with protective hSBA titers (prior to vaccination) and that vaccination had no effect on subsequent transmission of these disease-associated strains. . This suggests that such a transfer may affect baseline hSBA titers, and that persistence or recolonization after vaccination may contribute to an increase in the number of individuals with hSBA titer ≥LLOQ observed in adolescents 6 months after vaccination compared with young children and the older children participating in this study, who, as the inventors suggest, have a lower likelihood of colonization and a lower percentage of hSBA titers ≥LLOQ at baseline. Immunosistence studies using monovalent serogroup C meningococcal conjugate vaccine in infants and children similarly showed that protective immunity declined rapidly. However, even in the group of children whose percentage of hSBA titers >1:8 for serogroup C was only 46.9% 4 years after stopping the first dose, the subsequent booster dose produced >1:8 titers in 100% of individuals. 1 month and 1 year after the booster dose. This indicates that even individuals who were seronegative before the booster dose had a strong anamnestic response that persisted up to 1 year after the booster dose. Therefore, a booster response study and a persistence study that were conducted in individuals treated with the first series of divalent rLP2086, i.e. infants and older children, provided a different perspective on the value of a booster dose in protecting against IMZ in adolescents and adults.

In general, bivalent rLP2086 administered at the 0, 2 and 6 month schedule is highly immunogenic in infants and children ≥24 months to <10 years of age with protective immune responses that have been shown to be achieved. hSBA, in a large number of individuals after the introduction of the 3rd dose. The immune responses assessed in this study were expected to be similar to those observed in previous studies in adolescents 1 month after the second and third doses. The 3-dose regimen is expected to confer high levels of protective immunity in young children and in children ≥24 months to <10 years of age.

General conclusions

In conclusion, bivalent rLP2086 given to infants and children ≥24 months to <10 years of age on a 3-dose schedule for months 0, 2, and 6 elicits a robust immune response in most children after the second and third doses. while protective antibody titers were achieved after the third dose, as determined by hSBA. hSBA titers declined 6 months after the 3 dose series. The vaccine administered in this study was safe and well tolerated with an acceptable safety profile in infants and children ≥24 months to <10 years of age.

Example 20: Phase 2 Randomized, Blinded, Controlled Study to Describe the Immunogenicity, Safety, and Tolerability of Neisseria meningitidis Serological Group B Bivalent Recombinant Lipoprotein 2086 Vaccine 2086 (bivalent rLP2086) Administered to Healthy Children aged 12 to <18 months or 18 to <24 months (B1971017-syn).

Goals

The main objectives of the immunogenicity test are:

- Description of the immune response assessed by the human complement serum bactericidal assay (hSBA) performed on 4 primary Neisseria meningitidis serogroup B (MnB) strains, 2 of which express the LP2086 subfamily A protein and 2 express the B subfamily protein , as determined 1 month after the third vaccination with bivalent rLP2086, in healthy children aged 12 to <18 months at baseline.

- Description of the hSBA-assessed immune response performed on 4 primary MnB strains, 2 of which express the LP2086 subfamily A protein and 2 express the B subfamily protein, as determined 1 month after the third vaccination with bivalent rLP2086, in healthy aged children 18 to <24 months at baseline.

The main purpose of the safety analysis:

Evaluation of the safety profile of bivalent rLP2086 compared to control (Hepatitis A Virus Vaccine [HAV]), where such evaluation was performed on local reactions, systemic effects, adverse events (AE), serious adverse events (SAE), newly diagnosed chronic conditions (NDCMC), treatment-associated effects (MAE), and sudden AE in healthy children aged 12 to <18 months and 18 to <24 months at baseline and in both pooled age stratified groups.

Other purposes of the immunogenicity assay:

- Description of the immune response assessed by the hSBA assay performed on 4 primary MnB strains tested, 2 of which express the LP2086 subfamily A protein and 2 express the B subfamily protein, as determined 1 month after the third vaccination with bivalent rLP2086, in healthy children aged 12 to <24 months at baseline (i.e., in both pooled age stratified groups).

- Description of the immune response assessed by hSBA performed on 4 primary MnB strains tested, 2 of which express the LP2086 subfamily A protein and 2 express the B subfamily protein, as determined 1 month after the second vaccination and 6, 12, 24 , 36, and 48 months after the third vaccination in healthy children aged 12 to <18 months and 18 to <24 months at baseline and in both pooled age stratified groups.

Experimental objectives of the immunogenicity assay:

- Additional description of the immune response assessed by hSBA conducted for 4 primary MnB strains tested, 2 of which express the LP2086 subfamily A protein and 2 express the B subfamily protein, as determined 1 month after the second vaccination and 1.6, 12, 24, 36 and 48 months after the third vaccination with bivalent rLP2086 in healthy children aged 12 to <18 months and 18 to <24 months at baseline and in both pooled age stratified groups.

- Additional description of the hSBA-assessed immune response performed on the second MnB test strains expressing LP2086 subfamily A and B proteins at 1 month after the second vaccination and at 1, 6, 12, 24, 36 and 48 months after the third vaccination in healthy children aged 12 to <18 months and 18 to <24 months at baseline and in both pooled age stratified groups.

Methods

Study protocol:

This study is a randomized, actively controlled, multicenter, Phase 2, blinded method unknown to either the observer or the sponsor, randomly enrolling approximately 396 healthy infants, stratified by age, from 12 to <18 months or from 18 to <24 months in a 2:1 ratio who received bivalent rLP2086 (either 2 doses [60 mcg or 120 mcg]) or licensed childhood HAV vaccine (0.5 mL)/sterile saline injection (0.5 mL 0.85% sodium chloride).

The study was carried out in 2 stages. In Stage 1, the immunogenicity, safety and tolerability of the vaccine was assessed in 2 phases: a marker entry phase and an expanded participation phase. Stage 2 assessed the duration of the immune response to bivalent rLP2086.

For immunogenicity, all data up to 1 month post-vaccination 3 (Visit 7) are presented, except for additional data for the second MnB strain tested, pertaining to the experimental target. The final report includes all immunogenicity data obtained after completion of Stage 2 and safety data from the period after Visit 8 until the end of the study (Visit 13, 48 months after vaccination 3).

In accordance with the test program, the marker phase of inclusion in Stage 1 includes a total of four marker groups: 2 age-stratified groups for each dose (60 μg or 120 μg) of bivalent rLP2086. The marker groups, which included young children, consisted of individuals aged 12 to <15 months, and the marker groups, which included older children, consisted of individuals aged 18 to <24 months. According to the program, each of the 4 marker groups included approximately 33 individuals. The marker phase of inclusion in the test was supplemented with IRC data at predetermined time points, and this phase was completed in accordance with established norms. The 120 µg dose was not administered to children in the marker groups until the 60 µg dose was assessed by the IRC as safe and tolerable in the age-matched marker group. The 60 µg dose for younger children in the marker groups was not administered until this dose was assessed by the IRC as safe and tolerable in the 120 µg dose of the older children marker group.

Prior to Phase 1 of the extended inclusion phase, all IRC data were reviewed after vaccination 1 and entered into an electronic diary on day 7, and then SAE data were collected from marker group individuals. Based on the report, IRC data sampled for a dose of 120 μg of divalent rLP2086 were examined in the extended enrollment phase in Stage 1 for both age stratified groups. The expanded inclusion group, which included younger children (an additional 132 individuals), included children aged 12 to <18 months and was stratified by age into two subgroups: children 12 to <15 months and children 15 to <18 months. The expanded inclusion group, which included older children (an additional 132 individuals), included children aged 18 to <24 months during the expanded inclusion phase. The total duration of the study of individuals who completed only Stage 1 was approximately 18 months. The visit schedule for Stage 1 is presented in Table 23.

Stage 2 includes only those individuals who were randomly assigned and who received divalent rLP2086 (regardless of dose). The total duration of the study of individuals who completed Stage 2 was approximately 4.5 years (54 months). The visit schedule for Stage 2 is presented in Table 24.

Bivalent rLP2086 was administered at months 0, 2 and 6 (Visits 1, 4 and 6). Childhood HAV vaccine was administered at Months 0 and 6 (Visits 1 and 6) and saline was administered at Month 2 (Visit 4) while maintaining blinding.

Table 23. Scheme of visits in Stage 1 Visit number one 2 3 four 5 6 7 eight ^{9a 10a} Period of time Month 0 Week 1 Month 1 Month 2 Month 3 Month 6 Month 7 Month 12 Month 18 End of Stage 1 Description of the visit Vaccination
one Follow-up visit after vaccination 1 Phone contact Vaccination
2 Blood sampling after vaccination 2 Vaccination
3 Blood sampling after vaccination 3 Observation visit for month 6 ^b Blood sampling for persistence of antibodies ^b Phone contact Vaccination phase Observation phase Vaccination and 30 minute observation ^c X X X Blood sampling 5 ml X ^d X X ^Xe X Abbreviations: CRF = document in the form of a report; e-diary=electronic diary.
a. Visits 9 and 10 were not included in this clinical study data analysis report.
b. Related to vaccination 3.
c. Injection administered by a knowledgeable specialist; acute reactions assessed by an outside observer. The vaccination site is indicated in the source, CRF and in the electronic diary.
d. Blood was taken before vaccination and only after confirmation of inclusion in the study program.
e. The results of the analysis of the blood sample for immunogenicity at Visit 8 were not included in the primary analysis report in the clinical study. Source: Stage 2 study protocol outline.

Table 24. Scheme of visits in Stage 2 Visit number eleven 12 13 Period of time Month 30 Month 42 Month 54 Description of the visit (Time after vaccination 3) Immunogenicity 1
(24 months) Immunogenicity 2
(36 months) Immunogenicity 3
(48 months) Blood sampling 5 ml X X X Source: Stage 2 study protocol outline.

Administered vaccines : Bivalent rLP2086 (60 µg or 120 µg) was administered 3 times during the course of the study: with the first vaccination at Visit 1 (month 0), the second vaccination at Visit 4 (month 2), and the third vaccination at Visit 6 (month 6). Bivalent rLP2086 was administered by intramuscular injection into either the deltoid muscle or the anterior lateral thigh muscle. The HAV vaccine was administered twice during the entire course of the study: with the first vaccination at Visit 1 (month 0), and the third vaccination at Visit 6 (month 6). Saline was administered at the time of the second vaccination (month 2). The HAV vaccine/saline solution was administered by intramuscular injection into either the deltoid muscle or the anterior lateral thigh muscle. Investigational product was administered only by a qualified medical professional who is part of the study staff and is a third party. If the muscle mass of the deltoid muscle was unsuitable for intramuscular injection, then the thigh was chosen as the preferred injection site.

Immunogenicity Assessment : To facilitate immunogenicity testing, approximately 5 ml of blood was taken from individuals at the following time points during Stage 1: pre-vaccination 1 (Visit 1), 1 month post-vaccination 2 (Visit 5), 1 month post-vaccination 3 (Visit 7), 6 months after vaccination 3 (Visit 8), and 12 months after vaccination 3 (Visit 9). In general, 25 ml was taken over a period of 18 months. Local anesthesia/local anesthesia may be administered prior to blood sampling.

To determine the duration of the immune response, approximately 5 ml of blood was drawn from individuals in Stage 2 at the following time points: 2 years post-vaccination 3, 3 years post-vaccination 3, and 4 years post-vaccination 3. In total, 15 ml was taken over the period approximately 2.5 years.

To assess the immune response to bivalent rLP2086, functional antibodies were analyzed in hSBA using meningococcal strains of serogroup B. In hSBA, antibodies were detected in human serum that cause complement-dependent cytolysis of the meningococcal target strain. Four (4) MnB primary test strains, PMB80 (A22), PMB2001 (A56), PMB2948 (B24), and PMB2707 (B44), each expressing a variant factor H-binding protein (fHBP) heterologous to vaccine antigen components, were used in hSBA to determine immunogenicity endpoints in this study.

Due to serum volume limitation, 2 primary strains (PMB80 [A22] and PMB2948 [B24]) were tested at each time of blood sampling in one half of the individuals (in both age stratified groups), and the other 2 primary strains (PMB2001 [A56] and PMB2707 [B44]) were tested at every moment of blood sampling in the other half of the individuals.

Following the inclusion of all individuals in the study program (Visit 1), a team of statisticians from the Independent Statistical Center (ISC) outside the study program submitted 2 lists of individuals (randomly selected individuals, 50% of which were tested for PMB80 [A22]/ PMB2948 [B24], and the remaining 50% of individuals tested for PMB2001 [A56]/PMB2707 [B44]) by a sponsored sample analysis team. Both lists had the same randomization ratio (2:1) and the same age distribution as in the study protocol. The same pair of strains (PMB80 [A22]/PMB2948 [B24] or PMB2001 [A56]/PMB2707 [B44]) was tested on all visits for the same individuals.

After completion of testing for primary assays, and if serum was present in sufficient volume, additional testing could be performed to assess immune response to bivalent rLP2086: PMB80 (A22) and PMB2948 (B24) can be tested in serum samples taken from 50% individuals treated with divalent rLP2086 and these serum samples were first tested for PMB2001 (A56) and PMB2707 (B44). Conversely, PMB2001 (A56) and PMB2707 (B44) can be tested in serum samples taken from 50% of individuals treated with bivalent rLP2086, and these serum samples were first tested for PMB80 (A22) and PMB2948 (B24). Testing for secondary strains may be performed.

Statistical methods:

The primary endpoints of immunogenicity are:

- Number of individuals achieving hSBA titer ≥ lower limit(s) of quantification (LLOQ) 1 month after the third vaccination for each of the 4 primary MnB strains tested in healthy children aged 12 to <18 months at the beginning of the study.

- Number of individuals achieving hSBA titer ≥LLOQ 1 month after the third vaccination for each of the 4 primary MnB strains tested in healthy children aged 18 to <24 months at baseline.

All secondary immunogenicity endpoints for the entire study are described in this application, but this Example presents the results of immunogenicity endpoints that were obtained from visits 1-7 only. Subsequent endpoints were analyzed.

The following endpoint refers to results obtained for healthy subjects aged 12 to <24 months (i.e., in both pooled age stratified groups) at baseline:

- Number of individuals with hSBA titers ≥LLOQ for each of the 4 primary MnB strains tested at 1 month after the third vaccination with bivalent rLP2086 and at 6, 12, 24, 36 and 48 months after the third vaccination with bivalent rLP2016.

The following endpoints refer to results obtained for healthy individuals aged 12 to <18 months and 18 to <24 months at baseline and in both pooled age stratified groups:

- Number of individuals with hSBA titers ≥LLOQ for each of the 4 primary MnB strains tested 1 month after the second vaccination with divalent rLP2086.

- Number of individuals with hSBA titers ≥LLOQ, ≥1:4, ≥1:8, ≥1:16, ≥1:32, ≥1:64 and ≥1:128 for each of the 4 primary MnB strains per eligible visit to take a blood sample.

- Geometric mean hSBA titers (GMT) for each of the 4 primary MnB strains tested at each eligible blood sampling visit.

All experimental endpoints defined below refer to hSBA results for all healthy individuals aged 12 to <18 months or 18 to <24 months at baseline and for the two pooled age stratified groups where said individuals received divalent rLP2086 and were tested for the appropriate strain at the appropriate time:

- Number of individuals with hSBA titers ≥LLOQ, ≥1:4, ≥1:8, ≥1:16, ≥1:32, ≥1:64 and ≥1:128 at each appropriate time of blood sampling.

- GMT hSBA for each of the 4 primary MnB strains at each eligible blood sampling visit.

- Number of individuals achieving at least a 4-fold increase in hSBA titer from baseline 1 month after the third vaccination with divalent rLP2086 for each of the 4 primary test strains:

- For individuals with a baseline hSBA titer below the limit of detection (LOD) or with an hSBA titer <1:4, a 4-fold response was defined as hSBA titer≥1:16.

- For individuals with a baseline hSBA titer ≥LOD (ie, hSBA titer ≥1:4) and < LLOQ, a 4-fold response was defined as an hSBA titer that is ≥4 times the LLOQ.

- For individuals with a baseline hSBA titer≥LLOQ, a 4-fold response was defined as an hSBA titer that is ≥4 times the baseline titer.

The following end points were determined if sufficient serum was available to test each individual for all 4 primary strains and/or secondary strains.

- Number of individuals achieving hSBA titer ≥LLOQ for the combination of all 4 primary test strains (PMB80 [A22], PMB2948 [B24], PMB2001 [A56] and PMB2707 [B44]) 1 month after the third vaccination with bivalent rLP2086. This only applies to those individuals in whom all 4 primary strains have been tested.

- For hSBA, the following additional experimental analyzes were carried out for secondary strains of MnB:

- GMT hSBA for each secondary MnB strain tested, 1 month after the second and third vaccinations and/or at each time of blood sampling.

- Number of individuals with hSBA titer ≥LLOQ for each secondary MnB strain 1 month after the second and third vaccinations and/or at each time of blood sampling.

Primary Endpoint Analysis

The primary analysis to achieve the main goals is to determine the number of individuals with hSBA titer ≥LLOQ 1 month after the third vaccination for each of the 4 primary MnB strains tested in healthy children aged 12 to <18 months and 18 to <24 months, in the beginning of the study, respectively. To achieve this goal, a group assessed for immunogenicity was used and percentages and confidence intervals (CI) were presented.

Secondary Endpoint Analysis

All analyzes performed for the mITT group were considered secondary analyses.

Secondary analyzes also include the percentages of individuals with hSBA titers ≥LLOQ for each of the 4 primary MnB strains tested 1 month after the third vaccination for both immunogenicity groups and for the mITT group.

In this Example, the percentage of individuals with hSBA titers ≥LLOQ for each of the 4 primary MnB strains tested at 1 month after the second vaccination and 1 month after the third vaccination was determined for the immunogenicity group and for the mITT group. In the final analysis, the percentage of individuals with hSBA titers ≥LLOQ for each of the 4 primary MnB strains tested at 6, 12, 24, 36 and 48 months after the third vaccination was analyzed for the immunogenicity group and for the mITT group.

The percentage of individuals with hSBA titers ≥LLOQ 1:4, 1:8, 1:16, 1:32, 1:64, 1:128 for each of the 4 primary MnB strains tested at each eligible blood sampling visit was analyzed for group evaluated for immunogenicity and for the mITT group.

The GMT for each of the primary MnB strains tested at each eligible blood sampling visit was summarized for the immunogenicity group and for the mITT group.

Analysis of experimental endpoints

The number of individuals who achieved at least a 4-fold increase in hSBA titers from baseline 1 month after the second and third vaccinations with divalent rLP2086 were systematized for the immunogenicity group and for the mITT group.

Subgroup analysis

Some immunogenicity and safety endpoints were separately classified by sex, country, and age stratification.

results

Distribution of individuals and demographics:

A total of 396 individuals aged 12 to <24 months were selected for this study. Of all selected individuals, 44 individuals received 60 μg of bivalent rLP2086, 220 individuals received 120 μg of bivalent rLP2086, and 132 individuals received HAV vaccine/saline. 198 individuals were randomly assigned to each age stratified group (12 to <18 months and 18 to <24 months).

Of 396 randomly selected individuals, 385 (97.2%) individuals completed the vaccination phase (from vaccination in study 1 [Visit 1] up to 1 month after vaccination 3 [Visit 7]) of this study. A total of 386 (97.5%) individuals completed the follow-up phase (Visit 7 to 6 months post-vaccination 3 [Visit 8]). Overall, a total of 381 (96.2%) individuals completed all study visits up to the follow-up visit at month 6 (Visit 8) and after study completion, status was similar for individuals in each age stratified group (189 [95, 5%] of individuals and 192 [97.0%] of individuals in an age stratified group of 12 to <18 months and 18 to <24 months, respectively).

Overall, 52.8% of the individuals were female and the majority of the individuals were Indo-European (94.4%) and non-Hispanic/Latino (99.5%). The mean age (SD) of individuals at first vaccination was 17.3 (3.61) months (range 12 to 23 months). The demographics were largely similar for the vaccine groups.

The characteristics of the ITT and mITT groups were similar to those of the safety group.

Immunogenicity results:

The main objectives of this study are to characterize the immune response in individuals to bivalent rLP2086, as determined by hSBA, against 4 primary MnB strains tested, 2 of which expressed the LP2086 subfamily A protein and 2 expressed the LP2086 subfamily B protein, as determined through 1 month after the third vaccination in healthy individuals aged 12 to <18 months and 18 to <24 months. The second objective is to describe immune responses for patients in a pooled age stratified group (12 to <24 months). The primary goal endpoints are the number of individuals in each age stratified group who achieved hSBA titers ≥LLOQ for each of the 4 primary MnB strains 1 month after the third vaccination.

A sustained immune response was observed at both doses in children aged 12 to <18 months and in children aged 18 to <24 months, and in a pooled age stratified group (12 to <24 months) 1 month after administration of the third doses of divalent rLP2086, as evidenced by the number of individuals whose hSBA titer was ≥LLOQ (1:8 for A56, B24, and B44; 1:16 for A22) for each of the 4 primary MnB strains tested. For the 60 μg group, the number of individuals achieving hSBA titer ≥LLOQ ranged from 88.9% to 100.0% for younger children (12 to <18 months) and from 81.8% up to 100.0% for older children (18 to <24 months) after 3 doses. For the 120 µg group, the number of individuals achieving hSBA titer ≥LLOQ ranged from 71.1% to 100.0% for children aged 12 to <18 months and from 72.0% to 100. 0% for children aged 18 to <24 months after 3 doses. For the pooled age stratified group, the number of individuals with hSBA titer ≥LLOQ for each of the 4 primary MnB strains tested 1 month after the third vaccination ranged from 85.0% to 100.0% for the 60 µg group and from 71.6% to 100.0% for the 120 µg group. These data were also supported by high GMT (ranging from 4.0 to 8.5 at baseline to 15.1-171.4 at 1 month post-vaccination 3) and the number of individuals achieving hSBA titers ≥1:4 (71.1% - 100%) or ≥1:16 (63.6% - 100%) vs. each of the 4 primary MnB strains tested after 3 doses of bivalent rLP2086 versus baseline for both age stratified groups and for all doses. In addition, the number of individuals in the pooled age stratified groups whose hSBA titer was ≥4 compared to baseline 1 month after the third vaccination for each of the 4 primary MnB strains tested ranged from 67.4% to 100.0% for both dose-dependent groups. In conclusion, 3 doses of 60 µg or 120 µg of divalent rLP2086 given at month 0, 2, and 6 schedules induced robust immune responses in children aged 12 to <24 months (in separate and pooled age stratified group).

The second objective of the study is to characterize immune responses 1 month after a second dose of bivalent rLP2086 as assessed by ≥LLOQ responses, determined hSBA titers and hSBA GMT for 2 age stratified groups and pooled age stratified group. For the pooled age stratified group, the number of individuals with hSBA titer ≥LLOQ after the second dose of bivalent rLP2086 (administered 2 months after the first dose) ranged from 57.9% to 94.7% for individuals in the 60 µg group, and from 33.7% to 100.0% for individuals in the 120 μg group. Similar results were obtained for 2 separate age stratified groups, and no significant differences were observed between the stratified groups of younger and older age. These data were further supported by an increase in GMT (range 7.2 to 110.6) from baseline, and the number of individuals who achieved hSBA titer ≥1:4 (36.0% - 100%) or ≥ 1:16 (32.6% - 100%) vs. each of the 4 primary MnB strains tested after 2 doses of bivalent rLP2086 versus baseline for the pooled age stratified group treated with both doses. Similar results were obtained for 2 separate age stratified groups. In addition, the number of individuals in the pooled age stratified group whose hSBA titer was ≥4 times higher than baseline 1 month after the second vaccination for each of the 4 primary MnB strains tested ranged from 30.2% to 98, 9%. In conclusion, 2 doses of 60 μg or 120 μg of divalent rLP2086 administered 2 months apart induced immune responses in children aged 12 to <24 months (in a separate and pooled age stratified group). In general, at doses of 60 µg and 120 µg, bivalent rLP2086 given in 3 doses on a schedule for months 0, 2, and 6 elicits a robust immune response in children aged 12 to <24 months with protective antibody titers achievable as determined by hSBA, in a large number of individuals after the administration of the third dose.

Conclusions:

In conclusion, at the 60 µg and 120 µg doses, bivalent rLP2086 given to children 12 to <24 months of age on a schedule for months 0, 2, and 6 produces protective antibody titers after the third dose, as determined with hSBA. The vaccine administered in this study was safe and well tolerated in children aged 12 to <24 months with an acceptable safety profile.

Example 21: Phase 2 randomized, blinded, controlled trial to characterize the immunogenicity, safety, and tolerability of Neisseria meningitidis serogroup B recombinant lipoprotein 2086 bivalent vaccine (bivalent rLP2086) administered to healthy children aged 12 to <18 months or 18 to <24 months (B1971035-CSR).

The Phase 2 study was conducted in 2 stages. Stage 1 was designed and performed to evaluate the safety, tolerability, and immunogenicity of bivalent rLP2086 in healthy children 12 to <24 months of age up to 12 months after the last experimental vaccination (Visit 10). Stage 1 consisted of a trial entry marker phase, which included 4 marker groups, and an extended entry phase. In the marker group, bivalent rLP2086 was administered at 2 doses (60 μg or 120 μg) in 2 age stratified groups (ages 12 to <15 months and 18 to <24 months). Dose selection for the extended inclusion phase was based on a report submitted by the National Control Committee (IRC) to assess the safety profile at 2 doses. Stage 2 was planned to assess the duration of the immune response up to 4 years after the last experimental vaccination.

Main objectives of the immunogenicity assay

- Description of immune response assessed by hSBA assay performed on 4 primary MnB strains, 2 of which express LP2086 subfamily A protein and 2 express LP2086 subfamily B protein, as determined 1 month after the third vaccination with bivalent rLP2086, in healthy children aged 12 to <18 months at baseline.

- Description of the immune response assessed by hSBA performed on 4 primary MnB strains, 2 of which express the LP2086 subfamily A protein and 2 express the LP2086 subfamily B protein, as determined 1 month after the third vaccination with bivalent rLP2086, in healthy children at age 18 to <24 months at baseline.

Other purposes of the immunogenicity assay:

- Description of immune response assessed by hSBA assay performed on 4 primary MnB strains tested, 2 of which express LP2086 subfamily A protein and 2 express LP2086 subfamily B protein, as determined 1 month after third vaccination with bivalent rLP2086, in healthy subjects children aged 12 to <24 months at baseline (i.e., in both pooled age stratified groups).

- Description of the immune response assessed by hSBA conducted for 4 primary MnB strains tested, 2 of which express the LP2086 subfamily A protein and 2 express the LP2086 subfamily B protein, as determined 1 month after the second vaccination and 6, 12, 24, 36 and 48 months after the third vaccination in healthy children aged 12 to <18 months and 18 to <24 months at baseline and in both pooled age stratified groups.

Experimental objectives of the immunogenicity assay:

- Additional description of the immune response assessed by hSBA conducted for 4 primary MnB strains tested, 2 of which express LP2086 subfamily A protein and 2 express LP2086 subfamily B protein, as determined 1 month after the second vaccination and 1.6 , 12, 24, 36 and 48 months after the third vaccination with bivalent rLP2086 in healthy children aged 12 to <18 months and 18 to <24 months at baseline and in both pooled age stratified groups.

- Additional description of the hSBA-assessed immune response performed on the second MnB test strains expressing LP2086 subfamily A and B proteins at 1 month after the second vaccination and at 1, 6, 12, 24, 36 and 48 months after the third vaccination in healthy children aged 12 to <18 months and 18 to <24 months at baseline and in both pooled age stratified groups.

Study plan

General protocol and study design

This study is a randomized, actively controlled, multicenter, Phase 2, blinded method unknown to either the observer or the sponsor, randomly enrolling approximately 396 healthy infants, stratified by age, from 12 to <18 months or from 18 to <24 months in a 2:1 ratio who received bivalent rLP2086 (2 doses [60 mcg or 120 mcg]) or licensed HAV childhood vaccine (0.5 ml)/sterile saline injection (0.5 ml 0.5 ml) 85% sodium chloride). The study was carried out in 2 stages. In Stage 1, the immunogenicity, safety and tolerability of the vaccine was evaluated in 2 phases: a marker trial entry phase and an extended entry phase. Stage 2 assessed the duration of the immune response to bivalent rLP2086.

In accordance with the test program, the marker phase of inclusion in Stage 1 includes a total of four marker groups: 2 age stratified groups for each dose (60 μg or 120 μg) of bivalent rLP2086. The marker groups, which included younger children, consisted of individuals aged 12 to <15 months, and the marker groups, which included older children, consisted of individuals aged 18 to <24 months. According to the program, each of the 4 marker groups included approximately 33 individuals. The marker phase of inclusion in the test was supplemented with IRC data at predetermined time points and this phase was completed in accordance with the established norms. In the 120 μg marker group, this dose was not administered until the 60 μg dose was assessed by the IRC as safe and tolerable in the same age marker group. In the marker group of younger children at the 120 mcg dose, this dose was not administered until this dose was assessed by the IRC as safe and tolerable in the marker group of the older children who were given the dose of 120 mcg.

Prior to Stage 1 of the expanded inclusion phase, all IRC data were reviewed after vaccination 1 and entered into an electronic diary on day 7, and then SAE data were taken from individuals in the marker group. Based on the report, IRC data sampled for a dose of 120 μg of divalent rLP2086 were examined in the extended enrollment phase in Stage 1 for both age stratified groups. The expanded inclusion group, which included younger children (an additional 132 individuals), included children aged 12 to <18 months and was stratified by age into two subgroups: children 12 to <15 months and children 15 to <18 months. The expanded inclusion group, which included older children (an additional 132 individuals), included children aged 18 to <24 months during the expanded inclusion phase. The total duration of the study of individuals who completed only Stage 1 was approximately 18 months.

Stage 2 includes only those individuals who were randomly assigned and who received divalent rLP2086 (regardless of dose). The total duration of the study of individuals who completed Stage 2 was approximately 4.5 years (54 months).

Bivalent rLP2086 was administered at months 0, 2 and 6 (Visits 1, 4 and 6). Childhood HAV vaccine was administered at Months 0 and 6 (Visits 1 and 6) and saline was administered at Month 2 (Visit 4) while maintaining blinding.

Discussion of the study protocol, including the choice of control groups

This 2-stage study evaluated the safety, tolerability, and immunogenicity of bivalent rLP2086 at 2 doses (60 µg and 120 µg) in healthy children aged 12 to <24 months.

In this study, the HAV vaccine (given at months 0 and 6) was chosen as the control. Compared to other vaccines recommended for this age group, the HAV vaccine has a better tolerability profile. In addition, the HAV vaccine provides an advantage to individuals who have an increased risk of hepatitis A virus infection, either during future travel or other environmental conditions. The general recommended HAV vaccine schedule is to give 2 doses at months 0 and 6. In this study, saline was administered at month 2 to ensure the study was blinded.

Administered Vaccines

Bivalent rLP2086 (60 µg or 120 µg) was administered 3 times during the course of the study: with the first vaccination at Visit 1 (month 0), the second vaccination at Visit 4 (month 2), and the third vaccination at Visit 6 (month 6). Bivalent rLP2086 was administered by intramuscular injection into either the deltoid muscle or the anterior lateral thigh muscle. The HAV vaccine was administered twice during the course of the study: with the first vaccination at Visit 1 (month 0) and the third vaccination at Visit 6 (month 6). Saline was administered at the time of the second vaccination (month 2). The HAV vaccine/saline solution was administered by intramuscular injection into either the deltoid muscle or the anterior lateral thigh muscle. Investigational product was administered only by a qualified medical professional who is part of the study staff and is a third party. If the muscle mass of the deltoid muscle was unsuitable for intramuscular injection, then the thigh was chosen as the preferred injection site. The insertion site (eg, left/right arm/thigh) was specified in the explanatory documents or in the CRF report.

Identity of investigational product(s)

Bivalent rLP2086 (containing 30 μg [60 μg dose] or 60 μg [120 μg dose] of each of the purified rLP2086 subfamily A and subfamily B proteins adsorbed to aluminum in a sterile buffered isotonic suspension) was prepared at a dose of 0.5 ml for injection.

The licensed pediatric HAV vaccine was formulated as a 0.5 ml injection.

The placebo is a sterile saline solution for injection (0.85% sodium chloride) supplied in a dose of 0.5 ml.

Experimental products (bivalent rLP2086, HAV vaccine and saline) were supplied by the sponsor to each study site. Investigational vaccines were packaged and labeled as an experimental product in accordance with current regulations and requirements approved by local and judicial regulatory authorities. Each experimental product was labeled with a unique kit number.

Choosing a vaccination schedule

Bivalent rLP2086 (60 μg or 120 μg) was administered on a schedule for months 0, 2 and 6. A control group of individuals received an HAV vaccine for months 0 and month 6 and a saline injection for month 2 to enable the study to be blinded.

Serum bactericidal activity assay using divalent rLP2086 - Primary test strains

To assess the immune response to bivalent rLP2086, functional antibodies were analyzed in hSBA using meningococcal strains of serogroup B. In hSBA, antibodies were detected in human serum that cause complement-dependent cytolysis of the meningococcal target strain. Four (4) MnB primary test strains, PMB80 (A22), PMB2001 (A56), PMB2948 (B24), and PMB2707 (B44), each expressing a variant factor H-binding protein (fHBP) heterologous to vaccine antigen components, were used in hSBA to determine immunogenicity endpoints in this study.

Due to serum volume limitation, 2 primary strains (PMB80 [A22] and PMB2948 [B24]) were tested at each time of blood sampling in one half of the individuals (in both age stratified groups), and the other 2 primary strains (PMB2001 [A56] and PMB2707 [B44]) were tested at every moment of blood sampling in the other half of the individuals.

Following the inclusion of all individuals in the study program (Visit 1), a team of statisticians from the Independent Statistical Center (ISC) outside the study program submitted 2 lists of individuals (randomly selected individuals, 50% of which were tested for PMB80 [A22]/ PMB2948 [B24], and the remaining 50% of individuals tested for PMB2001 [A56]/PMB2707 [B44]) by a sponsored sample analysis team. Both lists had the same randomization ratio (2:1) and the same age distribution as in the study protocol. The same pair of strains (PMB80 [A22]/PMB2948 [B24] or PMB2001 [A56]/PMB2707 [B44]) was tested on all visits for the same individuals.

Additional tests

After completion of testing for primary assays, and if sufficient serum was present, additional testing could be performed to assess immune response to bivalent rLP2086: PMB80 (A22) and PMB2948 (B24) could be tested in serum samples taken from 50% individuals treated with divalent rLP2086 and these serum samples were first tested for PMB2001 (A56) and PMB2707 (B44). Conversely, PMB2001 (A56) and PMB2707 (B44) could be tested in serum samples taken from 50% of individuals treated with bivalent rLP2086, and these serum samples were first tested for PMB80 (A22) and PMB2948 (B24). Testing for secondary strains may be performed.

Immunogenicity assay

Comparison of Points of Interest and Endpoints - Primary Endpoints of Immunogenicity

The primary endpoints of immunogenicity are:

- Number of individuals achieving hSBA titer ≥ lower limit(s) of quantification (LLOQ) 1 month after the third vaccination for each of the 4 primary MnB strains tested in healthy children aged 12 to <18 months at the beginning of the study.

- Number of individuals achieving hSBA titer ≥LLOQ 1 month after the third vaccination for each of the 4 primary MnB strains tested in healthy children aged 18 to <24 months at baseline.

Comparison of Points of Interest and Endpoints - Secondary Endpoints of Immunogenicity

All secondary immunogenicity endpoints for the entire study are described in this application, but this Example presents the results of immunogenicity endpoints that were obtained only at visits 1-7 (Vaccination 1-3 months after vaccination 3).

The following endpoint refers to outcomes in healthy individuals aged 12 to <24 months (i.e., pooled age stratified group) at baseline:

- Number of individuals with hSBA titers ≥LLOQ for each of the 4 primary MnB strains tested at 1 month after the third vaccination with bivalent rLP2086 and at 6, 12, 24, 36 and 48 months after the third vaccination with bivalent rLP2016.

The following endpoints refer to outcomes in healthy subjects aged 12 to <18 months and 18 to <24 months at baseline and in the pooled age stratified group:

- Number of individuals with hSBA titers ≥LLOQ for each of the 4 primary MnB strains tested 1 month after the second vaccination with divalent rLP2086.

- Number of individuals with hSBA titers ≥LLOQ, ≥1:4, ≥1:8, ≥1:16, ≥1:32, ≥1:64 and ≥1:128 for each of the 4 primary MnB strains per eligible visit to take a blood sample.

- Geometric mean hSBA titers (GMT) for each of the 4 primary MnB strains tested at each eligible blood sampling visit.

Experimental Immunogenicity Endpoints

All experimental endpoints defined below refer to hSBA results for all healthy subjects aged 12 to <18 months or 18 to <24 months at baseline and in the pooled age stratified group where said individuals received divalent rLP2086 and were tested on the right strain at the right time:

- Number of individuals with hSBA titers ≥LLOQ, ≥1:4, ≥1:8, ≥1:16, ≥1:32, ≥1:64 and ≥1:128 at each appropriate time of blood sampling.

- GMT hSBA for each of the 4 primary MnB strains at each eligible blood sampling visit.

- Number of individuals achieving at least a 4-fold increase in hSBA titer from baseline 1 month after the third vaccination with divalent rLP2086 for each of the 4 primary test strains:

- For individuals with a baseline hSBA titer below the limit of detection (LOD) or with an hSBA titer <1:4, a 4-fold response was defined as hSBA titer≥1:16.

- For individuals with a baseline hSBA titer ≥LOD (ie, hSBA titer ≥1:4) and < LLOQ, a 4-fold response was defined as an hSBA titer that is ≥4 times the LLOQ.

- For individuals with a baseline hSBA titer≥LLOQ, a 4-fold response was defined as an hSBA titer that is ≥4 times the baseline titer.

The following endpoints were considered if sufficient serum was available to test each individual for all 4 primary strains and/or secondary strains.

- Number of individuals achieving hSBA titer ≥LLOQ for the combination of all 4 primary test strains (PMB80 [A22], PMB2948 [B24], PMB2001 [A56], and PMB2707 [B44]) 1 month after the third vaccination with bivalent rLP2086 . This only applies to those individuals in whom all 4 primary strains have been tested.

- For hSBA, the following additional experimental analyzes were carried out for secondary strains of MnB:

- GMT hSBA for each secondary MnB strain tested, 1 month after the second and third vaccinations and/or at each time of blood sampling.

- Number of individuals with hSBA titer ≥LLOQ, for each secondary MnB strain, 1 month after the second and third vaccinations and/or at each time of blood sampling.

Analyzed groups

Modified treatment group

All randomly selected individuals for whom at least 1 valid and definite probability analysis result was obtained were included in the modified treatment group (mITT). This series of assays was used to conduct an immunogenicity assay. Individuals were tested on the respective experimental product for which they were randomly assigned to the mITT Group.

Analysis Methods

The control group and each dose group, which included different groups with the same age stratification, were pooled for analysis. All analyzes for immunogenicity were systematized separately for each age stratified group, as well as for the general group.

This study was not conducted to test any hypothesis; therefore, an analysis method was used in this study to evaluate the primary, secondary, and experimental endpoints.

LLOQ is 1:16 for PMB80 (A22), 1:8 for PMB2001 (A56), 1:8 for PMB2707 (B44) and 1:8 for PMB2948 (B24).

To calculate GMT, hSBA results below LLOQ were reported as 0.5 × LLOQ for the primary analysis.

Primary Endpoint Analysis

The primary analysis to achieve the main goals is to determine the number of individuals with hSBA titer ≥LLOQ 1 month after the third vaccination, for each of the 4 primary MnB strains tested in healthy children aged 12 to <18 months and 18 to <24 months, at the start of the study, respectively. To achieve this goal, a group assessed for immunogenicity was used and percentages and confidence intervals (CI) were presented.

Secondary Endpoint Analysis

All analyzes performed for the mITT group were considered secondary analyses.

Secondary endpoint analyzes also include the percentage of individuals with hSBA titers ≥LLOQ for each of the 4 primary MnB strains tested 1 month after the third vaccination for the immunogenicity group and for the mITT group.

In this Example, the percentage of individuals with hSBA titers ≥LLOQ for each of the 4 primary MnB strains tested at 1 month post-second vaccination and 1 month post-vaccination 3 was determined for the immunogenicity group and for the mITT group, and these data are presented in report. The same analysis was scheduled for subsequent time periods (6, 12, 24, 36 and 48 months after the third vaccination).

Percentage of individuals with hSBA titers ≥LLOQ 1:4, 1:8, 1:16, 1:32, 1:64, 1:128 for each of the 4 primary MnB strains tested at 1 month post vaccination 2 and 1 month post vaccination 3 vaccinations were determined for the immunogenicity group and for the mITT group, and these data are presented in the report. The same analysis was scheduled for subsequent time periods (6, 12, 24, 36 and 48 months after vaccination 3).

The GMT for each of the primary tested MnB strains at 1 month after the second vaccination and 1 month after the third vaccination was systematized for the immunogenicity group and for the mITT group, and these data are presented in the report. The same analysis was scheduled for subsequent time periods (6, 12, 24, 36 and 48 months after vaccination 3).

Analysis of experimental endpoints

The number of individuals who achieved at least a 4-fold increase in hSBA titers from baseline 1 month after the second and third vaccinations with divalent rLP2086 were systematized for the immunogenicity group and for the mITT group.

Inverse Cumulative Distribution Curves

Empirical reverse cumulative distribution curves (RCDC) were also analyzed for each of the 4 primary MnB strains tested and 1 month post-vaccination 2 and 1 month post-vaccination 3 for the immunogenicity group.

Assessment of immunogenicity

Analyzed groups

The group assessed for immunogenicity is the primary immunogenicity test group. The mITT group was used as an additional group for immunogenicity analysis.

A total of 348 (87.9%) individuals were included in the group assessed for immunogenicity, and 48 (12.1%) individuals were excluded from this group. Individuals may be excluded from immunogenicity evaluation groups for more than 1 reason. A total of 31 (7.8%) individuals were excluded from the group assessed for immunogenicity because they did not have blood drawn before or after vaccination (including individuals who were not sampled and those who had were taken outside the protocol-specific window), 13 (3.3%) subjects were not or could not be included in the study before vaccination or 1 month after vaccination at Visit 3, 11 (2.8% ) individuals did not receive the vaccine because all vaccination visits were selective, and 16 (4.0%) individuals were prohibited from administering the vaccine or receiving treatment. Overall, the immunogenicity group included only 84.3% of individuals aged 12 to <18 months and 91.4% of individuals aged 18 to <24 months.

Immunogenicity Test Results

The results of the primary analysis, including all immunogenicity data up to 1 month post-vaccination 3 (Visit 7), are presented in the following sections. Immunogenicity endpoint analysis was performed based on hSBA results for PMB2001 (A56) and PMB2707 (B44) strains in one half of individuals and for PMB80 (A22) and PMB2948 (B24) strains in the other half of individuals.

Primary and secondary endpoints

Number of individuals achieving hSBA titer≥LLOQ

The primary immunogenicity endpoints are the number of individuals aged 12 months to <18 months (at baseline) and aged 18 to <24 months (at baseline) with hSBA titer ≥LLOQ for each of the 4 primary MnB strains tested 1 month after the third vaccination with bivalent rLP2086. Secondary endpoints are the number of all individuals in pooled age stratified groups with hSBA titer ≥LLOQ for each of the 4 primary MnB strains tested 1 month after the third vaccination with bivalent rLP2086, together with the number of individuals in single and pooled age stratified groups with hSBA titer ≥ LLOQ for each of the 4 primary MnB strains tested 1 month after the second vaccination with divalent rLP2086.

The number of individuals in each age stratified group with hSBA titer≥LLOQ for each of the 4 primary MnB strains tested is shown in the Table for the immunogenicity group.

For the pooled age stratified group, the number of individuals who achieved hSBA titer≥LLOQ at baseline was 0.0% and 3.1% for PMB80 (A22); 0.0% and 1.1% for PMB2001 (A56); 4.8% and 2.1% for PMB2948 (B24); and 0.0% and 1.1% for PMB2707 (B44) for the 60 µg and 120 µg dose groups, respectively.

At 1 month post-vaccination 2, the number of individuals achieving hSBA titer≥LLOQ for individuals stratified 12 to <18 months of age was 90.0% and 64.4% for PMB80 (A22); 100.0% and 100.0% for PMB2001 (A56); 70.0% and 23.8% for PMB2948 (B24); and 77.8% and 72.3% for PMB2707 (B44) for the 60 µg and 120 µg dose groups, respectively. For individuals with an age stratification of 18 to <24 months, the number of individuals who achieved hSBA titer ≥LLOQ was 66.7% and 84.0% for PMB80 (A22); 90.0% and 100.0% for PMB2001 (A56); 44.4% and 43.2% for PMB2948 (B24); and 60.0% and 63.8% for PMB2707 (B44) for the 60 µg and 120 µg dose groups, respectively. Overall, for the pooled age stratified group, the number of individuals who achieved hSBA titer≥LLOQ at 1 month post-vaccination 2 was 78.9% and 74.7% for PMB80 (A22); 94.7% and 100.0% for PMB2001 (A56); 57.9% and 33.7% for PMB2948 (B24); and 68.4% and 68.1% for PMB2707 (B44), for the 60 µg and 120 µg dose groups, respectively.

At 1 month post-vaccination 3, the number of individuals achieving hSBA titer≥LLOQ for individuals stratified 12 to <18 months of age was 88.9% and 91.1% for PMB80 (A22); 100.0% and 100.0% for PMB2001 (A56); 88.9% and 71.1% for PMB2948 (B24); and 88.9% and 87.2% for PMB2707 (B44), for the 60 µg and 120 µg dose groups, respectively. For individuals with an age stratification of 18 to <24 months, the number of individuals who achieved hSBA titer ≥LLOQ was 90.9% and 88.2% for PMB80 (A22); 100.0% and 100.0% for PMB2001 (A56); 81.8% and 72.0% for PMB2948 (B24); and 90.0% and 85.1% for PMB2707 (B44), for the 60 µg and 120 µg dose groups, respectively. Overall, for the pooled age stratified group, the number of individuals achieving hSBA titer≥LLOQ at 1 month post-vaccination 3 was 90.0% and 89.6% for PMB80 (A22); 100.0% and 100.0% for PMB2001 (A56); 85.0% and 71.6% for PMB2948 (B24); and 89.5% and 86.2% for PMB2707 (B44), for the 60 µg and 120 µg dose groups, respectively.

Overall, the number of individuals in the HAV/saline group that achieved hSBA titer≥LLOQ did not change over a period of time from baseline (Table 25).

The results for the mITT group were similar to those for the immunogenicity group.

The results of subgroup analyzes to determine the number of individuals who achieved hSBA titer≥LLOQ for each of the 4 primary MnB strains tested were evaluated for the gender and country immunogenicity assessment group. There were no clinically significant differences in subgroup analyses.

Table 25. Individuals with hSBA Titers ≥ LLOQ for Primary Strains - Immunogenicity Evaluation Group Vaccine group (randomized) 60 mcg rLP2086 120 mcg rLP2086 HAV/saline solution Taking a sample of a strain (variant) at a given point in time for an age stratified group N ^a n ^b (%) (95% CI) ^c N ^a n ^b (%) (95% CI) ^c N ^a n ^b (%) (95% CI) ^c PMB80(A22) Before vaccination 1 12 to <24 months twenty 0 (0.0) (0.0, 16.8) 97 3 (3.1) (0.6, 8.8) 61 one (1.6) (0.0, 8.8) 12 to <18 months 9 0 (0.0) (0.0, 33.6) 46 one (2.2) (0.1, 11.5) 31 0 (0.0) (0.0, 11.2) 18 to <24 months eleven 0 (0.0) (0.0, 28.5) 51 2 (3.9) (0.5, 13.5) thirty one (3.3) (0.1, 17.2) 1 month after vaccination 2 12 to <24 months 19 fifteen (78.9) (54.4, 93.9) 95 71 (74.7) (64.8, 83.1) 59 one (1.7) (0.0, 9.1) 12 to <18 months ten 9 (90.0) (55.5, 99.7) 45 29 (64.4) (48.8, 78.1) thirty 0 (0.0) (0.0, 11.6) 18 to <24 months 9 6 (66.7) (29.9, 92.5) fifty 42 (84.0) (70.9, 92.8) 29 one (3.4) (0.1, 17.8) 1 month after vaccination 3 12 to <24 months twenty eighteen (90.0) (68.3, 98.8) 96 86 (89.6) (81.7, 94.9) 60 3 (5.0) (1.0, 13.9) 12 to <18 months 9 eight (88.9) (51.8, 99.7) 45 41 (91.1) (78.8, 97.5) 31 one (3.2) (0.1, 16.7) 18 to <24 months eleven ten (90.9) (58.7, 99.8) 51 45 (88.2) (76.1, 95.6) 29 2 (6.9) (0.8, 22.8) PMB2001(A56) Before vaccination 1 12 to <24 months 19 0 (0.0) (0.0, 17.6) 95 one (1.1) (0.0, 5.7) 53 0 (0.0) (0.0, 6.7) 12 to <18 months 9 0 (0.0) (0.0, 33.6) 46 0 (0.0) (0.0, 7.7) 24 0 (0.0) (0.0, 14.2) 18 to <24 months ten 0 (0.0) (0.0, 30.8) 49 one (2.0) (0.1, 10.9) 29 0 (0.0) (0.0, 11.9) 1 month after vaccination 2 12 to <24 months 19 eighteen (94.7) (74.0, 99.9) 95 95 (100.0) (96.2, 100.0) 52 0 (0.0) (0.0, 6.8) 12 to <18 months 9 9 (100.0) (66.4, 100.0) 47 47 (100.0) (92.5, 100.0) 23 0 (0.0) (0.0, 14.8) 18 to <24 months ten 9 (90.0) (55.5, 99.7) 48 48 (100.0) (92.6, 100.0) 29 0 (0.0) (0.0, 11.9) 1 month after vaccination 3 12 to <24 months 19 19 (100.0) (82.4, 100.0) 95 95 (100.0) (96.2, 100.0) 54 one (1.9) (0.0, 9.9) 12 to <18 months 9 9 (100.0) (66.4, 100.0) 47 47 (100.0) (92.5, 100.0) 24 0 (0.0) (0.0, 14.2) 18 to <24 months ten ten (100.0) (69.2, 100.0) 48 48 (100.0) (92.6, 100.0) thirty one (3.3) (0.1, 17.2) PMB2948 (B24) Before vaccination 1 12 to <24 months 21 one (4.8) (0.1, 23.8) 97 2 (2.1) (0.3, 7.3) 61 one (1.6) (0.0, 8.8) 12 to <18 months ten 0 (0.0) (0.0, 30.8) 46 one (2.2) (0.1, 11.5) 31 0 (0.0) (0.0, 11.2) 18 to <24 months eleven one (9.1) (0.2, 41.3) 51 one (2.0) (0.0, 10.4) thirty one (3.3) (0.1, 17.2) 1 month after vaccination 2 12 to <24 months 19 eleven (57.9) (33.5, 79.7) 86 29 (33.7) (23.9, 44.7) 59 one (1.7) (0.0, 9.1) 12 to <18 months ten 7 (70.0) (34.8, 93.3) 42 ten (23.8) (12.1, 39.5) thirty 0 (0.0) (0.0, 11.6) 18 to <24 months 9 four (44.4) (13.7, 78.8) 44 19 (43.2) (28.3, 59.0) 29 one (3.4) (0.1, 17.8) 1 month after vaccination 3 12 to <24 months twenty 17 (85.0) (62.1, 96.8) 95 68 (71.6) (61.4, 80.4) 60 3 (5.0) (1.0, 13.9) 12 to <18 months 9 eight (88.9) (51.8, 99.7) 45 32 (71.1) (55.7, 83.6) 31 one (3.2) (0.1, 16.7) 18 to <24 months eleven 9 (81.8) (48.2, 97.7) fifty 36 (72.0) (57.5, 83.8) 29 2 (6.9) (0.8, 22.8) PMB2707 (B44) Before vaccination 1 12 to <24 months 19 0 (0.0) (0.0, 17.6) 95 one (1.1) (0.0, 5.7) 54 0 (0.0) (0.0, 6.6) 12 to <18 months 9 0 (0.0) (0.0, 33.6) 46 one (2.2) (0.1, 11.5) 24 0 (0.0) (0.0, 14.2) 18 to <24 months ten 0 (0.0) (0.0, 30.8) 49 0 (0.0) (0.0, 7.3) thirty 0 (0.0) (0.0, 11.6) 1 month after vaccination 2 12 to <24 months 19 13 (68.4) (43.4, 87.4) 94 64 (68.1) (57.7, 77.3) 52 0 (0.0) (0.0, 6.8) 12 to <18 months 9 7 (77.8) (40.0, 97.2) 47 34 (72.3) (57.4, 84.4) 23 0 (0.0) (0.0, 14.8) 18 to <24 months ten 6 (60.0) (26.2, 87.8) 47 thirty (63.8) (48.5, 77.3) 29 0 (0.0) (0.0, 11.9) 1 month after vaccination 3 12 to <24 months 19 17 (89.5) (66.9, 98.7) 94 81 (86.2) (77.5, 92.4) 54 0 (0.0) (0.0, 6.6) 12 to <18 months 9 eight (88.9) (51.8, 99.7) 47 41 (87.2) (74.3, 95.2) 24 0 (0.0) (0.0, 14.2) 18 to <24 months ten 9 (90.0) (55.5, 99.7) 47 40 (85.1) (71.7, 93.8) thirty 0 (0.0) (0.0, 11.6) Abbreviation: hSBA=serum bactericidal activity using human complement; LLOQ=lower limit of quantification
Note: LLOQ=1:16 for A22; 1:8 for A56, B24, and B44
a. N=number of individuals with confirmed and determined hSBA titers for a given strain.
b. n=number of individuals with an observed hSBA titer ≥ LLOQ for a given strain at a given time.
c. Exact two-tailed CI based on the observed number of individuals using the method of Klopper and Pearson

GMT hSBA

The secondary endpoint is hSBA GMT for each of the 4 primary MnB strains tested for individuals 12 to <18 months of age, 18 to <24 months of age, and for the pooled age stratified group at baseline, 1 month post-second vaccination, and 1 month post-vaccination. one month after the third vaccination with bivalent rLP2086. Table 26 shows the hSBA GMTs for the 4 primary MnB strains for the group assessed for immunogenicity.

For the pooled age stratified group, hSBA GMT at baseline was 8.0 and 8.4 for PMB80 (A22); 4.0 and 4.1 for PMB2001 (A56); 4.4 and 4.1 for PMB2948 (B24); and 4.0 and 4.0 for PMB2707 (B44) for the 60 μg and 120 μg dose groups, respectively.

At 1 month post-vaccination 2, the GMT hSBA of the age stratified group 12 to <18 months was 42.2 and 24.6 for PMB80 (A22); 101.6 and 117.2 for PMB2001 (A56); 10.6 and 6.0 for PMB2948 (B24); and 23.5 and 22.1 for PMB2707 (B44) for the 60 µg and 120 µg dose groups, respectively. For the age stratified group 18 to <24 months, hSBA GMT were 23.5 and 36.8 for PMB80 (A22); 68.6 and 104.6 for PMB2001 (A56); 6.9 and 8.5 for PMB2948 (B24); and 21.1 and 17.0 for PMB2707 (B44) for the 60 µg and 120 µg dose groups, respectively. Overall, at 1 month post-vaccination 2, age stratified group hSBA GMTs were 32.0 and 30.4 for PMB80 (A22); 82.6 and 110.6 for PMB2001 (A56); 8.6 and 7.2 for PMB2948 (B24); and 22.2 and 19.4 for PMB2707 (B44) dosed at 60 µg and 120 µg, respectively.

At 1 month post-vaccination 2, the GMT hSBA of the age stratified group 12 to <18 months was 80.6 and 63.0 for PMB80 (A22); 109.7 and 190.6 for PMB2001 (A56); 20.2 and 15.8 for PMB2948 (B24); and 29.6 and 46.3 for PMB2707 (B44) for the 60 µg and 120 µg dose groups, respectively. For the age stratified group 18 to <24 months, hSBA GMT were 82.3 and 71.4 for PMB80 (A22); 181.0 and 154.4 for PMB2001 (A56); 17.0 and 14.5 for PMB2948 (B24); and 34.3 and 44.9 for PMB2707 (B44) for the 60 µg and 120 µg dose groups, respectively. Overall, at 1 month post-vaccination 3, age stratified group hSBA GMTs were 81.6 and 67.3 for PMB80 (A22); 142.8 and 171.4 for PMB2001 (A56); 18.4 and 15.1 for PMB2948 (B24); and 32.0 and 45.6 for PMB2707 (B44) for the 60 μg and 120 μg dose groups, respectively.

In general, hSBA GMT for individuals in the HAV/saline group did not change over a period of time from baseline.

Table 26. GMT hSBA for Primary Strains - Group Assessed for Immunogenicity Vaccine group (randomized) 60 mcg rLP2086 120 mcg rLP2086 HAV/saline solution Taking a sample of a strain (variant) at a given point in time for an age stratified group N ^a GMT ^b (95% CI) ^c N ^a GMT ^b (95% CI) ^c N ^a GMT ^b (95% CI) ^c PMB80(A22) Before vaccination 1 12 to <24 months twenty 8.0 (NE, NE) 97 8.4 (7.9, 9.0) 61 8.1 (7.9, 8.3) 12 to <18 months 9 8.0 (NE, NE) 46 8.5 (7.5, 9.6) 31 8.0 (NE, NE) 18 to <24 months eleven 8.0 (NE, NE) 51 8.3 (7.8, 8.9) thirty 8.2 (7.8, 8.6) 1 month after vaccination 2 12 to <24 months 19 32.0 (19.7, 52.0) 95 30.4 (24.3, 38.1) 59 8.3 (7.7, 8.9) 12 to <18 months ten 42.2 (22.6, 79.1) 45 24.6 (17.8, 34.2) thirty 8.0 (NE, NE) 18 to <24 months 9 23.5 (10.1, 54.9) fifty 36.8 (26.9, 50.3) 29 8.6 (7.4, 10.0) 1 month after vaccination 3 12 to <24 months twenty 81.6 (46.6, 142.8) 96 67.3 (53.7, 84.3) 60 8.6 (7.9, 9.3) 12 to <18 months 9 80.6 (30.9, 210.7) 45 63.0 (44.5, 89.3) 31 8.6 (7.5, 9.8) 18 to <24 months eleven 82.3 (36.5, 185.8) 51 71.4 (52.7, 96.6) 29 8.6 (7.7, 9.6) PMB2001(A56) Before vaccination 1 12 to <24 months 19 4.0 (NE, NE) 95 4.1 (3.9, 4.3) 53 4.0 (NE, NE) 12 to <18 months 9 4.0 (NE, NE) 46 4.0 (NE, NE) 24 4.0 (NE, NE) 18 to <24 months ten 4.0 (NE, NE) 49 4.2 (3.8, 4.5) 29 4.0 (NE, NE) 1 month after vaccination 2 12 to <24 months 19 82.6 (51.4, 132.9) 95 110.6 (92.0, 133.0) 52 4.0 (NE, NE) 12 to <18 months 9 101.6 (64.0, 161.2) 47 117.2 (89.7, 153.0) 23 4.0 (NE, NE) 18 to <24 months ten 68.6 (28.2, 166.8) 48 104.6 (80.4, 136.0) 29 4.0 (NE, NE) 1 month after vaccination 3 12 to <24 months 19 142.8 (85.5, 238.6) 95 171.4 (141.6, 207.4) 54 4.2 (3.8, 4.5) 12 to <18 months 9 109.7 (70.4, 171.1) 47 190.6 (146.9, 247.4) 24 4.0 (NE, NE) 18 to <24 months ten 181.0 (68.6, 477.9) 48 154.4 (116.3, 205.1) thirty 4.3 (3.7, 4.9) PMB2948 (B24) Before vaccination 1 12 to <24 months 21 4.4 (3.6, 5.4) 97 4.1 (4.0, 4.3) 61 4.2 (3.8, 4.6) 12 to <18 months ten 4.0 (NE, NE) 46 4.1 (3.9, 4.4) 31 4.0 (NE, NE) 18 to <24 months eleven 4.8 (3.2, 7.4) 51 4.1 (3.9, 4.3) thirty 4.4 (3.6, 5.3) 1 month after vaccination 2 12 to <24 months 19 8.6 (6.1, 12.2) 86 7.2 (5.9, 8.7) 59 4.1 (3.9, 4.4) 12 to <18 months ten 10.6 (6.2, 18.0) 42 6.0 (4.7, 7.8) thirty 4.0 (NE, NE) 18 to <24 months 9 6.9 (4.1, 11.5) 44 8.5 (6.4, 11.3) 29 4.3 (3.7, 5.0) 1 month after vaccination 3 12 to <24 months twenty 18.4 (11.8, 28.6) 95 15.1 (12.3, 18.6) 60 4.3 (3.9, 4.8) 12 to <18 months 9 20.2 (11.1, 36.6) 45 15.8 (11.4, 21.8) 31 4.3 (3.7, 4.9) 18 to <24 months eleven 17.0 (8.2, 35.5) fifty 14.5 (11.1, 19.1) 29 4.4 (3.8, 5.0) PMB2707 (B44) Before vaccination 1 12 to <24 months 19 4.0 (NE, NE) 95 4.0 (4.0, 4.1) 54 4.0 (NE, NE) 12 to <18 months 9 4.0 (NE, NE) 46 4.1 (3.9, 4.2) 24 4.0 (NE, NE) 18 to <24 months ten 4.0 (NE, NE) 49 4.0 (NE, NE) thirty 4.0 (NE, NE) 1 month after vaccination 2 12 to <24 months 19 22.2 (11.2, 43.9) 94 19.4 (15.1, 24.9) 52 4.0 (NE, NE) 12 to <18 months 9 23.5 (9.3, 59.4) 47 22.1 (15.5, 31.6) 23 4.0 (NE, NE) 18 to <24 months ten 21.1 (6.5, 68.3) 47 17.0 (11.8, 24.4) 29 4.0 (NE, NE) 1 month after vaccination 3 12 to <24 months 19 32.0 (18.3, 55.8) 94 45.6 (35.2, 59.0) 54 4.0 (NE, NE) 12 to <18 months 9 29.6 (11.6, 75.8) 47 46.3 (31.6, 67.8) 24 4.0 (NE, NE) 18 to <24 months ten 34.3 (15.0, 78.2) 47 44.9 (31.3, 64.5) thirty 4.0 (NE, NE) Abbreviations: GMT=geometric mean titer; hSBA = serum bactericidal activity assay using human complement; LLOQ=lower limit of quantification; NE=not rated
Note: LLOQ=1:16 for A22; 1:8 for A56, B24 and B44. Titers below LLOQ mean 0.5 x LLOQ for analysis.
a. N=number of individuals with confirmed and determined hSBA titers for a given strain
b. GMT was calculated for all individuals with confirmed and determined hSBA titers at a given point in time.
c. CI means inverse transform of confidence levels based on Student's distribution for the mean logarithm of hSBA titers

The results for the mITT group were similar to those for the immunogenicity group.

The results of subgroup analyzes to determine the GMT of hSBA for each of the 4 primary MnB strains tested were evaluated for the gender and country immunogenicity assessment group. There were no clinically significant differences in subgroup analyses.

Defined hSBA titers

The secondary immunogenicity endpoint is the number of individuals aged 12 to <18 months, 18 to <24 months, and in the pooled age stratified group who achieved hSBA titers ≥1:4, ≥1:8, ≥1:16 , ≥1:32, ≥1:64 and ≥1:128 for each of the 4 primary test strains at baseline 1 month after the second vaccination and 1 month after the third vaccination with divalent rLP2086.

The number of individuals who achieved the determined hSBA titers for the 4 primary MnB strains is shown in Table 27 for the immunogenicity evaluation group.

The results obtained for individuals who achieved hSBA titers ≥1:4 and ≥1:16 are described below. hSBA titer ≥1:4 is widely recognized as a correlate of protection against IMZ; however, a more conservative hSBA titer of ≥1:16 is considered to be indicative of a 4-fold vaccine effect for pre-vaccination seronegative individuals.

In general, the number of individuals in the HAV/saline treated group that achieved specific hSBA titers did not change over a period of time from baseline.

The results for the mITT group were similar to those for the immunogenicity group.

hSBA-titer≥1:4

At 1 month after vaccination 2, the number of individuals aged 12 to <18 months who achieved hSBA titer ≥1:4 was 90.0% and 64.4% for PMB80 (A22); 100.0% and 100.0% for PMB2001 (A56); 70.0% and 28.6% for PMB2948 (B24); and 77.8% and 72.3% for PMB2707 (B44) for the 60 µg and 120 µg dose groups, respectively. The number of individuals aged 18 to <24 months who achieved hSBA titer ≥1:4 was 66.7% and 86.0% for PMB80 (A22); 100.0% and 100.0% for PMB2001 (A56); 44.4% and 43.2% for PMB2948 (B24); and 70.0% and 63.8% for PMB2707 (B44) for the 60 µg and 120 µg dose groups, respectively. Overall, for the pooled age stratified group, the number of individuals who achieved hSBA titer ≥1:4 at 1 month post-vaccination 2 was 78.9% and 75.8% for PMB80 (A22); 100.0% and 100.0% for PMB2001 (A56); 57.9% and 36.0% for PMB2948 (B24); and 73.7% and 68.1% for PMB2707 (B44) for the 60 µg and 120 µg dose groups, respectively.

At 1 month after vaccination 3, the number of individuals aged 12 to <18 months who achieved hSBA titer ≥1:4 was 88.9% and 91.1% for PMB80 (A22); 100.0% and 100.0% for PMB2001 (A56); 88.9% and 71.1% for PMB2948 (B24); and 88.9% and 87.2% for PMB2707 (B44) for the 60 µg and 120 µg dose groups, respectively. The number of individuals aged 18 to <24 months who achieved hSBA titer ≥1:4 was 90.9% and 88.2% for PMB80 (A22); 100.0% and 100.0% for PMB2001 (A56); 81.8% and 72.0% for PMB2948 (B24); and 90.0% and 87.2% for PMB2707 (B44) for the 60 µg and 120 µg dose groups, respectively. Overall, for the pooled age stratified group, the number of individuals achieving hSBA titer ≥1:4 at 1 month post-vaccination 3 was 90.0% and 89.6% for PMB80 (A22); 100.0% and 100.0% for PMB2001 (A56); 85.0% and 71.6% for PMB2948 (B24); and 89.5% and 87.2% for PMB2707 (B44) for the 60 µg and 120 µg dose groups, respectively.

hSBA titer≥1:16

At 1 month after vaccination 2, the number of individuals aged 12 to <18 months who achieved hSBA titer ≥1:16 was 90.0% and 64.4% for PMB80 (A22); 100.0% and 100.0% for PMB2001 (A56); 60.0% and 21.4% for PMB2948 (B24); and 77.8% and 72.3% for PMB2707 (B44) for the 60 µg and 120 µg dose groups, respectively. The number of individuals aged 18 to <24 months who achieved hSBA titer ≥1:16 was 66.7% and 86.0% for PMB80 (A22); 90.0% and 100.0% for PMB2001 (A56); 33.3% and 43.2% for PMB2948 (B24); and 60.0% and 61.7% for PMB2707 (B44) for the 60 µg and 120 µg dose groups, respectively. Overall, for the pooled age stratified group, the number of individuals achieving hSBA titer ≥1:16 at 1 month post-vaccination 2 was 78.9% and 74.7% for PMB80 (A22); 94.7% and 100.0% for PMB2001 (A56); 47.4% and 32.6% for PMB2948 (B24); and 68.4% and 67.0% for PMB2707 (B44) for the 60 µg and 120 µg dose groups, respectively.

At 1 month after vaccination 3, the number of individuals aged 12 to <18 months who achieved hSBA titer ≥1:16 was 88.9% and 91.1% for PMB80 (A22); 100.0% and 97.9% for PMB2001 (A56); 88.9% and 66.7% for PMB2948 (B24); and 77.8% and 87.2% for PMB2707 (B44) for the 60 µg and 120 µg dose groups, respectively. The number of individuals aged 18 to <24 months who achieved hSBA titer ≥1:16 was 90.9% and 88.2% for PMB80 (A22); 100.0% and 100.0% for PMB2001 (A56); 63.6% and 68.0% for PMB2948 (B24); and 90.0% and 85.1% for PMB2707 (B44) for the 60 µg and 120 µg dose groups, respectively. Overall, for the pooled age stratified group, the number of individuals achieving hSBA titer ≥1:16 at 1 month post-vaccination 3 was 90.0% and 89.6% for PMB80 (A22); 100.0% and 98.9% for PMB2001 (A56); 75.0% and 67.4% for PMB2948 (B24); and 84.2% and 86.2% for PMB2707 (B44) for the 60 µg and 120 µg dose groups, respectively.

Table 27 Individuals Achieving Specific hSBA Titers for Primary Strains Group Assessed for Immunogenicity Vaccine group (randomized) 60 mcg rLP2086 120 mcg rLP2086 HAV/saline solution Taking a sample of a strain (variant) at a given point in time for an age stratified group Titer N ^a n ^b % (95% CI) ^c N ^a n ^b % (95% CI) ^c N ^a n ^b % (95% CI) ^c PMB80(A22) Before vaccination 1 12 to <24 months four twenty 0 0.0 (0.0, 16.8) 97 four 4.1 (1.1, 10.2) 61 one 1.6 (0.0, 8.8) eight twenty 0 0.0 (0.0, 16.8) 97 3 3.1 (0.6, 8.8) 61 one 1.6 (0.0, 8.8) 16 twenty 0 0.0 (0.0, 16.8) 97 3 3.1 (0.6, 8.8) 61 one 1.6 (0.0, 8.8) 32 twenty 0 0.0 (0.0, 16.8) 97 2 2.1 (0.3, 7.3) 61 0 0.0 (0.0, 5.9) 64 twenty 0 0.0 (0.0, 16.8) 97 one 1.0 (0.0, 5.6) 61 0 0.0 (0.0, 5.9) 128 twenty 0 0.0 (0.0, 16.8) 97 one 1.0 (0.0, 5.6) 61 0 0.0 (0.0, 5.9) 12 to <18 months four 9 0 0.0 (0.0, 33.6) 46 2 4.3 (0.5, 14.8) 31 0 0.0 (0.0, 11.2) eight 9 0 0.0 (0.0, 33.6) 46 one 2.2 (0.1, 11.5) 31 0 0.0 (0.0, 11.2) 16 9 0 0.0 (0.0, 33.6) 46 one 2.2 (0.1, 11.5) 31 0 0.0 (0.0, 11.2) 32 9 0 0.0 (0.0, 33.6) 46 one 2.2 (0.1, 11.5) 31 0 0.0 (0.0, 11.2) 64 9 0 0.0 (0.0, 33.6) 46 one 2.2 (0.1, 11.5) 31 0 0.0 (0.0, 11.2) 128 9 0 0.0 (0.0, 33.6) 46 one 2.2 (0.1, 11.5) 31 0 0.0 (0.0, 11.2) 18 to <24 months four eleven 0 0.0 (0.0, 28.5) 51 2 3.9 (0.5, 13.5) thirty one 3.3 (0.1, 17.2) eight eleven 0 0.0 (0.0, 28.5) 51 2 3.9 (0.5, 13.5) thirty one 3.3 (0.1, 17.2) 16 eleven 0 0.0 (0.0, 28.5) 51 2 3.9 (0.5, 13.5) thirty one 3.3 (0.1, 17.2) 32 eleven 0 0.0 (0.0, 28.5) 51 one 2.0 (0.0, 10.4) thirty 0 0.0 (0.0, 11.6) 64 eleven 0 0.0 (0.0, 28.5) 51 0 0.0 (0.0, 7.0) thirty 0 0.0 (0.0, 11.6) 128 eleven 0 0.0 (0.0, 28.5) 51 0 0.0 (0.0, 7.0) thirty 0 0.0 (0.0, 11.6) 1 month after vaccination 2 12 to <24 months four 19 fifteen 78.9 (54.4, 93.9) 95 72 75.8 (65.9, 84.0) 59 one 1.7 (0.0, 9.1) eight 19 fifteen 78.9 (54.4, 93.9) 95 72 75.8 (65.9, 84.0) 59 one 1.7 (0.0, 9.1) 16 19 fifteen 78.9 (54.4, 93.9) 95 71 74.7 (64.8, 83.1) 59 one 1.7 (0.0, 9.1) 32 19 12 63.2 (38.4, 83.7) 95 56 58.9 (48.4, 68.9) 59 one 1.7 (0.0, 9.1) 64 19 7 36.8 (16.3, 61.6) 95 33 34.7 (25.3, 45.2) 59 one 1.7 (0.0, 9.1) 128 19 four 21.1 (6.1, 45.6) 95 13 13.7 (7.5, 22.3) 59 0 0.0 (0.0, 6.1) 12 to <18 months four ten 9 90.0 (55.5, 99.7) 45 29 64.4 (48.8, 78.1) thirty 0 0.0 (0.0, 11.6) eight ten 9 90.0 (55.5, 99.7) 45 29 64.4 (48.8, 78.1) thirty 0 0.0 (0.0, 11.6) 16 ten 9 90.0 (55.5, 99.7) 45 29 64.4 (48.8, 78.1) thirty 0 0.0 (0.0, 11.6) 32 ten eight 80.0 (44.4, 97.5) 45 23 51.1 (35.8, 66.3) thirty 0 0.0 (0.0, 11.6) 64 ten 5 50.0 (18.7, 81.3) 45 13 28.9 (16.4, 44.3) thirty 0 0.0 (0.0, 11.6) 128 ten 2 20.0 (2.5, 55.6) 45 5 11.1 (3.7, 24.1) thirty 0 0.0 (0.0, 11.6) 18 to <24 months four 9 6 66.7 (29.9, 92.5) fifty 43 86.0 (73.3, 94.2) 29 one 3.4 (0.1, 17.8) eight 9 6 66.7 (29.9, 92.5) fifty 43 86.0 (73.3, 94.2) 29 one 3.4 (0.1, 17.8) 16 9 6 66.7 (29.9, 92.5) fifty 42 84.0 (70.9, 92.8) 29 one 3.4 (0.1, 17.8) 32 9 four 44.4 (13.7, 78.8) fifty 33 66.0 (51.2, 78.8) 29 one 3.4 (0.1, 17.8) 64 9 2 22.2 (2.8, 60.0) fifty twenty 40.0 (26.4, 54.8) 29 one 3.4 (0.1, 17.8) 128 9 2 22.2 (2.8, 60.0) fifty eight 16.0 (7.2, 29.1) 29 0 0.0 (0.0, 11.9) 1 month after vaccination 3 12 to <24 months four twenty eighteen 90.0 (68.3, 98.8) 96 86 89.6 (81.7, 94.9) 60 four 6.7 (1.8, 16.2) eight twenty eighteen 90.0 (68.3, 98.8) 96 86 89.6 (81.7, 94.9) 60 four 6.7 (1.8, 16.2) 16 twenty eighteen 90.0 (68.3, 98.8) 96 86 89.6 (81.7, 94.9) 60 3 5.0 (1.0, 13.9) 32 twenty 17 85.0 (62.1, 96.8) 96 81 84.4 (75.5, 91.0) 60 2 3.3 (0.4, 11.5) 64 twenty fourteen 70.0 (45.7, 88.1) 96 64 66.7 (56.3, 76.0) 60 one 1.7 (0.0, 8.9) 128 twenty ten 50.0 (27.2, 72.8) 96 42 43.8 (33.6, 54.3) 60 0 0.0 (0.0, 6.0) 12 to <18 months four 9 eight 88.9 (51.8, 99.7) 45 41 91.1 (78.8, 97.5) 31 2 6.5 (0.8, 21.4) eight 9 eight 88.9 (51.8, 99.7) 45 41 91.1 (78.8, 97.5) 31 2 6.5 (0.8, 21.4) 16 9 eight 88.9 (51.8, 99.7) 45 41 91.1 (78.8, 97.5) 31 one 3.2 (0.1, 16.7) 32 9 eight 88.9 (51.8, 99.7) 45 37 82.2 (67.9, 92.0) 31 one 3.2 (0.1, 16.7) 64 9 6 66.7 (29.9, 92.5) 45 27 60.0 (44.3, 74.3) 31 one 3.2 (0.1, 16.7) 128 9 four 44.4 (13.7, 78.8) 45 17 37.8 (23.8, 53.5) 31 0 0.0 (0.0, 11.2) 18 to <24 months four eleven ten 90.9 (58.7, 99.8) 51 45 88.2 (76.1, 95.6) 29 2 6.9 (0.8, 22.8) eight eleven ten 90.9 (58.7, 99.8) 51 45 88.2 (76.1, 95.6) 29 2 6.9 (0.8, 22.8) 16 eleven ten 90.9 (58.7, 99.8) 51 45 88.2 (76.1, 95.6) 29 2 6.9 (0.8, 22.8) 32 eleven 9 81.8 (48.2, 97.7) 51 44 86.3 (73.7, 94.3) 29 one 3.4 (0.1, 17.8) 64 eleven eight 72.7 (39.0, 94.0) 51 37 72.5 (58.3, 84.1) 29 0 0.0 (0.0, 11.9) 128 eleven 6 54.5 (23.4, 83.3) 51 25 49.0 (34.8, 63.4) 29 0 0.0 (0.0, 11.9) PMB2001(A56) Before vaccination 1 12 to <24 months four 19 0 0.0 (0.0, 17.6) 95 2 2.1 (0.3, 7.4) 53 one 1.9 (0.0, 10.1) eight 19 0 0.0 (0.0, 17.6) 95 one 1.1 (0.0, 5.7) 53 0 0.0 (0.0, 6.7) 16 19 0 0.0 (0.0, 17.6) 95 one 1.1 (0.0, 5.7) 53 0 0.0 (0.0, 6.7) 32 19 0 0.0 (0.0, 17.6) 95 one 1.1 (0.0, 5.7) 53 0 0.0 (0.0, 6.7) 64 19 0 0.0 (0.0, 17.6) 95 0 0.0 (0.0, 3.8) 53 0 0.0 (0.0, 6.7) 128 19 0 0.0 (0.0, 17.6) 95 0 0.0 (0.0, 3.8) 53 0 0.0 (0.0, 6.7) 12 to <18 months four 9 0 0.0 (0.0, 33.6) 46 0 0.0 (0.0, 7.7) 24 one 4.2 (0.1, 21.1) eight 9 0 0.0 (0.0, 33.6) 46 0 0.0 (0.0, 7.7) 24 0 0.0 (0.0, 14.2) 16 9 0 0.0 (0.0, 33.6) 46 0 0.0 (0.0, 7.7) 24 0 0.0 (0.0, 14.2) 32 9 0 0.0 (0.0, 33.6) 46 0 0.0 (0.0, 7.7) 24 0 0.0 (0.0, 14.2) 64 9 0 0.0 (0.0, 33.6) 46 0 0.0 (0.0, 7.7) 24 0 0.0 (0.0, 14.2) 128 9 0 0.0 (0.0, 33.6) 46 0 0.0 (0.0, 7.7) 24 0 0.0 (0.0, 14.2) 18 to <24 months four ten 0 0.0 (0.0, 30.8) 49 2 4.1 (0.5, 14.0) 29 0 0.0 (0.0, 11.9) eight ten 0 0.0 (0.0, 30.8) 49 one 2.0 (0.1, 10.9) 29 0 0.0 (0.0, 11.9) 16 ten 0 0.0 (0.0, 30.8) 49 one 2.0 (0.1, 10.9) 29 0 0.0 (0.0, 11.9) 32 ten 0 0.0 (0.0, 30.8) 49 one 2.0 (0.1, 10.9) 29 0 0.0 (0.0, 11.9) 64 ten 0 0.0 (0.0, 30.8) 49 0 0.0 (0.0, 7.3) 29 0 0.0 (0.0, 11.9) 128 ten 0 0.0 (0.0, 30.8) 49 0 0.0 (0.0, 7.3) 29 0 0.0 (0.0, 11.9) 1 month after vaccination 2 12 to <24 months four 19 19 100.0 (82.4, 100.0) 95 95 100.0 (96.2, 100.0) 52 one 1.9 (0.0, 10.3) eight 19 eighteen 94.7 (74.0, 99.9) 95 95 100.0 (96.2, 100.0) 52 0 0.0 (0.0, 6.8) 16 19 eighteen 94.7 (74.0, 99.9) 95 95 100.0 (96.2, 100.0) 52 0 0.0 (0.0, 6.8) 32 19 eighteen 94.7 (74.0, 99.9) 95 91 95.8 (89.6, 98.8) 52 0 0.0 (0.0, 6.8) 64 19 16 84.2 (60.4, 96.6) 95 82 86.3 (77.7, 92.5) 52 0 0.0 (0.0, 6.8) 128 19 9 47.4 (24.4, 71.1) 95 54 56.8 (46.3, 67.0) 52 0 0.0 (0.0, 6.8) 12 to <18 months four 9 9 100.0 (66.4, 100.0) 47 47 100.0 (92.5, 100.0) 23 one 4.3 (0.1, 21.9) eight 9 9 100.0 (66.4, 100.0) 47 47 100.0 (92.5, 100.0) 23 0 0.0 (0.0, 14.8) 16 9 9 100.0 (66.4, 100.0) 47 47 100.0 (92.5, 100.0) 23 0 0.0 (0.0, 14.8) 32 9 9 100.0 (66.4, 100.0) 47 45 95.7 (85.5, 99.5) 23 0 0.0 (0.0, 14.8) 64 9 9 100.0 (66.4, 100.0) 47 41 87.2 (74.3, 95.2) 23 0 0.0 (0.0, 14.8) 128 9 four 44.4 (13.7, 78.8) 47 28 59.6 (44.3, 73.6) 23 0 0.0 (0.0, 14.8) 18 to <24 months four ten ten 100.0 (69.2, 100.0) 48 48 100.0 (92.6, 100.0) 29 0 0.0 (0.0, 11.9) eight ten 9 90.0 (55.5, 99.7) 48 48 100.0 (92.6, 100.0) 29 0 0.0 (0.0, 11.9) 16 ten 9 90.0 (55.5, 99.7) 48 48 100.0 (92.6, 100.0) 29 0 0.0 (0.0, 11.9) 32 ten 9 90.0 (55.5, 99.7) 48 46 95.8 (85.7, 99.5) 29 0 0.0 (0.0, 11.9) 64 ten 7 70.0 (34.8, 93.3) 48 41 85.4 (72.2, 93.9) 29 0 0.0 (0.0, 11.9) 128 ten 5 50.0 (18.7, 81.3) 48 26 54.2 (39.2, 68.6) 29 0 0.0 (0.0, 11.9) 1 month after vaccination 3 12 to <24 months four 19 19 100.0 (82.4, 100.0) 95 95 100.0 (96.2, 100.0) 54 5 9.3 (3.1, 20.3) eight 19 19 100.0 (82.4, 100.0) 95 95 100.0 (96.2, 100.0) 54 one 1.9 (0.0, 9.9) 16 19 19 100.0 (82.4, 100.0) 95 94 98.9 (94.3, 100.0) 54 one 1.9 (0.0, 9.9) 32 19 eighteen 94.7 (74.0, 99.9) 95 91 95.8 (89.6, 98.8) 54 one 1.9 (0.0, 9.9) 64 19 17 89.5 (66.9, 98.7) 95 85 89.5 (81.5, 94.8) 54 0 0.0 (0.0, 6.6) 128 19 13 68.4 (43.4, 87.4) 95 79 83.2 (74.1, 90.1) 54 0 0.0 (0.0, 6.6) 12 to <18 months four 9 9 100.0 (66.4, 100.0) 47 47 100.0 (92.5, 100.0) 24 2 8.3 (1.0, 27.0) eight 9 9 100.0 (66.4, 100.0) 47 47 100.0 (92.5, 100.0) 24 0 0.0 (0.0, 14.2) 16 9 9 100.0 (66.4, 100.0) 47 46 97.9 (88.7, 99.9) 24 0 0.0 (0.0, 14.2) 32 9 9 100.0 (66.4, 100.0) 47 46 97.9 (88.7, 99.9) 24 0 0.0 (0.0, 14.2) 64 9 9 100.0 (66.4, 100.0) 47 44 93.6 (82.5, 98.7) 24 0 0.0 (0.0, 14.2) 128 9 5 55.6 (21.2, 86.3) 47 42 89.4 (76.9, 96.5) 24 0 0.0 (0.0, 14.2) 18 to <24 months four ten ten 100.0 (69.2, 100.0) 48 48 100.0 (92.6, 100.0) thirty 3 10.0 (2.1, 26.5) eight ten ten 100.0 (69.2, 100.0) 48 48 100.0 (92.6, 100.0) thirty one 3.3 (0.1, 17.2) 16 ten ten 100.0 (69.2, 100.0) 48 48 100.0 (92.6, 100.0) thirty one 3.3 (0.1, 17.2) 32 ten 9 90.0 (55.5, 99.7) 48 45 93.8 (82.8, 98.7) thirty one 3.3 (0.1, 17.2) 64 ten eight 80.0 (44.4, 97.5) 48 41 85.4 (72.2, 93.9) thirty 0 0.0 (0.0, 11.6) 128 ten eight 80.0 (44.4, 97.5) 48 37 77.1 (62.7, 88.0) thirty 0 0.0 (0.0, 11.6) PMB2948 (B24) Before vaccination 1 12 to <24 months four 21 one 4.8 (0.1, 23.8) 97 2 2.1 (0.3, 7.3) 61 one 1.6 (0.0, 8.8) eight 21 one 4.8 (0.1, 23.8) 97 2 2.1 (0.3, 7.3) 61 one 1.6 (0.0, 8.8) 16 21 one 4.8 (0.1, 23.8) 97 2 2.1 (0.3, 7.3) 61 one 1.6 (0.0, 8.8) 32 21 one 4.8 (0.1, 23.8) 97 0 0.0 (0.0, 3.7) 61 one 1.6 (0.0, 8.8) 64 21 0 0.0 (0.0, 16.1) 97 0 0.0 (0.0, 3.7) 61 one 1.6 (0.0, 8.8) 128 21 0 0.0 (0.0, 16.1) 97 0 0.0 (0.0, 3.7) 61 0 0.0 (0.0, 5.9) 12 to <18 months four ten 0 0.0 (0.0, 30.8) 46 one 2.2 (0.1, 11.5) 31 0 0.0 (0.0, 11.2) eight ten 0 0.0 (0.0, 30.8) 46 one 2.2 (0.1, 11.5) 31 0 0.0 (0.0, 11.2) 16 ten 0 0.0 (0.0, 30.8) 46 one 2.2 (0.1, 11.5) 31 0 0.0 (0.0, 11.2) 32 ten 0 0.0 (0.0, 30.8) 46 0 0.0 (0.0, 7.7) 31 0 0.0 (0.0, 11.2) 64 ten 0 0.0 (0.0, 30.8) 46 0 0.0 (0.0, 7.7) 31 0 0.0 (0.0, 11.2) 128 ten 0 0.0 (0.0, 30.8) 46 0 0.0 (0.0, 7.7) 31 0 0.0 (0.0, 11.2) 18 to <24 months four eleven one 9.1 (0.2, 41.3) 51 one 2.0 (0.0, 10.4) thirty one 3.3 (0.1, 17.2) eight eleven one 9.1 (0.2, 41.3) 51 one 2.0 (0.0, 10.4) thirty one 3.3 (0.1, 17.2) 16 eleven one 9.1 (0.2, 41.3) 51 one 2.0 (0.0, 10.4) thirty one 3.3 (0.1, 17.2) 32 eleven one 9.1 (0.2, 41.3) 51 0 0.0 (0.0, 7.0) thirty one 3.3 (0.1, 17.2) 64 eleven 0 0.0 (0.0, 28.5) 51 0 0.0 (0.0, 7.0) thirty one 3.3 (0.1, 17.2) 128 eleven 0 0.0 (0.0, 28.5) 51 0 0.0 (0.0, 7.0) thirty 0 0.0 (0.0, 11.6) 1 month after vaccination 2 12 to <24 months four 19 eleven 57.9 (33.5, 79.7) 86 31 36.0 (26.0, 47.1) 59 one 1.7 (0.0, 9.1) eight 19 eleven 57.9 (33.5, 79.7) 86 29 33.7 (23.9, 44.7) 59 one 1.7 (0.0, 9.1) 16 19 9 47.4 (24.4, 71.1) 86 28 32.6 (22.8, 43.5) 59 one 1.7 (0.0, 9.1) 32 19 one 5.3 (0.1, 26.0) 86 12 14.0 (7.4, 23.1) 59 one 1.7 (0.0, 9.1) 64 19 0 0.0 (0.0, 17.6) 86 3 3.5 (0.7, 9.9) 59 0 0.0 (0.0, 6.1) 128 19 0 0.0 (0.0, 17.6) 86 one 1.2 (0.0, 6.3) 59 0 0.0 (0.0, 6.1) 12 to <18 months four ten 7 70.0 (34.8, 93.3) 42 12 28.6 (15.7, 44.6) thirty 0 0.0 (0.0, 11.6) eight ten 7 70.0 (34.8, 93.3) 42 ten 23.8 (12.1, 39.5) thirty 0 0.0 (0.0, 11.6) 16 ten 6 60.0 (26.2, 87.8) 42 9 21.4 (10.3, 36.8) thirty 0 0.0 (0.0, 11.6) 32 ten one 10.0 (0.3, 44.5) 42 four 9.5 (2.7, 22.6) thirty 0 0.0 (0.0, 11.6) 64 ten 0 0.0 (0.0, 30.8) 42 one 2.4 (0.1, 12.6) thirty 0 0.0 (0.0, 11.6) 128 ten 0 0.0 (0.0, 30.8) 42 one 2.4 (0.1, 12.6) thirty 0 0.0 (0.0, 11.6) 18 to <24 months four 9 four 44.4 (13.7, 78.8) 44 19 43.2 (28.3, 59.0) 29 one 3.4 (0.1, 17.8) eight 9 four 44.4 (13.7, 78.8) 44 19 43.2 (28.3, 59.0) 29 one 3.4 (0.1, 17.8) 16 9 3 33.3 (7.5, 70.1) 44 19 43.2 (28.3, 59.0) 29 one 3.4 (0.1, 17.8) 32 9 0 0.0 (0.0, 33.6) 44 eight 18.2 (8.2, 32.7) 29 one 3.4 (0.1, 17.8) 64 9 0 0.0 (0.0, 33.6) 44 2 4.5 (0.6, 15.5) 29 0 0.0 (0.0, 11.9) 128 9 0 0.0 (0.0, 33.6) 44 0 0.0 (0.0, 8.0) 29 0 0.0 (0.0, 11.9) 1 month after vaccination 3 12 to <24 months four twenty 17 85.0 (62.1, 96.8) 95 68 71.6 (61.4, 80.4) 60 3 5.0 (1.0, 13.9) eight twenty 17 85.0 (62.1, 96.8) 95 68 71.6 (61.4, 80.4) 60 3 5.0 (1.0, 13.9) 16 twenty fifteen 75.0 (50.9, 91.3) 95 64 67.4 (57.0, 76.6) 60 3 5.0 (1.0, 13.9) 32 twenty eight 40.0 (19.1, 63.9) 95 34 35.8 (26.2, 46.3) 60 one 1.7 (0.0, 8.9) 64 twenty 3 15.0 (3.2, 37.9) 95 13 13.7 (7.5, 22.3) 60 0 0.0 (0.0, 6.0) 128 twenty one 5.0 (0.1, 24.9) 95 2 2.1 (0.3, 7.4) 60 0 0.0 (0.0, 6.0) 12 to <18 months four 9 eight 88.9 (51.8, 99.7) 45 32 71.1 (55.7, 83.6) 31 one 3.2 (0.1, 16.7) eight 9 eight 88.9 (51.8, 99.7) 45 32 71.1 (55.7, 83.6) 31 one 3.2 (0.1, 16.7) 16 9 eight 88.9 (51.8, 99.7) 45 thirty 66.7 (51.0, 80.0) 31 one 3.2 (0.1, 16.7) 32 9 four 44.4 (13.7, 78.8) 45 17 37.8 (23.8, 53.5) 31 one 3.2 (0.1, 16.7) 64 9 one 11.1 (0.3, 48.2) 45 eight 17.8 (8.0, 32.1) 31 0 0.0 (0.0, 11.2) 128 9 0 0.0 (0.0, 33.6) 45 one 2.2 (0.1, 11.8) 31 0 0.0 (0.0, 11.2) 18 to <24 months four eleven 9 81.8 (48.2, 97.7) fifty 36 72.0 (57.5, 83.8) 29 2 6.9 (0.8, 22.8) eight eleven 9 81.8 (48.2, 97.7) fifty 36 72.0 (57.5, 83.8) 29 2 6.9 (0.8, 22.8) 16 eleven 7 63.6 (30.8, 89.1) fifty 34 68.0 (53.3, 80.5) 29 2 6.9 (0.8, 22.8) 32 eleven four 36.4 (10.9, 69.2) fifty 17 34.0 (21.2, 48.8) 29 0 0.0 (0.0, 11.9) 64 eleven 2 18.2 (2.3, 51.8) fifty 5 10.0 (3.3, 21.8) 29 0 0.0 (0.0, 11.9) 128 eleven one 9.1 (0.2, 41.3) fifty one 2.0 (0.1, 10.6) 29 0 0.0 (0.0, 11.9) PMB2707 (B44) Before vaccination 1 12 to <24 months four 19 0 0.0 (0.0, 17.6) 95 one 1.1 (0.0, 5.7) 54 0 0.0 (0.0, 6.6) eight 19 0 0.0 (0.0, 17.6) 95 one 1.1 (0.0, 5.7) 54 0 0.0 (0.0, 6.6) 16 19 0 0.0 (0.0, 17.6) 95 0 0.0 (0.0, 3.8) 54 0 0.0 (0.0, 6.6) 32 19 0 0.0 (0.0, 17.6) 95 0 0.0 (0.0, 3.8) 54 0 0.0 (0.0, 6.6) 64 19 0 0.0 (0.0, 17.6) 95 0 0.0 (0.0, 3.8) 54 0 0.0 (0.0, 6.6) 128 19 0 0.0 (0.0, 17.6) 95 0 0.0 (0.0, 3.8) 54 0 0.0 (0.0, 6.6) 12 to <18 months four 9 0 0.0 (0.0, 33.6) 46 one 2.2 (0.1, 11.5) 24 0 0.0 (0.0, 14.2) eight 9 0 0.0 (0.0, 33.6) 46 one 2.2 (0.1, 11.5) 24 0 0.0 (0.0, 14.2) 16 9 0 0.0 (0.0, 33.6) 46 0 0.0 (0.0, 7.7) 24 0 0.0 (0.0, 14.2) 32 9 0 0.0 (0.0, 33.6) 46 0 0.0 (0.0, 7.7) 24 0 0.0 (0.0, 14.2) 64 9 0 0.0 (0.0, 33.6) 46 0 0.0 (0.0, 7.7) 24 0 0.0 (0.0, 14.2) 128 9 0 0.0 (0.0, 33.6) 46 0 0.0 (0.0, 7.7) 24 0 0.0 (0.0, 14.2) 18 to <24 months four ten 0 0.0 (0.0, 30.8) 49 0 0.0 (0.0, 7.3) thirty 0 0.0 (0.0, 11.6) eight ten 0 0.0 (0.0, 30.8) 49 0 0.0 (0.0, 7.3) thirty 0 0.0 (0.0, 11.6) 16 ten 0 0.0 (0.0, 30.8) 49 0 0.0 (0.0, 7.3) thirty 0 0.0 (0.0, 11.6) 32 ten 0 0.0 (0.0, 30.8) 49 0 0.0 (0.0, 7.3) thirty 0 0.0 (0.0, 11.6) 64 ten 0 0.0 (0.0, 30.8) 49 0 0.0 (0.0, 7.3) thirty 0 0.0 (0.0, 11.6) 128 ten 0 0.0 (0.0, 30.8) 49 0 0.0 (0.0, 7.3) thirty 0 0.0 (0.0, 11.6) 1 month after vaccination 2 12 to <24 months four 19 fourteen 73.7 (48.8, 90.9) 94 64 68.1 (57.7, 77.3) 52 0 0.0 (0.0, 6.8) eight 19 13 68.4 (43.4, 87.4) 94 64 68.1 (57.7, 77.3) 52 0 0.0 (0.0, 6.8) 16 19 13 68.4 (43.4, 87.4) 94 63 67.0 (56.6, 76.4) 52 0 0.0 (0.0, 6.8) 32 19 eleven 57.9 (33.5, 79.7) 94 53 56.4 (45.8, 66.6) 52 0 0.0 (0.0, 6.8) 64 19 6 31.6 (12.6, 56.6) 94 23 24.5 (16.2, 34.4) 52 0 0.0 (0.0, 6.8) 128 19 2 10.5 (1.3, 33.1) 94 ten 10.6 (5.2, 18.7) 52 0 0.0 (0.0, 6.8) 12 to <18 months four 9 7 77.8 (40.0, 97.2) 47 34 72.3 (57.4, 84.4) 23 0 0.0 (0.0, 14.8) eight 9 7 77.8 (40.0, 97.2) 47 34 72.3 (57.4, 84.4) 23 0 0.0 (0.0, 14.8) 16 9 7 77.8 (40.0, 97.2) 47 34 72.3 (57.4, 84.4) 23 0 0.0 (0.0, 14.8) 32 9 5 55.6 (21.2, 86.3) 47 29 61.7 (46.4, 75.5) 23 0 0.0 (0.0, 14.8) 64 9 3 33.3 (7.5, 70.1) 47 13 27.7 (15.6, 42.6) 23 0 0.0 (0.0, 14.8) 128 9 one 11.1 (0.3, 48.2) 47 5 10.6 (3.5, 23.1) 23 0 0.0 (0.0, 14.8) 18 to <24 months four ten 7 70.0 (34.8, 93.3) 47 thirty 63.8 (48.5, 77.3) 29 0 0.0 (0.0, 11.9) eight ten 6 60.0 (26.2, 87.8) 47 thirty 63.8 (48.5, 77.3) 29 0 0.0 (0.0, 11.9) 16 ten 6 60.0 (26.2, 87.8) 47 29 61.7 (46.4, 75.5) 29 0 0.0 (0.0, 11.9) 32 ten 6 60.0 (26.2, 87.8) 47 24 51.1 (36.1, 65.9) 29 0 0.0 (0.0, 11.9) 64 ten 3 30.0 (6.7, 65.2) 47 ten 21.3 (10.7, 35.7) 29 0 0.0 (0.0, 11.9) 128 ten one 10.0 (0.3, 44.5) 47 5 10.6 (3.5, 23.1) 29 0 0.0 (0.0, 11.9) 1 month after vaccination 3 12 to <24 months four 19 17 89.5 (66.9, 98.7) 94 82 87.2 (78.8, 93.2) 54 0 0.0 (0.0, 6.6) eight 19 17 89.5 (66.9, 98.7) 94 81 86.2 (77.5, 92.4) 54 0 0.0 (0.0, 6.6) 16 19 16 84.2 (60.4, 96.6) 94 81 86.2 (77.5, 92.4) 54 0 0.0 (0.0, 6.6) 32 19 12 63.2 (38.4, 83.7) 94 72 76.6 (66.7, 84.7) 54 0 0.0 (0.0, 6.6) 64 19 7 36.8 (16.3, 61.6) 94 55 58.5 (47.9, 68.6) 54 0 0.0 (0.0, 6.6) 128 19 four 21.1 (6.1, 45.6) 94 thirty 31.9 (22.7, 42.3) 54 0 0.0 (0.0, 6.6) 12 to <18 months four 9 eight 88.9 (51.8, 99.7) 47 41 87.2 (74.3, 95.2) 24 0 0.0 (0.0, 14.2) eight 9 eight 88.9 (51.8, 99.7) 47 41 87.2 (74.3, 95.2) 24 0 0.0 (0.0, 14.2) 16 9 7 77.8 (40.0, 97.2) 47 41 87.2 (74.3, 95.2) 24 0 0.0 (0.0, 14.2) 32 9 5 55.6 (21.2, 86.3) 47 35 74.5 (59.7, 86.1) 24 0 0.0 (0.0, 14.2) 64 9 four 44.4 (13.7, 78.8) 47 27 57.4 (42.2, 71.7) 24 0 0.0 (0.0, 14.2) 128 9 2 22.2 (2.8, 60.0) 47 fourteen 29.8 (17.3, 44.9) 24 0 0.0 (0.0, 14.2) 18 to <24 months four ten 9 90.0 (55.5, 99.7) 47 41 87.2 (74.3, 95.2) thirty 0 0.0 (0.0, 11.6) eight ten 9 90.0 (55.5, 99.7) 47 40 85.1 (71.7, 93.8) thirty 0 0.0 (0.0, 11.6) 16 ten 9 90.0 (55.5, 99.7) 47 40 85.1 (71.7, 93.8) thirty 0 0.0 (0.0, 11.6) 32 ten 7 70.0 (34.8, 93.3) 47 37 78.7 (64.3, 89.3) thirty 0 0.0 (0.0, 11.6) 64 ten 3 30.0 (6.7, 65.2) 47 28 59.6 (44.3, 73.6) thirty 0 0.0 (0.0, 11.6) 128 ten 2 20.0 (2.5, 55.6) 47 16 34.0 (20.9, 49.3) thirty 0 0.0 (0.0, 11.6) Abbreviation: hSBA=serum bactericidal activity using human complement;
a. N=number of individuals with confirmed and determined hSBA titers for a given strain.
b. n=number of individuals with observed hSBA titer ≥ defined titer for a given strain at a given time.
c. Exact two-tailed CI based on the observed number of individuals using the method of Klopper and Pearson

Experimental Immunogenicity Endpoints

hSBA titer that is ≥4 times baseline titer

Table 28 shows the number of individuals with hSBA titers that are ≥4 times the baseline titer for the 4 primary MnB strains tested.

At 1 month post-vaccination 2, the number of individuals aged 12 to <18 months who achieved an hSBA titer ≥4 times baseline hSBA titer was 80.0% and 62.2% for PMB80 (A22) ; 100.0% and 97.9% for PMB2001 (A56); 60.0% and 19.0% for PMB2948 (B24); and 77.8% and 70.2% for PMB2707 (B44) for the 60 µg and 120 µg dose groups, respectively. For the age stratified group 18 to <24 months, the number of individuals who achieved an hSBA titer ≥4 times baseline hSBA titer was 66.7% and 80.0% for PMB80 (A22); 90.0% and 100.0% for PMB2001 (A56); 33.3% and 40.9% for PMB2948 (B24); and 60.0% and 61.7% for PMB2707 (B44) for the 60 µg and 120 µg dose groups, respectively. Overall, for the pooled age stratified group, the number of individuals who achieved an hSBA titer ≥4 times baseline hSBA titer at 1 month post-vaccination 2 was 73.7% and 71.6% for PMB80 (A22) ; 94.7% and 98.9% for PMB2001 (A56); 47.4% and 30.2% for PMB2948 (B24); and 68.4% and 66.0% for PMB2707 (B44) for the 60 µg and 120 µg dose groups, respectively.

At 1 month post-vaccination 3, the number of individuals aged 12 to <18 months who achieved an hSBA titer ≥4 times baseline hSBA titer was 77.8% and 86.7% for PMB80 (A22) ; 100.0% and 95.7% for PMB2001 (A56); 88.9% and 66.7% for PMB2948 (B24); and 77.8% and 85.1% for PMB2707 (B44) for the 60 µg and 120 µg dose groups, respectively. For the age stratified group 18 to <24 months, the number of individuals who achieved an hSBA titer ≥4 times baseline hSBA titer was 90.9% and 88.2% for PMB80 (A22); 100.0% and 100.0% for PMB2001 (A56); 63.6% and 68.0% for PMB2948 (B24); and 90.0% and 85.1% for PMB2707 (B44) for the 60 µg and 120 µg dose groups, respectively. Overall, for the pooled age stratified group, the number of individuals who achieved an hSBA titer ≥4 times baseline hSBA titer at 1 month post-vaccination 3 was 85.0% and 87.5% for PMB80 (A22) ; 100.0% and 97.9% for PMB2001 (A56); 75.0% and 67.4% for PMB2948 (B24); and 84.2% and 85.1% for PMB2707 (B44) for the 60 µg and 120 µg dose groups, respectively.

The number of individuals in the HAV/saline group (pooled age stratified group) who achieved an hSBA titer ≥4 times baseline hSBA titer 1 month post-vaccination 3 was 5.0% for PMB80 (A22), 1.9% for PMB2001 (A56), 3.3% for PMB2948 (B24), and 0.0% for PMB2707 (B44).

Table 28. Individuals with an increase in hSBA titer ≥ 4 times for primary strains - Group assessed for immunogenicity Vaccine group (randomized) 60 mcg rLP2086 120 mcg rLP2086 HAV/saline solution Taking a sample of a strain (variant) at a given point in time for an age stratified group N ^a n ^b (%) (%) (95% CI) ^c N ^a n ^b (%) (%) (95% CI) ^c N ^a n ^b (%) (%) (95% CI) ^c PMB80(A22) 1 month after vaccination 2 12 to <24 months 19 fourteen (73.7) (48.8, 90.9) 95 68 (71.6) (61.4, 80.4) 59 one (1.7) (0.0, 9.1) 12 to <18 months ten eight (80.0) (44.4, 97.5) 45 28 (62.2) (46.5, 76.2) thirty 0 (0.0) (0.0, 11.6) 18 to <24 months 9 6 (66.7) (29.9, 92.5) fifty 40 (80.0) (66.3, 90.0) 29 one (3.4) (0.1, 17.8) 1 month after vaccination 3 12 to <24 months twenty 17 (85.0) (62.1, 96.8) 96 84 (87.5) (79.2, 93.4) 60 3 (5.0) (1.0, 13.9) 12 to <18 months 9 7 (77.8) (40.0, 97.2) 45 39 (86.7) (73.2, 94.9) 31 one (3.2) (0.1, 16.7) 18 to <24 months eleven ten (90.9) (58.7, 99.8) 51 45 (88.2) (76.1, 95.6) 29 2 (6.9) (0.8, 22.8) PMB2001(A56) 1 month after vaccination 2 12 to <24 months 19 eighteen (94.7) (74.0, 99.9) 95 94 (98.9) (94.3, 100.0) 52 0 (0.0) (0.0, 6.8) 12 to <18 months 9 9 (100.0) (66.4, 100.0) 47 46 (97.9) (88.7, 99.9) 23 0 (0.0) (0.0, 14.8) 18 to <24 months ten 9 (90.0) (55.5, 99.7) 48 48 (100.0) (92.6, 100.0) 29 0 (0.0) (0.0, 11.9) 1 month after vaccination 3 12 to <24 months 19 19 (100.0) (82.4, 100.0) 95 93 (97.9) (92.6, 99.7) 54 one (1.9) (0.0, 9.9) 12 to <18 months 9 9 (100.0) (66.4, 100.0) 47 45 (95.7) (85.5, 99.5) 24 0 (0.0) (0.0, 14.2) 18 to <24 months ten ten (100.0) (69.2, 100.0) 48 48 (100.0) (92.6, 100.0) thirty one (3.3) (0.1, 17.2) PMB2948 (B24) 1 month after vaccination 2 12 to <24 months 19 9 (47.4) (24.4, 71.1) 86 26 (30.2) (20.8, 41.1) 59 0 (0.0) (0.0, 6.1) 12 to <18 months ten 6 (60.0) (26.2, 87.8) 42 eight (19.0) (8.6, 34.1) thirty 0 (0.0) (0.0, 11.6) 18 to <24 months 9 3 (33.3) (7.5, 70.1) 44 eighteen (40.9) (26.3, 56.8) 29 0 (0.0) (0.0, 11.9) 1 month after vaccination 3 12 to <24 months twenty fifteen (75.0) (50.9, 91.3) 95 64 (67.4) (57.0, 76.6) 60 2 (3.3) (0.4, 11.5) 12 to <18 months 9 eight (88.9) (51.8, 99.7) 45 thirty (66.7) (51.0, 80.0) 31 one (3.2) (0.1, 16.7) 18 to <24 months eleven 7 (63.6) (30.8, 89.1) fifty 34 (68.0) (53.3, 80.5) 29 one (3.4) (0.1, 17.8) PMB2707 (B44) 1 month after vaccination 2 12 to <24 months 19 13 (68.4) (43.4, 87.4) 94 62 (66.0) (55.5, 75.4) 52 0 (0.0) (0.0, 6.8) 12 to <18 months 9 7 (77.8) (40.0, 97.2) 47 33 (70.2) (55.1, 82.7) 23 0 (0.0) (0.0, 14.8) 18 to <24 months ten 6 (60.0) (26.2, 87.8) 47 29 (61.7) (46.4, 75.5) 29 0 (0.0) (0.0, 11.9) 1 month after vaccination 3 12 to <24 months 19 16 (84.2) (60.4, 96.6) 94 80 (85.1) (76.3, 91.6) 54 0 (0.0) (0.0, 6.6) 12 to <18 months 9 7 (77.8) (40.0, 97.2) 47 40 (85.1) (71.7, 93.8) 24 0 (0.0) (0.0, 14.2) 18 to <24 months ten 9 (90.0) (55.5, 99.7) 47 40 (85.1) (71.7, 93.8) thirty 0 (0.0) (0.0, 11.6) Abbreviations: hSBA = serum bactericidal activity assay using human complement; LLOQ=lower limit of quantification; LOD=limit of detection.
Note: LLOQ=1:16 for A22; 1:8 for A56, B24 and B44.
Note: A 4-fold increase is defined as follows: (1) For individuals with baseline hSBA titer below LOD (hSBA titer <1:4), response is defined as hSBA titer ≥1:16 or LLOQ (however high the titer is). was). (2) For individuals with a baseline hSBA titer ≥LOD and <LLOQ, response is defined as an hSBA titer that is ≥4 times the LLOQ. (3) For individuals with baseline hSBA titer r ≥ LLOQ, response is defined as an hSBA titer that is ≥4 times baseline titer.
a. For an hSBA titer that is ≥4 times baseline titer, N=number of individuals with valid and defined hSBA titers for a given strain at a particular time point and baseline.
b. For an hSBA titer that is ≥4 times baseline, n=number of individuals whose hSBA titer is ≥4 times baseline for the strain.
c. Exact two-tailed CI based on the observed number of individuals using the method of Klopper and Pearson

Similar results were obtained for the mITT group.

The results of subgroup analyzes to determine the number of individuals who achieved a ≥4-fold increase in hSBA titer for each of the 4 primary MnB strains tested were evaluated for the gender and country immunogenicity assessment group. There were no clinically significant differences in subgroup analyses.

Inverse Cumulative Distribution Curves for Primary MnB Test Strains

RCDC for the number of individuals with hSBA response (≥LLOQ) for each of the 4 primary MnB strains and at each blood sampling point for the pooled age stratified group was estimated for PMB80 [A22], PMB2001 [A56], PMB2948 [B24], and PMB2707 [ B44]. RCDC showed that immune responses after vaccination 2 and vaccination 3 were higher for the groups treated with divalent rLP2086 compared to the group that was injected with HAV/saline solution. Immune responses in groups treated with divalent rLP2086 increased with each vaccination.

Conclusions on immunogenicity

The main objective of this study is to characterize the immune response to bivalent rLP2086, as determined by hSBA, against 4 primary MnB strains tested, 2 of which expressed LP2086 subfamily A protein and 2 expressed LP2086 subfamily B protein, as determined 1 month after third vaccination in healthy individuals aged 12 to <18 months and 18 to <24 months. The second objective is to describe immune responses for patients in a pooled age stratified group (12 to <24 months). The primary goal endpoints are the number of individuals in each age stratified group who achieved hSBA titers ≥LLOQ for each of the 4 primary MnB strains 1 month after the third vaccination.

A sustained immune response was observed with both doses in children aged 12 to <18 months and in children aged 18 to <24 months, as well as in children of the pooled age stratified group (12 to <24 months) after 1 month after the third dose of divalent rLP2086, as evidenced by the number of individuals whose hSBA titer was ≥LLOQ (1:8 for A56, B24 and B44; 1:16 for A22) for each of the 4 primary MnB strains tested. For the 60 µg group, the number of individuals achieving hSBA titer ≥LLOQ ranged from 88.9% to 100.0% for younger children (12 to <18 months) and from 81.8% up to 100.0% for older children (18 to <24 months) after 3 doses. For the 120 µg group, the number of individuals achieving hSBA titer ≥LLOQ ranged from 71.1% to 100.0% for children aged 12 to <18 months, and from 72.0% to 100 0% for children aged 18 to <24 months after 3 doses. The number of individuals in this pooled age stratified group with hSBA titer ≥LLOQ for each of the 4 primary MnB strains tested 1 month after the third vaccination ranged from 85.0% to 100.0%, in the group administered 60 μg, and from 71.6% to 100.0% in the 120 μg group. These data were also supported by high GMT (ranging from 4.0-8.5 at baseline to 15.1-171.4 at 1 month post-vaccination 3) and the number of individuals achieving hSBA titer ≥1:4 (71.1% - 100%) or ≥1:16 (63.6% - 100%) vs. each of the 4 primary MnB strains tested after 3 doses of bivalent rLP2086 compared to baseline for both age stratified groups and within groups who received the indicated doses. In addition, the number of individuals in the pooled age stratified groups whose hSBA titer was ≥4 higher than baseline 1 month after the third vaccination for each of the 4 primary MnB strains tested ranged from 67.4% to 100, 0% in both groups administered these doses. In conclusion, 3 doses of 60 µg or 120 µg of bivalent rLP2086 given at months 0, 2 and 6 induced robust immune responses in children 12 to <24 months of age (in separate and pooled age stratified groups). ).

The second objective of the study is to characterize immune responses 1 month after a second dose of bivalent rLP2086 as assessed by ≥LLOQ responses, determined hSBA titers and hSBA GMT for 2 age stratified groups and pooled age stratified group. For the pooled age stratified group, the number of individuals with hSBA titer ≥LLOQ following the second dose of bivalent rLP2086 (2 months after the first dose) ranged from 57.9% to 94.7%, for individuals in the 60 µg group, and from 33.7% to 100.0% for individuals in the 120 μg group. Similar results were obtained for two separate age stratified groups, and no significant differences were observed between the younger and older stratified groups. These data were further supported by an increase in GMT (range 7.2 to 110.6) from baseline, and the number of individuals who achieved hSBA titer ≥1:4 (36.0% - 100%) or ≥ 1:16 (32.6% - 100%) vs. each of the 4 primary MnB strains tested after 2 doses of divalent rLP2086 versus baseline with both doses for the pooled age stratified group. Similar results were obtained for 2 separate age stratified groups. In addition, the number of individuals in the pooled age stratified group whose hSBA titer was ≥4-fold higher than baseline 1 month after the second vaccination for each of the 4 primary MnB strains tested ranged from 30.2% to 98 ,9%. In conclusion, 2 doses of 60 μg or 120 μg of divalent rLP2086 given 2 months apart induced robust immune responses in children aged 12 to <24 months (in separate and pooled age stratified groups).

Overall, at a dose of 60 µg or 120 µg, bivalent rLP2086 given in 3 doses on a schedule for months 0, 2, and 6 elicited a robust immune response in children aged 12 to <24 months with protective antibody titers achievable , as determined by hSBA, in a large number of individuals after the administration of the third dose.

Discussion and general conclusions

Discussion of immunogenicity. The results of this Phase 2 immunogenicity study on a 3-dose schedule (months 0, 2, and 6 schedule) of bivalent rLP2086 (at 2 doses) administered to children 12 to <24 months of age are consistent with previous studies with the participation of adolescents and young people who were administered a dose of 120 mcg.

Immunogenic responses to vaccination with bivalent rLP2086 were assessed with hSBA for validity using 4 MnB primary test strains, each expressing fHBP variants heterologous to vaccine component antigens, against more stringent criteria than the generally accepted correlate of protection (hSBA titer ≥1: four). Based on hSBA titer ≥LLOQ for the 4 primary MnB strains tested at 1 month post-vaccination 3, infants in this study (who were given any of these doses) had immune responses similar to those seen in adolescents (from 10 years to <19 years) in study B1971009 and in infants and older children in study B1971017 (≥24 months to <10 years), where the number of individuals with hSBA titer ≥LLOQ after the third vaccination ( schedule for months 0, 2, and 6) ranged from 71.6% to 100% for the 120 mcg group (aged 12 to <24 months) in this study, from 87.1% to 99 .5% in study B1971009 and 79.1% to 100.0% in study B1971017. There were no significant differences in the number of individuals with hSBA titer ≥LLOQ for the 4 primary test strains at 1 month post-vaccination 3 in these 3 studies, although study B1971009 included a much larger number of adolescents with hSBA titer ≥ LLOQ before vaccination compared with titers in infants in this study. Bivalent rLP2086 appears to be highly immunogenic in individuals in the 12 to <24 month group and appears to have a protective effect against MnB infection similar to that expected in adolescents based on the hSBA correlate of protection. After administration of 3 doses to individuals in separate age stratified groups and in both combined age stratified groups, the responses to the 60 μg dose were not significantly different from the responses to the 120 μg dose.

With respect to other targets, namely immune responses in this study for a pooled age stratified group (12 to <24 months) 1 month after a second dose of bivalent rLP2086 (any dose), the number of individuals who achieved hSBA titer ≥LLOQ, ranged from 33.7% to 100.0% compared with the number of adolescents (10 years to <19 years) participating in the B1971009 study who received 2 doses of bivalent rLP2086 with an interval of 2 months, which ranged from 64.2% to 99.1%, and compared to the number of younger and older children (≥24 months to <10 years) who participated in study B1971017, who ranged from 48.5% to 100 .0%. For the age stratified group and pooled age stratified group, immune responses after administration of 2 doses of bivalent rLP2086 were not significantly different from those observed after administration of 2 doses of 120 μg of bivalent rLP2086. It should be noted that the smaller the sample size of the 60 μg group, the more difficult it is to obtain accurate results when comparing responses for 60 μg and 120 μg doses.

In general, at a dose of 60 µg or 120 µg, bivalent rLP2086 given at the 0, 2, and 6 month dosing schedule is highly immunogenic in children 12 to <24 months of age with protective immune responses achieved as was determined using hSBA, in a large number of individuals after the introduction of the third dose. The immune responses assessed in this study were similar to those observed in previous studies in adolescents and children 1 month after the third dose. The 3 dose regimen provides high levels of protective immunity in children aged 12 to <24 months.

General conclusions. In conclusion, bivalent rLP2086 given to children aged 12 to <24 months on a 60 µg or 120 µg dosing schedule for months 0, 2, and 6 produces protective antibody titers after the third dose, as determined by hSBA. The vaccine administered in this study was safe and well tolerated with an acceptable safety profile in children aged 12 to <24 months.

Example 22 Evaluation of Immunogenicity of Pentavalent Mn Vaccine and Trumenba® Vaccine Neisseria meningitidis serogroup B in CBA/J mice

The immune response to Neisseria meningitidis serogroup B fHBP following vaccination with bivalent fHBP MnB vaccine, Trumenba or bivalent fHBP MnB vaccine formulated with ACWY quadrivalent polysaccharide conjugate vaccine (pentavalent Mn ABCWY) was assessed in CBA/J mice. Groups of CBA/J mice were immunized with 3 different vaccines: pentavalent vaccine (ABCYW), Trumenba® (MnB) and Nimenrix® (ACYW) (Table 29).

Table 29 - Study protocol: Doses for each vaccine Doses, mcg/0.25 ml Dilution factor pentavalent Mn(ABCWY) TRUMENBA®
(B) NIMENRIX® (ACWY) AlPO _four (diluent) one 8+1.33 eight 1.33 125 2 4+0.67 four 0.67 125 four 2+0.33 2 0.33 125 eight 1+0.17 one 0.17 125

For each group, CBA/J mice (25/group) were immunized subcutaneously in the back of the neck by administering a 2-fold dose of the appropriate vaccine (Table 29). Mice were primed with vaccine at time 0 and boosted 2 weeks later. Serum was collected every other day (PD2) at week 3 for testing with two different serum bactericidal assays using human complement (hSBA). One hSBA used a strain expressing fHBP subfamily A (M98250771) and the other used a strain expressing fHBP subfamily B (CDC1127).

In hSBA, antibody-dependent complement-mediated bactericidal activity was determined against N meningitidis strains of serogroup B. Briefly, test serum at the appropriate dilution was mixed in 96-well microtiter assay plates with freshly prepared bacterial cultures of N meningitidis B strains (subfamilies A or B) and with human complement. The assay plates were placed on an orbital shaker and mixed for 30 minutes in a high humidity incubator (37°C/5% CO ₂ ). Then, aliquots of the assay reaction mixture from each well were transferred to 96-well filter plates to enumerate surviving bacteria.

Vaccination response rates were calculated as the percentage of mice per group for each dose (n=25) that responded in hSBA. When tested at a predetermined dilution level, mouse serum samples that killed ≥50% meningococcal bacteria in the T ₃₀ control wells were considered responders. Control wells T ₃₀ contained bacteria and complement, but did not contain test serum and were evaluated at the end of the 30-minute incubation for analysis.

Table 30 and Table 31 show the comparable dose-dependent levels of responses induced by administration of TRUMENBA® or pentavalent Mn for both N. meningitidis serotype B subfamily A and subfamily B strains. As expected, NIMENRIX™ did not induce a functional immune response to strains M&B.

Table 30 - hSBA responses to subfamily A (% of respondents) Dilution factor ^a TRUMENBA NIMENRIX pentavalent eight 24% 0% eight% four 40% 0% 16% 2 52% 0% 56% one 80% 0% 92% ^a See appropriate doses in Table 29

Table 31 - hSBA responses to subfamily B (% of respondents) Dilution factor ^a TRUMENBA NIMENRIX pentavalent eight 28% 0% 36% four 56% 0% 60% 2 60% 0% 76% one 72% 0% 76% ^a See appropriate doses in Table 29

The following paragraphs describe additional embodiments of the invention:

C1. Composition containing a polypeptide and a capsular saccharide conjugateNeisseria meningitidis serological group A (MenA); capsular saccharide conjugateNeisseria meningitidis serogroup C (MenC); capsular saccharide conjugateNeisseria meningitidis serogroup W₁₃₅ (MenW); and capsular saccharide conjugateNeisseria meningitidis serogroup Y (MenY).

C2. The composition according to C1, wherein the MenA capsular saccharide is conjugated to a carrier protein; the MenC capsular saccharide is conjugated to a carrier protein; the MenW capsular saccharide is conjugated to a carrier protein; and the MenY capsular saccharide is conjugated to a carrier protein.

C3. A composition according to C1, wherein the composition further comprises a capsular saccharide conjugate of Neisseria meningitidis serogroup X (MenX).

C4. The composition according to C1, wherein the polypeptide is factor H binding protein (fHBP).

C5. The composition of item C1, wherein the polypeptide comprises an amino acid sequence that is at least 70% identical to the amino acid sequence shown in any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQID NO: 60, SEQ ID NO: 61 and SEQ ID NO: 62.

C6. Composition containing:

a. the first lipidized polypeptide containing the amino acid sequence shown in SEQ ID NO:1;

b. a second lipidized polypeptide containing the amino acid sequence shown in SEQ ID NO:2; and

With. Neisseria meningitidis serogroup A (MenA) capsular saccharide conjugated to a carrier protein.

C7. Composition containing:

a. the first lipidized polypeptide containing the amino acid sequence shown in SEQ ID NO:1;

b. a second lipidized polypeptide containing the amino acid sequence shown in SEQ ID NO:2; and

With. capsular saccharide of Neisseria meningitidis serogroup C (MenC) conjugated to a carrier protein.

C8. Composition containing:

a. the first lipidized polypeptide containing the amino acid sequence shown in SEQ ID NO:1;

b. a second lipidized polypeptide containing the amino acid sequence shown in SEQ ID NO:2; and

With. capsular saccharide of Neisseria meningitidis serogroup W-135 (MenW) conjugated to a carrier protein.

C9. Composition containing:

a. the first lipidized polypeptide containing the amino acid sequence shown in SEQ ID NO:1;

b. a second lipidized polypeptide containing the amino acid sequence shown in SEQ ID NO:2; and

With. Neisseria meningitidis serogroup Y (MenY) capsular saccharide conjugated to a carrier protein.

C10. Composition containing

a. the first lipidized polypeptide containing the amino acid sequence shown in SEQ ID NO:1;

b. a second lipidized polypeptide containing the amino acid sequence shown in SEQ ID NO:2; and

With. capsular saccharide of Neisseria meningitidis serogroup A (MenA) conjugated to a carrier protein.

d. capsular saccharide of Neisseria meningitidis serogroup C (MenC) conjugated to a carrier protein.

e. Neisseria meningitidis serogroup W ₁₃₅ (MenW) capsular saccharide conjugated to a carrier protein.

f. Neisseria meningitidis serogroup Y (MenY) capsular saccharide conjugated to a carrier protein.

C11. Composition containing

a. the first lipidized polypeptide containing the amino acid sequence shown in SEQ ID NO:1;

b. a second lipidized polypeptide containing the amino acid sequence shown in SEQ ID NO:2; and

With. Neisseria meningitidis serogroup A (MenA) capsular saccharide conjugated to a tetanus toxoid (TT) carrier protein;

d. Neisseria meningitidis serogroup C (MenC) capsular saccharide conjugated to a tetanus toxoid (TT) carrier protein;

e. Neisseria meningitidis serogroup W ₁₃₅ (MenW) capsular saccharide conjugated to a tetanus toxoid (TT) carrier protein; and

f. Neisseria meningitidis serogroup Y (MenY) capsular saccharide conjugated to a tetanus toxoid (TT) carrier protein.

C12. A composition according to C1, wherein the MenA capsular saccharide is conjugated to an adipic acid dihydrazide (ADH) linker via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry, and wherein the linker is conjugated to a tetanus toxoid (TT) carrier protein by a carbodiimide chemistry (conjugate MenA _AH -TT).

C13. The composition according to paragraph C1, wherein the MenC capsular saccharide is conjugated to an ADH linker via the chemical compound 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, and wherein the linker is conjugated to a tetanus toxoid (TT) carrier protein by a carbodiimide chemical method (MenC _AH -TT conjugate ).

C14. A composition according to C1, wherein the MenW capsular saccharide is directly conjugated to a tetanus toxoid (TT) carrier protein via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry in the absence of a linker (MenW-TT conjugate).

C15. A composition according to C1, wherein the MenY capsular saccharide is directly conjugated to a tetanus toxoid (TT) carrier protein via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry in the absence of a linker (MenY-TT conjugate).

C16. Composition in accordance with paragraph C1, also containing Tris-HCl.

C17. Composition according to C1, also containing sodium chloride.

C18. Composition according to point C1, also containing sucrose.

C19. Composition according to C1, also containing histidine.

C20. Composition according to point C1, also containing polysorbate 80.

C21. Composition according to C1, also containing aluminium.

C22. Composition according to C1, also containing aluminum phosphate.

C23. The composition in accordance with paragraph C1, where the composition contains approximately 120 μg/ml of the first polypeptide; approximately 120 μg/ml of the second polypeptide; approximately 0.5 mg/ml aluminum as aluminum phosphate; about 0.02 mg polysorbate-80; about 10 mM histidine; and approximately 150 mm sodium chloride.

C24. The composition in accordance with paragraph C1, where the composition contains approximately 60 μg of the first polypeptide; approximately 60 μg of the second polypeptide; approximately 5 μg MenA capsular saccharide conjugated to approximately 7.5 μg TT; approximately 5 μg MenC capsular saccharide conjugated to approximately 7.5 μg TT; approximately 5 μg MenW capsular saccharide conjugated to approximately 3.75 μg TT; approximately 5 μg MenY capsular saccharide conjugated to approximately 3.25 μg TT; approximately 97 µg Tris-HCl, pH 6.8 ± 0.3; 4.69-4.71 mg sodium chloride; approximately 28 mg sucrose; approximately 0.78 mg L-histidine; about 0.02 mg polysorbate-80; approximately 0.25 mg aluminum; and also contains 0.5 ml of water per dose.

C25. A composition according to C1, wherein the composition is suitable for administration to a patient 12 to <18 months of age, or 18 to <24 months of age.

C26. A composition according to C1, wherein the composition is suitable for administration to a patient between 18 and <24 months of age.

C27. The composition in accordance with paragraph C1, where the composition is suitable for administration to a patient aged ≥24 months to <10 years.

C28. A composition according to C20, wherein the composition contains at least 0.010 mg of polysorbate-80 and a maximum of 0.018 mg of polysorbate-80.

C29. Composition in accordance with paragraph C20, where the composition contains at least 0.01 mg of polysorbate-80 and a maximum of 0.02 mg of polysorbate-80.

C30. The composition according to paragraph C1, where the composition also does not contain a polypeptide having a sequence that is less than 100% identical to the sequence of SEQ ID NO:1.

C31. A composition according to C1, wherein the first polypeptide has a total of 258 amino acids.

C32. The composition according to paragraph C1, where the composition also does not contain a polypeptide having a sequence that is less than 100% identical to the sequence of SEQ ID NO:2.

C33. A composition according to C1, wherein the second polypeptide has a total of 261 amino acids.

C34. A composition according to C1, wherein the composition contains a maximum of two lipidized polypeptides.

C35. A composition according to C1, wherein the composition does not contain a fusion protein.

C36. A composition according to C1, wherein the composition does not contain a chimeric protein.

C37. A composition according to C1, wherein the composition does not contain a fusion protein.

C38. Composition in accordance with paragraph C1, where the composition is not lyophilized.

C39. Composition in accordance with paragraph C1, where the composition does not contain formaldehyde.

C40. A composition according to C1, wherein the composition does not contain diphtheria toxoid or CRM.

C41. A composition according to C1, wherein the composition does not contain a MenA capsular saccharide in the absence of an adipic acid dihydrazide (ADH) linker.

C42. A composition according to C1, wherein the composition does not contain the MenC capsular saccharide in the absence of an adipic acid dihydrazide (ADH) linker.

C43. The composition according to paragraph C1, where the composition is a liquid composition.

C44. A composition according to C1, wherein the composition also does not contain any of the following immunogenic compositions: MENACTRA(R), MENVEO(R), ADACEL(R), HAVRIX(R), GARDASIL(R), REPEVAX, or any combination thereof.

C45. A composition according to C1, wherein the composition also does not contain a meningococcal polysaccharide A, C, Y and W-135 (MCV4) conjugate composition, wherein the carrier protein is a diphtheria toxoid.

C46. A composition in accordance with paragraph C1, wherein the composition also does not contain a meningococcal polysaccharide A, C, Y and W-135 (MCV4) conjugate composition, wherein the carrier protein is CRM ₁₉₇ .

C47. A composition according to C1, wherein the composition also does not contain a NIMENRIX vaccine.

C48. Composition in accordance with paragraph C1, where the composition also does not contain the NIMENRIX vaccine, where NIMENRIX contains a diluent consisting of sodium chloride and water.

C49. A kit containing (a) a first composition comprising a lipidized MenB subfamily A polypeptide rLP2086 and a lipidized MenB subfamily B polypeptide rLP2086; and (b) a second composition comprising a Neisseria meningitidis serogroup A (MenA) capsular saccharide conjugated to a tetanus toxoid (TT) carrier protein; Neisseria meningitidis serogroup C (MenC) capsular saccharide conjugated to a tetanus toxoid (TT) carrier protein; Neisseria meningitidis serogroup W ₁₃₅ (MenW) capsular saccharide conjugated to a tetanus toxoid (TT) carrier protein; and Neisseria meningitidis serogroup Y (MenY) capsular saccharide conjugated to a tetanus toxoid (TT) carrier protein.

C50. A kit according to C49, wherein the first composition is a liquid composition and the second composition is a lyophilized composition.

C51. A kit according to C49, wherein the lyophilized composition does not contain polysorbate 80.

C52. A kit according to C49, wherein the kit also does not include any of the following immunogenic compositions: MENACTRA(R), MENVEO(R), ADACEL(R), HAVRIX(R), GARDASIL(R), REPEVAX, or any combination thereof.

C53. A kit according to paragraph C49, wherein the kit also does not include a meningococcal polysaccharide A, C, Y, and W-135 (MCV4) conjugate composition, wherein the carrier protein is a diphtheria toxoid.

C54. A kit according to C49, wherein the kit also does not include a meningococcal polysaccharide A, C, Y, and W-135 (MCV4) conjugate composition, wherein the carrier protein is CRM ₁₉₇ .

C55. Kit in accordance with C49, where the kit also does not include NIMENRIX vaccine.

C56. Kit according to C49, where the kit also does not include NIMENRIX(R) vaccine, where NIMENRIX(R) contains a diluent consisting of sodium chloride and water.

C57. Set including:

a. liquid composition containing:

i. the first lipidized polypeptide containing the amino acid sequence shown in SEQ ID NO:1; and

ii. a second lipidized polypeptide containing the amino acid sequence shown in SEQ ID NO:2; and

b. a lyophilized composition containing:

i. Neisseria meningitidis serogroup A (MenA) capsular saccharide conjugated to an adipic acid dihydrazide (ADH) linker via the chemical compound 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, wherein the linker is conjugated to a tetanus toxoid (TT) carrier protein by a carbodiimide chemical method (MenA conjugate _AH -TT);

ii. Neisseria meningitidis serogroup C (MenC) capsular saccharide conjugated to an ADH linker via the chemical compound 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, wherein the linker is conjugated to a tetanus toxoid (TT) carrier protein by a carbodiimide chemical method (MenC _AH -TT conjugate) ;

iii. Neisseria meningitidis serogroup W ₁₃₅ (MenW) capsular saccharide directly conjugated to a tetanus toxoid (TT) carrier protein via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry in the absence of a linker (MenW-TT conjugate); and

iv. Neisseria meningitidis serogroup Y (MenY) capsular saccharide directly conjugated to a tetanus toxoid (TT) carrier protein via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry in the absence of a linker (MenY-TT conjugate).

C58. A kit according to C57, wherein the liquid composition also contains sodium chloride.

C59. A kit according to C57, wherein the liquid composition also contains L-histidine.

C60. A kit according to C57, wherein the liquid composition also contains polysorbate 80.

C61. A set according to C57, wherein the liquid composition also contains aluminum phosphate.

C62. A kit according to C57, where the liquid composition also does not contain Tris-HCl.

C63. A set according to C57, wherein the liquid composition also does not contain sucrose.

C64. A kit according to C57, wherein the lyophilized composition also contains sodium chloride.

C65. A kit according to C57, wherein the lyophilized composition does not contain polysorbate-80.

C66. A kit according to C60, wherein the liquid composition contains at least 0.010 mg of polysorbate-80 and a maximum of 0.018 mg of polysorbate-80.

C67. A kit according to C60, wherein the liquid composition contains at least 0.010 mg of polysorbate-80 and a maximum of 0.02 mg of polysorbate-80.

C68. An immunogenic composition comprising:

a. a liquid composition containing: (i) a first lipidized polypeptide containing the amino acid sequence shown in SEQ ID NO: 1; and (ii) a second lipidized polypeptide containing the amino acid sequence shown in SEQ ID NO: 2; and

b. a lyophilized composition containing:

i. Neisseria meningitidis serogroup A (MenA) capsular saccharide conjugated to an adipic acid dihydrazide (ADH) linker via the chemical compound 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, wherein the linker is conjugated to a tetanus toxoid (TT) carrier protein by a carbodiimide chemical method (MenA conjugate _AH -TT);

ii. Neisseria meningitidis serogroup C (MenC) capsular saccharide conjugated to an ADH linker via the chemical compound 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, wherein the linker is conjugated to a tetanus toxoid (TT) carrier protein by a carbodiimide chemical method (MenC _AH -TT conjugate) ;

iii. Neisseria meningitidis serogroup W ₁₃₅ (MenW) capsular saccharide directly conjugated to a tetanus toxoid (TT) carrier protein via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry in the absence of a linker (MenW-TT conjugate); and

iv. Neisseria meningitidis serogroup Y (MenY) capsular saccharide directly conjugated to a tetanus toxoid (TT) carrier protein via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry in the absence of a linker (MenY-TT conjugate).

C69. An immunogenic composition according to C68, wherein the lyophilized composition is reconstituted with a liquid composition.

C70. An immunogenic composition according to C68, wherein the liquid composition also contains histidine.

C71. An immunogenic composition according to C68, wherein the liquid composition also contains polysorbate-80.

C72. An immunogenic composition according to C68, wherein the liquid composition also contains aluminum phosphate.

C73. An immunogenic composition according to C68, wherein the liquid composition also contains sodium chloride.

C74. An immunogenic composition according to C68, wherein the composition is suitable for administration to a patient 12 to <18 months of age or 18 to <24 months of age.

C75. An immunogenic composition according to C68, wherein the composition is suitable for administration to a patient between 18 and <24 months of age.

C76. An immunogenic composition according to C68, wherein the composition is suitable for administration to a patient ≥24 months of age to <10 years of age.

C77. An immunogenic composition comprising:

a. a liquid composition comprising: (i) a first lipidized polypeptide containing the amino acid sequence shown in SEQ ID NO:1; and (ii) a second lipidized polypeptide containing the amino acid sequence shown in SEQ ID NO:2; and

b. a lyophilized composition containing:

i. Neisseria meningitidis serogroup A (MenA) capsular saccharide conjugated to an adipic acid dihydrazide (ADH) linker via the chemical compound 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, wherein the linker is conjugated to a tetanus toxoid (TT) carrier protein by a carbodiimide chemical method (MenA conjugate _AH -TT);

ii. Neisseria meningitidis serogroup C (MenC) capsular saccharide conjugated to an ADH linker via the chemical compound 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, wherein the linker is conjugated to a tetanus toxoid (TT) carrier protein by a carbodiimide chemical method (MenC _AH -TT conjugate) ;

iii. Neisseria meningitidis serogroup W ₁₃₅ (MenW) capsular saccharide directly conjugated to a tetanus toxoid (TT) carrier protein via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate chemistry in the absence of a linker (MenW-TT conjugate); and

iv. Neisseria meningitidis serogroup Y (MenY) capsular saccharide directly conjugated to a tetanus toxoid (TT) carrier protein via the chemical compound 1-cyano-4-dimethylaminopyridinium tetrafluoroborate in the absence of a linker (MenY-TT conjugate),

where the lyophilized composition is diluted with a liquid composition to obtain an immunogenic composition.

C78. An immunogenic composition according to C77, wherein the liquid composition also contains aluminum.

C79. An immunogenic composition according to C77, wherein the liquid composition also contains aluminum phosphate.

C80. An immunogenic composition according to C77, wherein the lyophilized composition also contains sodium chloride.

C81. An immunogenic composition according to C77, wherein the immunogenic composition contains at least 0.010 mg of polysorbate-80 and a maximum of 0.018 mg of polysorbate-80.

C82. An immunogenic composition according to C77, wherein the immunogenic composition contains at least 0.01 mg of polysorbate-80 and a maximum of 0.02 mg of polysorbate-80.

C83. An immunogenic composition according to C77, wherein the lyophilized composition does not contain aluminium.

C84. An immunogenic composition according to C78, wherein the first polypeptide and the second polypeptide are bound to aluminum.

C85. An immunogenic composition according to C78, wherein the first polypeptide and the second polypeptide are bound to aluminum in the immunogenic composition.

C86. An immunogenic composition according to C78, wherein the concentration of aluminum-bound polypeptides in the immunogenic composition does not decrease after 24 hours compared to the concentration of aluminum-bound polypeptides in the liquid composition prior to reconstitution of the lyophilized composition.

C87. An immunogenic composition according to C77, wherein the concentration of the MenA _AH -TT conjugate in the immunogenic composition does not decrease after 24 hours compared to the concentration of the MenA _AH -TT conjugate in the lyophilized composition.

C88. An immunogenic composition according to _C77 , wherein the concentration of the MenC AH-TT conjugate in the immunogenic composition does not decrease after 24 hours compared to the concentration of the MenC _AH -TT conjugate in the lyophilized composition.

C89. An immunogenic composition according to C77, wherein the concentration of the MenW-TT conjugate in the immunogenic composition does not decrease after 24 hours compared to the concentration of the MenW-TT conjugate in the lyophilized composition.

C90. An immunogenic composition according to C77, wherein the concentration of the MenY-TT conjugate in the immunogenic composition does not decrease after 24 hours compared to the concentration of the MenY-TT conjugate in the lyophilized composition.

C91. An immunogenic composition according to C86, wherein the concentration is reduced by a maximum of 1% after 24 hours compared to the corresponding concentration in the liquid composition before reconstitution.

C92. An immunogenic composition according to C86, wherein the concentration is reduced by a maximum of 5% after 24 hours compared to the corresponding concentration in the liquid composition before reconstitution.

C93. An immunogenic composition according to C86, wherein the concentration is reduced by a maximum of 10% after 24 hours compared to the corresponding concentration in the liquid composition before reconstitution.

C94. An immunogenic composition according to C87-C90, wherein the concentration is reduced by a maximum of 1% after 24 hours compared to the corresponding concentration in the lyophilized composition before reconstitution.

C95. An immunogenic composition according to C87-C90, wherein the concentration is reduced by a maximum of 5% after 24 hours compared to the corresponding concentration in the lyophilized composition before reconstitution.

C96. An immunogenic composition according to C87-C90, wherein the concentration is reduced by a maximum of 10% after 24 hours compared to the corresponding concentration in the lyophilized composition before reconstitution.

C97. An immunogenic composition according to C68 or C77, wherein the pH of the reconstituted immunogenic composition is less than the pH of the lyophilized composition when diluted with sodium chloride.

C98. A composition comprising: a) a first lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 and b) a second lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2.

C99. A composition according to C98, wherein the composition also contains polysorbate-80, aluminium, histidine and sodium chloride.

C100. A composition according to C98, wherein the composition contains 60 μg of the first lipidized polypeptide and 60 μg of the second lipidized polypeptide.

C101. A method of inducing a bactericidal immune response against a Neisseria meningitidis serogroup B subfamily A strain and against a Neisseria meningitidis serogroup B subfamily strain B in a human, said method comprising administering to the human an effective amount of the composition according to C1.

C102. A method of inducing a bactericidal immune response against a Neisseria meningitidis serogroup B subfamily A strain and against a Neisseria meningitidis serogroup B subfamily strain B in a human, said method comprising administering to the human an effective amount of the composition according to C68.

C103. A method of inducing a bactericidal immune response against a Neisseria meningitidis serogroup B subfamily A strain and against a Neisseria meningitidis serogroup B subfamily strain B in a human, said method comprising administering to the human an effective amount of the composition according to C77.

C104. A method for inducing a bactericidal immune response against a strain of Neisseria meningitidis serogroup A, Neisseria meningitidis serogroup C, Neisseria meningitidis serogroup W and/or Neisseria meningitidis serogroup Y in a human, said method comprising administering to the human an effective amount of the composition according to C1.

C105. A method of inducing a bactericidal immune response against a strain of Neisseria meningitidis serogroup A, Neisseria meningitidis serogroup C, Neisseria meningitidis serogroup W and/or Neisseria meningitidis serogroup Y in a human, said method comprising administering to the human an effective amount of the composition according to C68.

C106. A method of inducing a bactericidal immune response against a strain of Neisseria meningitidis serogroup A, Neisseria meningitidis serogroup C, Neisseria meningitidis serogroup W and/or Neisseria meningitidis serogroup Y in a human, said method comprising administering to the human an effective amount of the composition according to C77.

C107. A method for inducing a bactericidal immune response against a strain of Neisseria meningitidis serogroup A, Neisseria meningitidis serogroup B, Neisseria meningitidis serogroup C, Neisseria meningitidis serogroup W and/or Neisseria meningitidis serogroup Y in a human, said method comprising administering to a human an effective amount of the composition in accordance with paragraph C1.

C108. A method for inducing a bactericidal immune response against a strain of Neisseria meningitidis serogroup A, Neisseria meningitidis serogroup B, Neisseria meningitidis serogroup C, Neisseria meningitidis serogroup W and/or Neisseria meningitidis serogroup Y in a human, said method comprising administering to a human an effective amount of the composition in accordance with paragraph C68.

C109. A method for inducing a bactericidal immune response against a strain of Neisseria meningitidis serogroup A, Neisseria meningitidis serogroup B, Neisseria meningitidis serogroup C, Neisseria meningitidis serogroup W and/or Neisseria meningitidis serogroup Y in a human, said method comprising administering to a human an effective amount of the composition in accordance with paragraph C77.

C110. A method for inducing a bactericidal immune response against a strain of Neisseria meningitidis serogroup A, Neisseria meningitidis serogroup B, Neisseria meningitidis serogroup C, Neisseria meningitidis serogroup W, Neisseria meningitidis serogroup Y, and Neisseria meningitidis serogroup X in a human, said method comprising administering to a person an effective amount of the composition in accordance with paragraph C1.

C111. A method for inducing a bactericidal immune response against a strain of Neisseria meningitidis serogroup A, Neisseria meningitidis serogroup B, Neisseria meningitidis serogroup C, Neisseria meningitidis serogroup W, Neisseria meningitidis serogroup Y, and Neisseria meningitidis serogroup X in a human, said method comprising administering to a person an effective amount of the composition according to paragraph C68.

C112. A method for inducing a bactericidal immune response against a strain of Neisseria meningitidis serogroup A, Neisseria meningitidis serogroup B, Neisseria meningitidis serogroup C, Neisseria meningitidis serogroup W, Neisseria meningitidis serogroup Y, and Neisseria meningitidis serogroup X in a human, said method comprising administering to a person an effective amount of the composition according to paragraph C77.

C113. A method for inducing a bactericidal immune response against a strain of Neisseria meningitidis serogroup A, Neisseria meningitidis serogroup B, Neisseria meningitidis serogroup C, Neisseria meningitidis serogroup W, Neisseria meningitidis serogroup Y, and Neisseria meningitidis serogroup X in a human, said method comprising administering to a person an effective amount of the composition according to paragraph C98.

C114. A method of inducing a bactericidal immune response against a N. meningitidis serogroup C strain expressing factor H binding protein A10 in a human, the method comprising administering to a human a composition comprising a first lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1; a second lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2.

C115. A method for inducing a bactericidal immune response against a N. meningitidis serogroup W strain expressing factor H binding protein A10 in a human, wherein said method includes administering to a human a composition containing a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1.

C116. A method for inducing a bactericidal immune response against a N. meningitidis serogroup W strain expressing factor H binding protein A19 in a human, said method comprising administering to a human a composition containing a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1.

C117. A method for inducing a bactericidal immune response against a N. meningitidis strain of serogroup A expressing B16 protein that binds to factor H in a human, wherein the method includes administering to a human a composition containing a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2.

C118. A method for inducing a bactericidal immune response against a strain of N. meningitidis of serogroup Y expressing the B47 protein that binds to factor H in a human, wherein said method includes administering to a human a composition containing a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2.

C119. A method for inducing a bactericidal immune response against a strain of N. meningitidis serogroup C expressing factor H binding protein A10 in a human, said method comprising administering to a human a composition comprising a first lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1; the second lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO:2.

C120. A method for inducing a bactericidal immune response against a strain of N. meningitidis serogroup W expressing factor H binding protein A10 in a human, said method comprising administering to a human a composition comprising a first lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1; the second lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO:2.

C121. A method for inducing a bactericidal immune response against a N. meningitidis serogroup W strain expressing factor H binding protein A19 in a human, the method comprising administering to a human a composition comprising a first lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1; the second lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO:2.

C122. A method for inducing a bactericidal immune response against a strain of N. meningitidis serogroup A expressing factor H binding protein B16 in a human, said method comprising administering to a human a composition comprising a first lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1; the second lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO:2.

C123. A method of inducing a bactericidal immune response against a strain of N. meningitidis serogroup Y expressing the B47 factor H binding protein in a human, the method comprising administering to a human a composition comprising a first lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1; the second lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO:2.

C124. A method for inducing a bactericidal immune response against a strain of Neisseria meningitidis serogroup X in a human, comprising administering to a human an effective amount of a polypeptide that is at least 70% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 , SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO : 22, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32 , SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO : 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62.

C125. A method for inducing a bactericidal immune response against a strain of Neisseria meningitidis serogroup X in a human, comprising administering to the human an effective amount of the composition according to C1.

C126. A method of inducing a bactericidal immune response against a Neisseria meningitidis serogroup X strain in a human, comprising administering to the human an effective amount of a composition according to C68.

C127. A method of inducing a bactericidal immune response against a Neisseria meningitidis serogroup X strain in a human, comprising administering to the human an effective amount of the composition according to C77.

C128. A method of inducing a bactericidal immune response against a strain of Neisseria meningitidis serogroup X in a human, comprising administering to the human an effective amount of a composition according to C98.

C129. A method of inducing a bactericidal immune response against a Neisseria meningitidis serogroup X strain expressing factor H binding protein B49 in a human, said method comprising administering to a human a composition according to C98.

C130. A method for inducing a bactericidal immune response against a strain of N. meningitidis serogroup X expressing the B49 factor H binding protein in a human, the method comprising administering to a human a composition comprising a first lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1; the second lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO:2.

C131. The method according to any one of C101-C130, wherein the composition induces a bactericidal serum immunoglobulin titer that is at least 2 times the human serum immunoglobulin bactericidal titer after the first dose, as compared to the bactericidal human serum immunoglobulin titer before the first dose, as was determined under identical conditions in a serum bactericidal assay using human complement.

C132. The method according to any one of C101-C130, wherein the composition induces a bactericidal serum immunoglobulin titer that is at least 4 times the human serum immunoglobulin bactericidal titer after the first dose as compared to the bactericidal human serum immunoglobulin titer before the first dose, as was determined under identical conditions in a serum bactericidal assay using human complement.

C133. The method according to any one of C101-C130, wherein the composition induces a bactericidal serum immunoglobulin titer that is at least 8 times the human serum immunoglobulin bactericidal titer after the first dose, as compared to the bactericidal human serum immunoglobulin titer before the first dose, as was determined under identical conditions in a serum bactericidal assay using human complement.

C134. The method according to any one of C101-C132, wherein the age of the patient is 12 to <18 months or 18 to <24 months.

C135. The method according to any one of C101-C132, wherein the patient is 18 to <24 months of age.

C136. The method according to any one of C101-C132, wherein the age of the patient is ≥24 months to <10 years.

C137. A method for generating an immune response in a patient, comprising: (a) administering an immunogenic composition to a patient aged 12 to 18 months, wherein said immunogenic composition comprises a first lipidized polypeptide containing the amino acid sequence shown in SEQ ID NO: 1; and a second lipidized polypeptide containing the amino acid sequence shown in SEQ ID NO:2.

C138. A method for generating an immune response in a patient, comprising: (a) administering an immunogenic composition to a patient aged 18 to 24 months, wherein said immunogenic composition comprises a first lipidized polypeptide containing the amino acid sequence shown in SEQ ID NO: 1; and a second lipidized polypeptide containing the amino acid sequence shown in SEQ ID NO:2.

C139. A method for generating an immune response in a patient, comprising: (a) administering an immunogenic composition to a patient aged 24 months to 10 years, wherein said immunogenic composition comprises a first lipidized polypeptide containing the amino acid sequence shown in SEQ ID NO: 1; and a second lipidized polypeptide containing the amino acid sequence shown in SEQ ID NO:2.

C140. The method according to any one of C137-C139, wherein the composition also contains polysorbate-80, aluminum, histidine, and sodium chloride.

Claims (15)

1. An immunogenic composition capable of inducing an immune response against a strain of Neisseria meningitidis serogroup A, Neisseria meningitidis serogroup C, Neisseria meningitidis serogroup W and Neisseria meningitidis serogroup Y in humans, comprising: a) a liquid composition containing: (i) the first lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1; and (ii) a second lipidized polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 and aluminum; and b) a lyophilized composition comprising: i) Neisseria meningitidis serogroup A (MenA) capsular saccharide conjugated to an adipic acid dihydrozide (ADH) linker via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, wherein said linker is conjugated to tetanus toxoid (TT) by carbodiimide chemistry (MenAAH-TT conjugate); ii) Neisseria meningitidis serogroup C (MenC) capsular saccharide conjugated to an ADH linker via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate, wherein said linker is conjugated to tetanus toxoid (TT) via a carbodiimide chemical method (MenCAH-TT conjugate); iii) Neisseria meningitidis serogroup W135 (MenW) capsular saccharide directly conjugated to tetanus toxoid (TT) via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate in the absence of a linker (MenW-TT conjugate); and iv) Neisseria meningitidis serogroup Y (MenY) capsular saccharide directly conjugated to tetanus toxoid (TT) via 1-cyano-4-dimethylaminopyridinium tetrafluoroborate in the absence of a linker (MenY-TT conjugate), wherein the lyophilized composition is diluted with the liquid composition. 2. The immunogenic composition according to claim 1, wherein the aluminum contains aluminum phosphate. 3. An immunogenic composition according to claim 1, wherein the composition also contains polysorbate-80. 4. The immunogenic composition according to claim 1, wherein the composition does not contain aluminum. 5. The immunogenic composition of claim 1, wherein the first polypeptide and the second polypeptide bind to aluminum. 6. An immunogenic composition according to claim 1, wherein the composition also contains tris-HCl, sodium chloride, sucrose, L-histidine, polysorbate 80, and aluminum phosphate. 7. The immunogenic composition of claim 1, wherein the concentration of aluminum-bound polypeptides in the immunogenic composition is reduced by a maximum of 10% after 24 hours compared to the corresponding concentration of aluminum-bound polypeptides in the liquid composition prior to reconstitution of the lyophilized composition. 8. The immunogenic composition of claim 1, wherein the concentration of the MenAAH-TT conjugate in the immunogenic composition decreased by a maximum of 10% after 24 hours compared to the concentration of the MenAAH-TT conjugate in the lyophilized composition. 9. The immunogenic composition of claim 1, wherein the concentration of the MenCAH-TT conjugate in the immunogenic composition decreased by a maximum of 10% after 24 hours compared to the concentration of the MenCAH-TT conjugate in the lyophilized composition. 10. The immunogenic composition of claim 1, wherein the concentration of the MenW-TT conjugate in the immunogenic composition is reduced by a maximum of 10% after 24 hours compared to the concentration of the MenW-TT conjugate in the lyophilized composition. 11. The immunogenic composition of claim 1, wherein the concentration of the MenY-TT conjugate in the immunogenic composition decreased by a maximum of 10% after 24 hours compared to the concentration of the MenY-TT conjugate in the lyophilized composition. 12. A method of inducing an immune response against a strain of Neisseria meningitidis serogroup A, Neisseria meningitidis serogroup C, Neisseria meningitidis serogroup W, and Neisseria meningitidis serogroup Y in a human, wherein said method comprises administering to a human an effective amount of a composition according to any one of claims. 1-11. 13. The method of claim 12 wherein the age of the patient is 12 to <18 months or 18 to <24 months. 14. The method of claim 12 wherein the patient is 18 to <24 months of age. 15. The method of claim 12 wherein the age of the patient is 24 months to <10 years.

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