HK1188385B - Non-lipidated variants of neisseria meningitidis orf2086 antigens - Google Patents

Description

Non-lipidated variants of neisseria meningitidis ORF2086 antigen

This application claims priority from U.S. provisional application No. 61/381,837, filed on 9/10/2010, which is hereby incorporated by reference in its entirety.

Technical Field

The present invention relates to non-lipidated variants of neisseria meningitidis (neisserial meningitidis) ORF2086 antigens in immunogenic compositions as described herein. The invention also relates to methods of maintaining the conformation of a non-lipidated variant of neisseria meningitidis ORF2086 antigen. The invention further includes compositions and methods relating to improved expression of non-lipidated neisseria meningitidis ORF2086 antigens, as compared to the corresponding wild-type antigen.

Background

rLP2086 is a recombinant 28-kDa lipoprotein that induces cross-reactive bacterial antibodies against a number of neisseria meningitidis strains, including neisseria meningitidis serotype b (mnb) strains or, more precisely, serogroup b (mnb) strains. Based on deduced amino acid sequence homology, 2 different rLP2086 subfamilies a and B were identified. The 2 subfamilies were used to formulate 20, 60, 120 and 200 μ g/mL MnB-rLP2086 vaccine samples in 10mM histidine (pH6.0), 150mM NaCl and 0.5mg/mL aluminum and varying amounts of polysorbate 80 (PS-80). Native LP2086 is a lipoprotein. Fletcher et al, Infection & Immunity, Vol.72 (4):2088-2100(2004) demonstrated that in mice, rLP2086 with an amino-terminal lipid was more immunogenic than the non-lipidated form of the same protein. Other preclinical and clinical studies have demonstrated that the combination of these two lipidated proteins can provide a broad range of action (coverage) across the fHBP family. Neisseria meningitidis (meningococcal meningitis) is a devastating disease that kills children and adolescents within hours, regardless of the availability of antibiotics. There remains a need for suitable immunogenic compositions of serogroup B neisseria meningitidis.

Summary of The Invention

To meet these and other needs for neisseria meningitidis vaccines, other compositions have been evaluated to provide a range of action using non-lipidated variants of neisseria meningitidis ORF2086 polypeptides. A first aspect of the invention provides an immunogenic composition comprising a non-lipidated ORF2086 protein, wherein the ORF2086 protein is a B44, B02, B03, B22, B24, B09, a05, a04, a12, or a22 variant. In some embodiments, the ORF2086 protein is a B44, B22, B09, a05, a12, or a22 variant.

Another aspect of the invention provides an immunogenic composition comprising a non-lipidated ORF2086 protein subfamily B variant (P2086 subfamily B polypeptide). In some embodiments, the P2086 subfamily B polypeptide is a B44, B02, B03, B22, B24, or B09 variant. In some embodiments, the immunogenic composition further comprises a non-lipidated ORF2086 protein subfamily a variant (P2086 subfamily a polypeptide). In some embodiments, the P2086 subfamily a polypeptide is an a05, a04, a12, or a22 variant.

In some embodiments, the immunogenic composition further comprises an adjuvant. In some embodiments, the adjuvant is an aluminum adjuvant, a saponin (saponin), a CpG nucleotide sequence, or any combination thereof. In some embodiments, the aluminum adjuvant is AlPO₄、Al(OH)₃、Al₂(SO₄)₃Or alum. In some embodiments, the concentration of aluminum in the immunogenic composition is between 0.125 μ g/ml and 0.5 μ g/ml. In some embodiments, the concentration of aluminum in the immunogenic composition is 0.25 μ g/ml. In a preferred embodiment, the concentration of aluminum in the immunogenic composition is between 0.125mg/ml and 0.5 mg/ml. In some preferred embodiments, the concentration of aluminum in the immunogenic composition is 0.25 mg/ml.

In some embodiments, the concentration of saponin in the immunogenic composition is between 1 μ g/ml and 250 μ g/ml. In some embodiments, the concentration of saponin in the immunogenic composition is between 10 μ g/ml and 100 μ g/ml. In some embodiments, the concentration of saponin in the immunogenic composition is 10 μ g/ml. In some embodiments, immunogenic combinationsThe saponin concentration in the extract is 100 μ g/ml. In some embodiments, the saponin is QS-21(Agenus, Lexington, MA) or(CSLLimited,Parkville,Australia)。

In some embodiments, the immunogenic composition confers the ability to elicit an immunogenic response to neisseria meningitidis following administration of multiple doses of the immunogenic composition to a subject. In some embodiments, an immunogenic response is conferred after 2 doses are administered to the subject. In some embodiments, an immunogenic response is conferred after 3 doses are administered to the subject.

Another aspect of the invention provides a composition that confers increased immunogenicity to a non-lipidated P2086 antigen, wherein the composition comprises a saponin and at least one non-lipidated P2086 antigen. In some embodiments, the concentration of saponin in the immunogenic composition is between 1 μ g/ml and 250 μ g/ml. In some embodiments, the concentration of saponin in the immunogenic composition is between 10 μ g/ml and 100 μ g/ml. In some embodiments, the concentration of saponin in the immunogenic composition is 10 μ g/ml. In some embodiments, the concentration of saponin in the immunogenic composition is 100 μ g/ml. In some embodiments, the saponin is QS-21 or ISCOMATRIX.

In some embodiments, the composition further comprises aluminum. In some embodiments, the aluminum is AlPO₄、Al(OH)₃、Al₂(SO₄)₃Or alum. In some embodiments, the concentration of aluminum in the composition is between 0.125 μ g/ml and 0.5 μ g/ml. In some embodiments, the concentration of aluminum in the composition is 0.25 μ g/ml. In a preferred embodiment, the concentration of aluminum in the immunogenic composition is between 0.125mg/ml and 0.5 mg/ml. In some preferred embodiments, the concentration of aluminum in the immunogenic composition is 0.25mg/ml。

In some embodiments, the immunogenic composition confers the ability to elicit an immunogenic response to neisseria meningitidis following administration of multiple doses of the immunogenic composition to a subject. In some embodiments, an immunogenic response is conferred after 2 doses are administered to the subject. In some embodiments, an immunogenic response is conferred after 3 doses are administered to the subject.

In some embodiments, the non-lipidated P2086 antigen is a P2086 subfamily B polypeptide. In some embodiments, the P2086 subfamily B polypeptide is a B44, B02, B03, B22, B24, or B09 variant. In some embodiments, the non-lipidated P2086 antigen is a P2086 subfamily a polypeptide. In some embodiments, the P2086 subfamily a polypeptide is an a05, a04, a12, or a22 variant.

In some embodiments, the compositions comprise at least two non-lipidated P2086 antigens, wherein the two non-lipidated P2086 antigens are at least one non-lipidated P2086 subfamily a polypeptide and at least one non-lipidated P2086 subfamily B polypeptide. In some embodiments, the non-lipidated P2086 subfamily a polypeptide is an a05 variant and the non-lipidated P2086 subfamily B polypeptide is a B44 variant. In some embodiments, the non-lipidated P2086 subfamily a polypeptide is an a05 variant and the non-lipidated P2086 subfamily B polypeptide is a B22 variant. In some embodiments, the non-lipidated P2086 subfamily a polypeptide is an a05 variant and the non-lipidated P2086 subfamily B polypeptide is a B09 variant.

Another aspect of the invention provides a method of conferring immunity to a neisseria meningitidis bacterium in a subject, wherein the method comprises the step of administering to the subject an immunogenic composition comprising a non-lipidated P2086 subfamily B polypeptide. In some embodiments, the P2086 subfamily B polypeptide is a B44, B02, B03, B22, B24, or B09 variant. In some embodiments, the immunogenic composition further comprises a P2086 subfamily a polypeptide. In some embodiments, the P2086 subfamily a polypeptide is an a05, a04, a12, or a22 variant.

In some casesIn embodiments, the immunogenic composition further comprises an adjuvant. In some embodiments, the adjuvant is an aluminum adjuvant, a saponin, a CpG nucleotide sequence, or any combination thereof. In some embodiments, the aluminum adjuvant is AlPO₄、Al(OH)₃、Al₂(SO₄)₃Or alum. In some embodiments, the concentration of aluminum in the immunogenic composition is between 0.125 μ g/ml and 0.5 μ g/ml. In some embodiments, the concentration of aluminum in the immunogenic composition is 0.25 μ g/ml. In a preferred embodiment, the concentration of aluminum in the immunogenic composition is between 0.125mg/ml and 0.5 mg/ml. In some embodiments, the concentration of aluminum in the immunogenic composition is 0.25 mg/ml.

In some embodiments, the concentration of saponin in the immunogenic composition is between 1 μ g/ml and 250 μ g/ml. In some embodiments, the concentration of saponin in the immunogenic composition is between 10 μ g/ml and 100 μ g/ml. In some embodiments, the concentration of saponin in the immunogenic composition is 10 μ g/ml. In some embodiments, the concentration of saponin in the immunogenic composition is 100 μ g/ml. In some embodiments, the saponin is QS-21 or ISCOMATRIX.

In some embodiments, the immunogenic composition is administered to the subject in multiple doses in a dosing regimen. In some embodiments, the immunogenic composition is administered to the subject in 2 doses in a dosing regimen. In some embodiments, the immunogenic composition is administered to the subject in 3 doses in a dosing regimen.

Another aspect of the invention provides a method of producing a non-lipidated P2086 variant, comprising the steps of: (a) cloning the ORF2086 variant nucleic acid into an expression vector to produce an ORF2086 expression vector; (b) transforming a bacterium with the OFR2086 expression vector; (c) inducing expression of a P2086 variant from the ORF2086 expression vector; and (d) isolating the expressed P2086 variant protein; wherein the ORF2086 expression vector does not comprise lipidation control sequences. In some embodiments, the bacterium is escherichia coli (e. In some embodiments, expression is induced by the addition of IPTG.

In some embodiments, the codon encoding the N-terminal Cys of the P2086 variant is deleted. In some embodiments, the codon encoding the N-terminal Cys of the P2086 variant is mutated to produce an Ala, Gly, or Val codon. In some embodiments, the P2086 variant is an a05, B01, or B44 variant. In some embodiments, the P2086 variant is a B09 variant.

In some embodiments, the N-terminal tail is mutated to add Ser and Gly residues to extend the Gly/Ser stalk (talk) immediately downstream of the N-terminal Cys. In some embodiments, the total number of Gly and Ser residues in the Gly/Ser stem is at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12.

In some embodiments, the codons of the N-terminal tail of the P2086 variant are optimized by point mutagenesis. In some embodiments, the codon of the N-terminal tail of the ORF2086 variant is optimized by point mutagenesis such that the codon encoding the 5 th amino acid of the ORF2086 variant is 13-15100% identical to nucleotides of seq id No. 8 and the codon encoding the 13 th amino acid of the ORF2086 variant is 37-39100% identical to nucleotides of seq id No. 8. In some embodiments, the N-terminal tail of the non-lipidated ORF2086 variant is optimized to make the 5'45 nucleic acids 1-45100% identical to the nucleic acids of seq id No. 8. In some embodiments, the N-terminal tail of the non-lipidated ORF2086 variant is optimized to make the 5'42 nucleic acids 4-45100% identical to the nucleic acids of seq id No. 8. In some embodiments, the N-terminal tail of the non-lipidated ORF2086 variant is optimized such that the 5'39 nucleic acids are 4-42100% identical to the nucleic acids of seq id No. 8. In some embodiments, the N-terminal tail of the non-lipidated P2086 variant comprises at least one amino acid substitution compared to amino acids 1-15 of seq id No. 18. In some embodiments, the N-terminal tail of the non-lipidated P2086 variant comprises two amino acid substitutions as compared to amino acids 1-15 of seq id No. 18. In some embodiments, the N-terminal tail of the non-lipidated P2086 variant comprises at least one amino acid substitution compared to amino acids 2-15 of seq id No. 18. In some embodiments, the N-terminal tail of the non-lipidated P2086 variant comprises two amino acid substitutions as compared to amino acids 2-15 of seq id No. 18. In some embodiments, the amino acid substitution is a conservative amino acid substitution.

In one embodiment, the invention relates to a stable formulation of neisseria meningitidis ORF2086 subfamily B antigen in the form of an immunogenic composition. The invention also relates to methods of maintaining the conformation of neisseria meningitidis ORF2086 antigens and methods of determining the potency of neisseria meningitidis rLP2086 antigens.

In one aspect, the invention relates to a composition comprising an isolated non-pyruvylated non-lipidated ORF2086 polypeptide. In one embodiment, the composition is an immunogenic composition. In another embodiment, the polypeptide includes a deletion of an N-terminal Cys compared to a corresponding wild-type non-lipidated ORF2086 polypeptide. In one embodiment, the polypeptide comprises an amino acid sequence selected from the group consisting of: 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 and SEQ ID NO. 21, wherein the cysteine at position 1 is deleted. In another embodiment, the polypeptide comprises an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 44, SEQ ID NO. 49, SEQ ID NO. 50 and SEQ ID NO. 55.

In another embodiment, the polypeptide is encoded by a nucleotide sequence operably linked to an expression system, wherein the expression system is capable of expression in a bacterial cell. In one embodiment, the expression system is a plasmid expression system. In one embodiment, the bacterial cell is an escherichia coli cell. In another embodiment, the nucleotide sequence is linked to a regulatory sequence that controls the expression of the nucleotide sequence.

In another aspect, the invention relates to a composition comprising a non-pyruvylated non-lipidated ORF2086 polypeptide obtainable by a method. The method comprises expressing a nucleotide sequence encoding a polypeptide having an amino acid sequence selected from the group consisting of: 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21, wherein the cysteine at position 1 is deleted, wherein the nucleotide sequence is operably linked to an expression system capable of being expressed in bacterial cells. In one embodiment, the bacterial cell is E.coli.

In one aspect, the invention relates to a composition comprising an isolated polypeptide comprising the amino acid sequence set forth in SEQ ID NO. 49 and an isolated polypeptide comprising the amino acid sequence set forth in SEQ ID NO. 44. In one embodiment, the compositions described herein are immunogenic compositions. In another embodiment, the compositions described herein further include an ORF2086 subfamily a polypeptide from serogroup B neisseria meningitidis. In another embodiment, the compositions described herein elicit a bactericidal immune response in a mammal against an ORF2086 subfamily B polypeptide from serogroup B neisseria meningitidis.

In one aspect, the invention relates to an isolated polypeptide comprising the amino acid sequence shown in SEQ ID NO. 49. In another aspect, the invention relates to an isolated nucleotide sequence comprising SEQ ID NO. 46. In one aspect, the invention relates to an isolated nucleotide sequence comprising SEQ ID NO. 47. In one aspect, the invention relates to an isolated nucleotide sequence comprising SEQ ID NO. 48. In one aspect, the invention relates to an isolated polypeptide comprising the amino acid sequence shown in SEQ ID NO. 50. In one aspect, the invention relates to an isolated nucleotide sequence comprising SEQ ID NO. 45. In one aspect, the invention relates to an isolated polypeptide comprising the amino acid sequence shown in SEQ ID NO. 44.

In one aspect, the invention relates to a plasmid comprising a nucleotide sequence selected from the group consisting of seq id No. 46, seq id No. 47, seq id No. 48 and seq id No. 45, wherein the plasmid is capable of being expressed in a bacterial cell. In one embodiment, the bacterial cell is E.coli.

In one aspect, the invention relates to a method of eliciting in a mammal bactericidal antibodies specific for ORF2086 subfamily B serogroup B neisseria meningitidis. The method comprises administering to the mammal an effective amount of an isolated polypeptide comprising an amino acid sequence selected from the group consisting of seq id No. 44 and seq id No. 49, or a combination thereof.

In one aspect, the invention relates to a method of producing a polypeptide. The method comprises expressing in a bacterial cell a polypeptide comprising a sequence having greater than 90% identity to seq id No. 21, the sequence comprising at least one domain selected from the group consisting of: amino acids 13-18 of SEQ ID NO. 21, amino acids 21-34 of SEQ ID NO. 21 and amino acids 70-80 of SEQ ID NO. 21 or combinations thereof, wherein the sequence lacks the N-terminal cysteine. The method further comprises purifying the polypeptide. In one embodiment, the sequence further comprises at least one domain selected from the group consisting of: amino acids 96-116 of SEQ ID NO. 21, amino acids 158-170 of SEQ ID NO. 21, amino acids 172-185 of SEQ ID NO. 21, amino acids 187-199 of SEQ ID NO. 21, amino acids 213-224 of SEQ ID NO. 21, amino acids 226-237 of SEQ ID NO. 21, amino acids 239-248 of SEQ ID NO. 21, or combinations thereof. In one embodiment, the bacterial cell is E.coli.

In one aspect, the invention relates to an isolated polypeptide produced by a method comprising the methods described herein. In another aspect, the invention relates to an immunogenic composition produced by a method comprising the methods described herein.

In one aspect, the invention relates to an immunogenic composition comprising an ORF2086 subfamily B polypeptide from serogroup B neisseria meningitidis, wherein the polypeptide is non-pyruvylated non-lipidated B44. In one embodiment, the composition further comprises a second ORF2086 subfamily B polypeptide from serogroup B neisseria meningitidis, wherein the second polypeptide is non-pyruvylated non-lipidated B09. In one embodiment, the composition includes up to 3 ORF2086 subfamily B polypeptides. In another embodiment, the composition includes up to two ORF2086 subfamily B polypeptides. In one embodiment, the composition further comprises an ORF2086 subfamily a polypeptide. In another embodiment, a composition includes an a05 subfamily a polypeptide.

Brief Description of Drawings

FIG. 1: a P2086 variant nucleic acid sequence.

FIG. 2: p2086 variant amino acid sequence. The Gly/Ser stem in the N-terminal tail of each variant is underlined.

FIG. 3: structure of ORF2086 protein.

FIG. 4: removal of the N-terminal Cys resulted in loss of expression in e.

FIG. 5: effect of Gly/Ser stem length on non-lipidated ORF2086 variant expression. The sequence related to the protein variant labeled B01 is shown as SEQ ID NO 35. The sequence related to the protein variant labeled B44 is shown as SEQ ID NO 36. The sequence related to the protein variant labeled A05 is shown as SEQ ID NO 37. The sequence related to the protein variant labeled A22 is shown as SEQ ID NO 38. The sequence related to the protein variant labeled B22 is shown as SEQ ID NO 39. The sequence related to the protein variant labeled A19 is shown as SEQ ID NO 40.

FIG. 6: the expression of non-lipidated B09 was higher despite the shorter Gly/Ser stalk. The left 2 lanes illustrate the expression of the N-terminal Cys deletion B09 variant before and after induction. Lanes 3 and 4 illustrate the expression of the N-terminal Cys-positive B09 variant before and after induction. The rightmost lane is the molecular weight standard. The amino acid sequence shown below the image is shown as SEQ ID NO: 41. The nucleotide sequence representing the N-terminal Cys deletion A22 variant (referred to in the figure as "A22 _ 001") is shown as SEQ ID NO:42, with SEQ ID NO:42 being shown in the figure below SEQ ID NO: 41. The nucleotide sequence representing the N-terminal Cys deletion B22 variant (referred to in the figure as "B22 _ 001") is shown as SEQ ID NO: 52. The nucleotide sequence representing the N-terminal Cys deletion B09 variant (referred to in the figure as "B09 _ 004") is shown as SEQ ID NO: 53.

FIG. 7: codon optimization increased expression of the non-lipidated B22 and a22 variants. The left panel illustrates expression of the N-terminal Cys deletion B22 variant before (lanes 1 and 3) and after (lanes 2 and 4) IPTG induction. The right panel illustrates the expression of the N-terminal Cys deletion a22 variant before (lane 7) and after IPTG induction (lane 8). Lanes 5 and 6 are molecular weight standards.

FIG. 8: p2086 variant nucleic acid and amino acid sequences.

Serial number

SEQ ID NO 1 shows the DNA sequence of the Neisseria meningitidis serogroup B2086 variant A04 gene, including the codon encoding the N-terminal Cys.

SEQ ID NO 2 shows the DNA sequence of the Neisseria meningitidis serogroup B2086 variant A05 gene, including the codon encoding the N-terminal Cys.

SEQ ID NO 3 shows the DNA sequence of the Neisseria meningitidis serogroup B2086 variant A12 gene, including the codon encoding the N-terminal Cys.

SEQ ID NO. 4 shows the DNA sequence of the Neisseria meningitidis serogroup B2086 variant A12-2 gene, including the codon encoding the N-terminal Cys.

SEQ ID NO. 5 shows the DNA sequence of the Neisseria meningitidis serogroup B2086 variant A22 gene, including the codon encoding the N-terminal Cys.

SEQ ID NO 6 shows the DNA sequence of the Neisseria meningitidis serogroup B2086 variant B02 gene, including the codon encoding the N-terminal Cys.

SEQ ID NO 7 shows the DNA sequence of the Neisseria meningitidis serogroup B2086 variant B03 gene, including the codon encoding the N-terminal Cys.

SEQ ID NO 8 shows the DNA sequence of the Neisseria meningitidis serogroup B2086 variant B09 gene, including the codon encoding the N-terminal Cys.

SEQ ID NO 9 shows the DNA sequence of the Neisseria meningitidis serogroup B2086 variant B22 gene, including the codon encoding the N-terminal Cys.

SEQ ID NO 10 shows the DNA sequence of the Neisseria meningitidis serogroup B2086 variant B24 gene, including the codon encoding the N-terminal Cys.

SEQ ID NO 11 shows the DNA sequence of the Neisseria meningitidis serogroup B2086 variant B44 gene, including the codon encoding the N-terminal Cys.

SEQ ID NO. 12 shows the amino acid sequence of N.meningitidis serogroup B2086 variant A04, including an N-terminal Cys at amino acid position 1.

SEQ ID NO 13 shows the amino acid sequence of N.meningitidis serogroup B2086 variant A05, including an N-terminal Cys at amino acid position 1.

SEQ ID NO. 14 shows the amino acid sequence of N.meningitidis serogroup B2086 variant A12, including an N-terminal Cys at amino acid position 1.

SEQ ID NO. 15 shows the amino acid sequence of N.meningitidis serogroup B2086 variant A22, which includes an N-terminal Cys at amino acid position 1.

Seq id No. 16 shows the amino acid sequence of neisseria meningitidis serogroup B2086 variant B02, including an N-terminal Cys at amino acid position 1.

SEQ ID NO 17 shows the amino acid sequence of N.meningitidis serogroup B2086 variant B03, which includes an N-terminal Cys at amino acid position 1.

SEQ ID NO 18 shows the amino acid sequence of N.meningitidis serogroup B2086 variant B09, which includes an N-terminal Cys at amino acid position 1.

Seq id No. 19 shows the amino acid sequence of neisseria meningitidis serogroup B2086 variant B22, including an N-terminal Cys at amino acid position 1.

SEQ ID NO 20 shows the amino acid sequence of N.meningitidis serogroup B2086 variant B24, which includes an N-terminal Cys at amino acid position 1.

Seq id No. 21 shows the amino acid sequence of neisseria meningitidis serogroup B2086 variant B44, including an N-terminal Cys at amino acid position 1.

SEQ ID NO. 22 shows the DNA sequence of the forward primer shown in example 2.

SEQ ID NO. 23 shows the DNA sequence of the reverse primer shown in example 2.

SEQ ID NO. 24 shows the DNA sequence of the forward primers shown in Table 1 of example 2.

SEQ ID NO. 25 shows the DNA sequence of the reverse primer shown in Table 1 of example 2.

SEQ ID NO. 26 shows the DNA sequence of the forward primers shown in Table 1 of example 2.

SEQ ID NO. 27 shows the DNA sequence of the reverse primer shown in Table 1 of example 2.

SEQ ID NO 28 shows the DNA sequence of Gly/Ser stem shown in example 4.

SEQ ID NO:29 shows the amino acid sequence of Gly/Ser stem shown in example 4, which is encoded by, for example, SEQ ID NO: 28.

SEQ ID NO 30 shows the DNA sequence of Gly/Ser stem shown in example 4.

SEQ ID NO:31 shows the amino acid sequence of Gly/Ser stem shown in example 4, which is encoded by, for example, SEQ ID NO: 30.

SEQ ID NO 32 shows the DNA sequence of Gly/Ser stem shown in example 4.

SEQ ID NO 33 shows the amino acid sequence of Gly/Ser stem, which is encoded by, for example, SEQ ID NO 32 and SEQ ID NO 34.

SEQ ID NO 34 shows the DNA sequence of Gly/Ser stem shown in example 4.

SEQ ID NO 35 shows the N-terminal amino acid sequence of the N-terminus of the N.meningitidis serogroup B2086 variant B01 shown in FIG. 5.

SEQ ID NO 36 shows the N-terminal amino acid sequence of the N-terminus of the N.meningitidis serogroup B2086 variant B44 shown in FIG. 5.

SEQ ID NO 37 shows the N-terminal amino acid sequence of the N-terminus of N.meningitidis serogroup B2086 variant A05 shown in FIG. 5.

SEQ ID NO 38 shows the N-terminal amino acid sequence of the N-terminus of N.meningitidis serogroup B2086 variant A22 shown in FIG. 5.

SEQ ID NO 39 shows the N-terminal amino acid sequence of the N-terminus of the N.meningitidis serogroup B2086 variant B22 shown in FIG. 5.

SEQ ID NO 40 shows the N-terminal amino acid sequence of the N-terminus of the N.meningitidis serogroup B2086 variant A19 shown in FIG. 5.

SEQ ID NO:41 shows the N-terminal amino acid sequence of the N-terminal N-neisseria meningitidis serogroup B2086 variant shown in FIG. 6.

SEQ ID NO 42 shows the DNA sequence of the N-terminus of N.meningitidis serogroup B2086 variant A22 shown in FIG. 6. SEQ ID NO:43 shows a codon optimized DNA sequence of the Neisseria meningitidis serogroup B2086 variant B44 gene wherein the codon encoding the N-terminal cysteine has been deleted compared to SEQ ID NO: 11. Plasmid pDK087 includes SEQ ID NO: 43.

SEQ ID NO:44 shows the amino acid sequence of the non-pyruvylated non-lipidated Neisseria meningitidis serogroup B2086 variant B44. SEQ ID NO. 44 is identical to SEQ ID NO. 21, wherein the N-terminal cysteine at position 1 of SEQ ID NO. 21 is deleted. SEQID44 is encoded by, for example, SEQID NO: 43.

Seq id No. 45 shows a codon optimized DNA sequence of the neisseria meningitidis serogroup B2086 variant B09 gene, wherein the codon encoding the N-terminal cysteine has been deleted compared to seq id No. 8, and wherein the sequence comprises a codon encoding another Gly/Ser region. Plasmid pEB063 comprises SEQ ID NO: 45.

Seq id No. 46 shows a codon optimized DNA sequence of the neisseria meningitidis serogroup B2086 variant B09 gene wherein the codon encoding the N-terminal cysteine has been deleted compared to seq id No. 8. Plasmid pEB064 includes SEQ ID NO: 46.

Seq id No. 47 shows the codon optimized DNA sequence of the neisseria meningitidis serogroup B2086 variant B09 gene, wherein the codon encoding the N-terminal cysteine has been deleted compared to seq id No. 8. Plasmid pEB065 includes SEQ ID NO: 47.

SEQ ID NO 48 shows the DNA sequence of the Neisseria meningitidis serogroup B2086 variant B09 gene with the codon encoding the N-terminal cysteine deleted compared to SEQ ID NO 8. Plasmid pLA134 includes SEQ ID NO: 48.

SEQ ID NO. 49 shows the amino acid sequence of the non-pyruvylated non-lipidated Neisseria meningitidis serogroup B2086 variant B09. SEQ ID NO. 49 is identical to SEQ ID NO. 18, wherein the N-terminal cysteine at position 1 of SEQ ID NO. 18 is deleted. SEQ ID49 is encoded by a DNA sequence selected from the group consisting of, for example, SEQ ID NO 46, SEQ ID NO 47 and SEQ ID NO 48.

Seq id No. 50 shows the amino acid sequence of neisseria meningitidis serogroup B2086 variant B09, wherein the codon encoding the N-terminal cysteine has been deleted and wherein the sequence comprises a codon encoding another Gly/Ser region, as compared to seq id No. 18. SEQ ID NO. 50 is encoded by, for example, SEQ ID NO. 45.

SEQ ID NO:51 shows the DNA sequence of the Neisseria meningitidis serogroup B2086 variant B44 gene, with the codon encoding the N-terminal cysteine deleted compared to SEQ ID NO: 11. Plasmid pLN056 includes SEQ ID NO: 51.

SEQ ID NO 52 shows the N-terminal DNA sequence of the N-terminus of the N.meningitidis serogroup B2086 variant B22 shown in FIG. 6.

SEQ ID NO 53 shows the DNA sequence of the N-terminus of N.meningitidis serogroup B2086 variant B09 shown in FIG. 6.

SEQ ID NO:54 shows the DNA sequence of the Neisseria meningitidis serogroup B2086 variant A05 gene with the codon encoding the N-terminal cysteine deleted compared to SEQ ID NO: 2.

SEQ ID NO:55 shows the amino acid sequence of the non-pyruvylated non-lipidated Neisseria meningitidis serogroup B2086 variant A05. SEQ ID NO. 55 is identical to SEQ ID NO. 13, wherein the N-terminal cysteine at position 1 of SEQ ID NO. 13 is deleted. SEQ ID NO. 55 is encoded by, for example, SEQ ID NO. 54.

SEQ ID NO. 56 shows the amino acid sequence of the serine-glycine repeat sequence shown in example 7.

SEQ ID NO:57 shows the amino acid sequence of the non-pyruvylated non-lipidated Neisseria meningitidis serogroup B2086 variant B01. SEQ ID NO. 57 is identical to SEQ ID NO. 58, wherein the N-terminal cysteine at position 1 of SEQ ID NO. 58 is deleted.

SEQ ID NO:58 shows the amino acid sequence of N.meningitidis serogroup B2086 variant B01, which includes an N-terminal Cys at amino acid position 1.

SEQ ID NO 59 shows the amino acid sequence of N.meningitidis serogroup B2086 variant B15, which includes an N-terminal Cys at amino acid position 1.

Seq id No. 60 shows the amino acid sequence of neisseria meningitidis serogroup B2086 variant B16, including an N-terminal Cys at amino acid position 1.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patents, and other documents mentioned herein are incorporated by reference in their entirety.

It should be noted that, in the present invention, terms such as "comprising," "containing," and the like may have meanings assigned thereto in U.S. patent law; for example, it may mean "including" and the like. These terms are meant to include a particular component or group of components without excluding any other components. Terms such as "consisting essentially of …" have the meaning assigned thereto in U.S. patent law, for example, which allows for the inclusion of other ingredients or steps that do not detract from the novel or essential features of the invention, that is, which excludes other unrecited ingredients or steps that do detract from the novel or essential features of the invention, and which excludes ingredients or steps of the prior art (such as documents in the art that are cited or incorporated by reference herein), especially when this document is intended to define patentable (e.g., novel, nonobvious, inventive) embodiments as compared to the prior art, for example as compared to documents cited or incorporated by reference herein. Furthermore, the term "consisting of …" has the meaning assigned thereto in U.S. patent law; that is, these terms are closed terms. Thus, these terms are meant to include a particular component or group of components and exclude all other components.

Definition of

As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "the method" includes one or more methods and/or steps of the type described herein and/or which will become apparent to those of ordinary skill upon reading this disclosure, and so forth.

As used herein, the plural encompasses singular references unless the context clearly dictates otherwise. Thus, for example, reference to "the method" includes one or more methods and/or steps of the type described herein and/or which will become apparent to those of ordinary skill upon reading this disclosure, and so forth.

As used herein, "about" means within a statistically significant range of a value, such as a specified concentration range, time range, molecular weight, temperature, or pH value. Such a range may be within a given value or an order of magnitude of a range, typically within 20%, more typically within 10%, and even more typically within 5%. The tolerance for coverage by the term "about" will depend on the particular system under study and can be readily appreciated by one of ordinary skill. Whenever a range is recited in this application, every integer within the range is also contemplated as an embodiment of the invention.

Term(s) forAn "adjuvant" refers to a compound or mixture that enhances an immune response to an antigen as further described and exemplified herein. Non-limiting examples of adjuvants that can be used in the vaccines of the present invention include RIBI adjuvant system (RibiInc., Hamilton, Mont.), alum, mineral gels (such as aluminum hydroxide gels), oil-in-water emulsions, water-in-oil emulsions (e.g., Freund's complete and incomplete adjuvants), block copolymers (CytRx, AtlantaGa.), QS-21(Cambridge Biotech Inc., Cambridge Mass.), SAF-M (Chiron, Emeryville Calif.)Saponin, QuilA or other saponin moieties, monophosphoryl lipid a, and Avridine lipid-amine adjuvants.

An "antibody" is an immunoglobulin molecule capable of specifically binding to a target such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, unless the context indicates otherwise, the term is intended to encompass not only intact polyclonal or monoclonal antibodies, but also engineered antibodies (e.g., chimeric, humanized and/or derivatized to alter effector function, stability and other biological activity) and fragments thereof (such as Fab, Fab ', F (ab')2, Fv, single chain (ScFv) and domain antibodies, including shark and camelid antibodies), and fusion proteins comprising an antibody portion, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity), and antibody fragments, and any other modified configuration of an immunoglobulin molecule comprising an antigen recognition site. Antibodies include antibodies of any class, such as IgG, IgA, or IgM (or subclasses thereof), and antibodies need not be of any particular class. Depending on the antibody amino acid sequence of the constant domain of the heavy chain of the immunoglobulin, the immunoglobulins can be assigned to different classes. There are 5 major immunoglobulin classes: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2 in humans. The heavy chain constant domains corresponding to different immunoglobulin classes are called α, γ and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

An "antibody fragment" comprises only a portion of an intact antibody, wherein the portion preferably retains at least one, preferably most or all of the functions normally associated with the portion when present in an intact antibody.

The term "antigen" generally refers to a biomolecule, typically a protein, peptide, polysaccharide, lipid or conjugate, containing at least one epitope to which a cognate antibody (cognatentabody) can selectively bind; or in some cases, to an immunogenic substance that can stimulate the production of antibodies or T cell responses or both in an animal, including compositions that are injected or absorbed into an animal. The immune response may be raised against the entire molecule or against one or more different parts of the molecule (e.g., an epitope or hapten). The term may be used to refer to individual molecules or homogeneous or heterogeneous populations of antigenic molecules. Antigens are recognized by antibodies, T cell receptors, or other components with specific humoral and/or cellular immunity. The term "antigen" includes all relevant antigenic epitopes. Epitopes of a given antigen can be identified using a number of epitope mapping techniques well known in the art. See, e.g., epitopemppingprotocolsinmolecular biology, volume 66 (glenn e. morris eds, 1996) HumanaPress, Totowa, n.j. For example, a linear epitope can be determined by, for example, the following methods: a plurality of peptides are simultaneously synthesized on a solid support, wherein the peptides correspond to portions of a protein molecule, and the peptides are reacted with an antibody while still attached to the support. Such techniques are known in the art and are described, for example, in U.S. Pat. nos. 4,708,871; geysen et al (1984) Proc.Natl.Acad.Sci.USA81: 3998-4002; geysen et al (1986) molecular. Immunol.23:709-715, all of which are incorporated herein by reference in their entirety. Similarly, conformational epitopes can be identified by determining the spatial configuration of amino acids, such as by x-ray crystallography and 2-dimensional nuclear magnetic resonance, for example. See, e.g., epitomepgingprotocols (supra). Furthermore, for the purposes of the present invention, "antigen" may also be used to refer to a protein that includes modifications, such as deletions, additions, and substitutions (typically substantially conserved, but which may be non-conserved) made to the native sequence, so long as the protein maintains the ability to elicit an immune response. These modifications may be artificial, e.g., via site-directed mutagenesis or via specific synthetic procedures, or via genetic engineering methods; or may be episodic, such as via mutation of the host producing the antigen. Furthermore, the antigen may be derived, obtained or isolated from a microorganism, such as a bacterium, or may be the entire organism. Similarly, this definition also includes oligonucleotides or polynucleotides that express antigens, such as in nucleic acid immunization applications. Synthetic antigens such as polyepitopes, flanking epitopes and other recombinantly or synthetically derived antigens are also included (Bergmann et al (1993) eur.j. immunol.23: 27772781; Bergmann et al (1996) j. immunol.157: 32423249; Suhrbier, a. (1997) Immunol, and cell biol.75: 402408; Gardner et al (1998) 12th world AIDS conference (12th world AIDS conference), Geneva, Switzerland,1998, from 28 th month to 7 th month 3 th).

The term "conservative" amino acid substitution may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, tryptophan, and methionine; polar/neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. In some embodiments, conservative amino acid changes alter the primary sequence of the ORF2086 polypeptide, but do not alter the function of the molecule. When producing these mutants, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biological function on a polypeptide is generally understood in the art (Kyte and Doolittle,1982, J.mol.biol.,157(1): 105-32). It is known that certain amino acids may be substituted for other amino acids having similar hydropathic indices or scores and still result in polypeptides of similar biological activity. Each amino acid has been assigned a hydrophilicity index based on its hydrophobicity and charge characteristics. These indices are: isoleucine (+ 4.5); valine (+ 4.2); leucine (+ 3.8); phenylalanine (+ 2.8); cysteine/cystine (+ 2.5); methionine (+ 1.9); alanine (+ 1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

It is believed that the relative hydrophilic character of the amino acid residues determines the secondary and tertiary structure of the resulting polypeptide, which in turn defines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and the like. It is known in the art that an amino acid may be substituted for another amino acid with a similar hydropathic index and still obtain functionally equivalent polypeptides. Of these variations, amino acid substitutions having a hydropathic index within +/-2 are preferred, amino acid substitutions having a hydropathic index within +/-1 are particularly preferred, and amino acid substitutions having a hydropathic index within +/-0.5 are more particularly preferred.

Conservative amino acid substitutions or insertions may also be made on the basis of hydrophilicity. As described in U.S. patent No. 4,554,101, which is hereby incorporated by reference herein, the maximum local average hydrophilicity of a polypeptide, as governed by the hydrophilicity of its adjacent amino acids, correlates with the immunogenicity and antigenicity of the polypeptide, i.e., with the biological properties of the polypeptide. U.S. Pat. No. 4,554,101 states that the following hydrophilicity values have been assigned to each amino acid residue: arginine (+ 3.0); lysine (+ 3.0); aspartic acid (+3.0 ± 1); glutamic acid (+3.0 ± 1); serine (+ 0.3); asparagine (+ 0.2); glutamine (+ 0.2); glycine (0); proline (-0.5 ± 1); threonine (-0.4); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It will be appreciated that an amino acid may be substituted for another amino acid having a similar hydrophilicity value and still obtain a biologically equivalent and in particular immunologically equivalent polypeptide. Among these changes, amino acid substitutions having hydrophilicity values within ± 2 are preferred; particularly preferred are amino acid substitutions having a hydrophilicity value within ± 1; and even more particularly amino acid substitutions having a hydrophilicity value within ± 0.5. Exemplary substitutions that take into account various of the above features are well known to those skilled in the art and include (without limitation): arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

The term "effective immunogenic amount" as used herein refers to an amount of a polypeptide or a composition comprising a polypeptide effective to elicit an immune response in a vertebrate host. For example, an effective immunogenic amount of an rLP2086 protein of the invention is an amount effective to elicit an immune response in a vertebrate host. The particular "effective immunogenic dose or amount" will depend on the age, weight and medical condition of the host, as well as the method of administration. Suitable dosages are readily determined by those skilled in the art.

The term "Gly/Ser stalk" as used herein refers to the series of Gly and Ser residues immediately downstream of the N-terminal Cys residue of the protein encoded by ORF 2086. 5 to 12 Gly and Ser residues may be present in the Gly/Ser stem. Thus, the Gly/Ser stalk consists of amino acids between position 2 and between 7 and 13 of the protein encoded by ORF 2086. Preferably, the Gly/Ser stalk consists of the amino acids at position 2 and up to between 7 and 13 of the protein encoded by ORF 2086. The Gly/Ser stalk of the P2086 variants of the invention is represented by the underlined sequence in FIG. 2(SEQ ID NOs: 12-21). As shown herein, the length of the Gly/Ser stalk can affect the stability or expression of the non-lipidated P2086 variant. In an exemplary embodiment, the effect produced by affecting the length of the Gly/Ser stalk is compared to the corresponding wild-type variant.

The term "immunogenic" refers to an antigen or vaccine that is capable of eliciting a humoral immune response or a cell-mediated immune response, or both.

An "immunogenic amount" or an "immunologically effective amount" or "dose," each of which is used interchangeably herein, generally refers to an amount of an antigen or immunogenic composition sufficient to elicit an immune response, a cellular (T cell) response or a humoral (B cell or antibody) response, or both, as measured by standard assays known to those of skill in the art.

The term "immunogenic composition" relates to any pharmaceutical composition containing an antigen (e.g. a microorganism) or a component thereof, which composition can be used to elicit an immune response in a subject. The immunogenic compositions of the invention are useful for treating humans susceptible to infection by neisseria meningitidis by means of administering said immunogenic compositions via systemic transdermal or transmucosal route. These administrations may include injection via intramuscular (i.m.), intraperitoneal (i.p.), intradermal (i.d.), or subcutaneous routes; administration via a patch or other transdermal delivery device; or transmucosal administration to the oral/digestive, respiratory or genitourinary tract. In one embodiment, the immunogenic composition can be used in the manufacture of a vaccine or to elicit polyclonal or monoclonal antibodies that can be used to passively protect or treat a subject.

The optimal amounts of the components of a particular immunogenic composition can be determined by standard studies involving observing the appropriate immune response in a subject. After the initial vaccination, the subject may receive one or several booster immunizations at appropriate intervals.

The term "isolated" means that the substance is removed from its original environment (e.g., from its natural environment (if it is naturally occurring) or from its host organism (if it is a recombinant entity), or from one environment to a different environment). For example, an "isolated" protein or peptide is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free of chemical precursors or other chemicals that are present in the mixture at the time of chemical synthesis or otherwise as part of a chemical reaction. In the present invention, the protein may be isolated from the bacterial cells or cell fragments so that it is provided in a form suitable for use in the manufacture of an immunogenic composition. The term "isolated" or "isolating" may include purification, including, for example, purification methods of a protein as described herein. The phrase "substantially free of cellular material" includes preparations of a polypeptide or protein in which the polypeptide or protein is separated from cellular components of the cell from which it is isolated or recombinantly produced. Thus, a protein or peptide that is substantially free of cellular material includes preparations of capsular polysaccharides, proteins or peptides that are less than about 30%, 20%, 10%, 5%, 2.5% or 1% (by dry weight) contaminating protein or polysaccharide or other cellular material. When the polypeptide/protein is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation. When the polypeptide or protein is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from the chemical precursors or other chemicals involved in the synthesis of the protein or polysaccharide. Thus, these preparations of polypeptide or protein contain less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the relevant polypeptide/protein or polysaccharide fragment.

The term "N-terminal tail" as used herein refers to the N-terminal portion of the protein encoded by ORF2086, which attaches the protein to the cell membrane. The N-terminal tail is shown at the bottom of the side view structure in fig. 3. The N-terminal tail typically comprises the N-terminal 16 amino acids of the protein encoded by ORF 2086. In some embodiments, the N-terminal tail is amino acids 1-16 of any one of SEQ ID NOs 12-21. The term "ORF 2086" as used herein refers to the open reading frame 2086 from a bacterium of the Neisseria (Neisseria) species. Neisserial ORF2086, proteins encoded thereby, fragments of such proteins, and immunogenic compositions comprising such proteins are known in the art and described, for example, in WO2003/063766 and U.S. patent application publication nos. US20060257413 and US20090202593, each of which is hereby incorporated by reference in its entirety.

The term "P2086" generally refers to the protein encoded by ORF 2086. The former "P" of "2086" is an abbreviation for "protein". The P2086 protein of the invention can be a lipidated or non-lipidated protein. "LP 2086" and "P2086" generally refer to lipidated and non-lipidated forms, respectively, of the 2086 protein. The P2086 protein of the invention can be a recombinant protein. "rLP 2086" and "rP 2086" generally refer to lipidated and non-lipidated forms, respectively, of the recombinant 2086 protein. "2086" is also known as factor H binding protein (fHBP) because of its ability to bind to factor H.

The term "pharmaceutically acceptable carrier" as used herein is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with administration to a human or other vertebrate host. Typically, a pharmaceutically acceptable carrier is one approved by a regulatory agency of the federal, a state government or other regulatory agency, or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans, as well as non-human mammals. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. These pharmaceutical carriers can be sterile liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water, physiological saline and dextrose aqueous solutions, and glycerol solutions are useful as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. If desired, the compositions may also contain minor amounts of wetting agents, bulking agents, emulsifying agents, or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsions, sustained release formulations, and the like. Examples of suitable pharmaceutical carriers are described in "Remington's pharmaceutical sciences" of e.w. martin. The formulation should be suitable for the mode of administration. Suitable carriers will be apparent to those skilled in the art and will depend in large part on the route of administration.

By "protective" immune response is meant that the immunogenic composition is capable of eliciting a humoral or cell-mediated immune response that is useful for protecting a subject from infection. The protection provided need not be absolute protection, i.e. the infection need not be completely prevented or eradicated, if statistically significantly improved compared to a control population of subjects, e.g. infected animals, to which the vaccine or immunogenic composition has not been administered. Protection may be limited to mitigating the severity or rapidity of onset of symptoms of infection. In general, a "protective immune response" includes inducing an increase in the amount of antibodies specific for a particular antigen in at least 50% of subjects, including some increase in the extent of measurable functional antibody responses to each antigen. In particular instances, a "protective immune response" can include inducing a 2-fold or 4-fold increase in the amount of antibody specific for a particular antigen in at least 50% of subjects, including some degree of increase in a measurable functional antibody response to each antigen. In certain embodiments, an opsonophagocytic antibody (opsoniningantibody) is associated with a protective immune response. Thus, a protective immune response can be determined by measuring the percent reduction in bacterial count in a Serum Bactericidal Activity (SBA) assay or an opsonophagocytosis assay, such as the following assays. These assays are also known in the art. For neisseria meningitidis vaccines, for example, the SBA assay is an established surrogate for protection. In some embodiments, the bacterial count is reduced by at least 10%, 25%, 50%, 65%, 75%, 80%, 85%, 90%, 95%, or 95% or more compared to the bacterial count in the absence of the immunogenic composition.

The terms "protein", "polypeptide" and "peptide" refer to a polymer of amino acid residues and are not limited to the minimum length of the product. Thus, peptides, oligopeptides, dimers, multimers, and the like are included within this definition. Both full-length proteins and fragments thereof are encompassed by this definition. The terms also include modifications to the native sequence, such as deletions, additions and substitutions (typically substantially conservative, but which may be non-conservative), preferably such that the protein maintains the ability to elicit an immune response in the animal to which the protein is administered. Post-expression modifications such as glycosylation, acetylation, lipidation, phosphorylation, and the like are also included.

The term "recombinant" as used herein refers to any protein, polypeptide, or cell expressing a gene of interest produced by genetic engineering methods. The term "recombinant" as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. The proteins of the invention may be isolated from natural sources or produced by genetic engineering methods. "recombinant" as used herein additionally describes a nucleic acid molecule that, by virtue of its origin or manipulation, does not associate with all or a portion of the polynucleotide with which it is associated in nature. The term "recombinant" as used with respect to a host cell means a host cell that includes a recombinant polynucleotide.

The term "stabilizer" refers to a compound that binds to an antigen and maintains an epitope or immune responsiveness of the antigen for a period of time. Stabilizers are known in the art. Examples of stabilizers include multivalent cations such as calcium or aluminum.

The term "subject" refers to a mammal, bird, fish, reptile, or any other animal. The term "subject" also includes humans. The term "subject" also includes domestic pets. Non-limiting examples of domestic pets include: dogs, cats, pigs, rabbits, rats, mice, gerbils (gerbils), hamsters, guinea pigs (guineapig), ferrets, birds, snakes, lizards, fish, turtles, and frogs. The term "subject" also includes livestock animals. Non-limiting examples of livestock animals include: alpaca, bison, camel, cattle, deer, pig, horse, llama, mule, donkey, sheep, goat, rabbit, reindeer, yak, chicken, goose and turkey.

The term "mammal" as used herein refers to any mammal, such as a human, mouse, rabbit, non-human primate. In a preferred embodiment, the mammal is a human.

The terms "vaccine" or "vaccine composition" are used interchangeably to refer to a pharmaceutical composition comprising at least one immunogenic composition that induces an immune response in a subject.

General description

The present invention results from the following novel findings: the particular formulation and dosing regimen of the non-lipidated variant of P2086 elicits high bactericidal antibody titers such as the previous formulation of P2086 described, for example, in Fletcher et al, Infection & immunity, Vol.72 (4):2088-2100 (2004). Alternatively, the present invention results from the following novel findings: the particular formulation and dosing regimen of the non-lipidated variant of P2086 elicits higher bactericidal antibody titers than the commercial formulation of the lipidated LP2086 variant. It should be noted, however, that commercial formulations of lipidated LP2086 may not be currently available. Vaccines containing non-lipidated rP2086 variants were observed to have higher response rates (as determined by a 4-fold or 4-fold increase in SBA titers compared to baseline values) compared to lipidated rLP2086 vaccines. Formulations of non-lipidated P2086 variants elicited bactericidal antibodies against a broader spectrum of strains, including strains with similar (>92% identity) and different (<92% identity) LP2086 sequences.

The present invention also identifies previously unidentified difficulties expressing non-lipidated P2086 variants and provides methods to overcome these difficulties and novel compositions obtained thereby. Although plasmid constructs encoding non-lipidated P2086 variants provided robust expression of the non-lipidated variants, these variants were acylated with acetone at the N-terminal Cys. The possibility that acetonylation may hinder or reduce the manufacturing identity of the polypeptide. The inventors have additionally found that deletion of the N-terminal Cys in the sequence of the non-lipidated P2086 variant avoids acetonylation of the non-lipidated P2086 variant. Attempts to overcome pyruvylation by deletion of the codon for the N-terminal Cys would either abolish expression or result in expression of insoluble variants. Alternatively, removal of the N-terminal Cys from the non-lipidated P2086 variant may result in reduced expression of some variants. However, surprisingly, the inventors found that despite deletion of the N-terminal Cys residue, at least non-pyruvylated non-lipidated a05, B01, B09 and B44 variants could be expressed. In general, these polypeptides can be expressed without other modifications than Cys deletion compared to the corresponding wild-type sequence. See, e.g., examples 2 and 4. Furthermore, the inventors found that the non-pyruvylated non-lipidated variant was surprisingly immunogenic and that it unexpectedly elicited bactericidal antibodies.

Thus, the present invention provides two methods to overcome or reduce the likelihood of these difficulties of expressing non-lipidated variants. However, other methods are contemplated by the present invention. The first method is to change the length of the Gly/Ser stalk in the N-terminal tail immediately downstream of the N-terminal Cys. The second approach is codon optimization within the N-terminal tail. However, the invention encompasses optimization of other codons. These methods result in enhanced expression of soluble non-lipidated P2086 variants. For example, in one embodiment, the increased expression of the soluble non-lipidated P2086 variant is compared to the expression of a corresponding wild-type non-lipidated variant.

Isolated polypeptides

The inventors have surprisingly found that an isolated non-pyruvylated non-lipidated ORF2086 polypeptide. The inventors have additionally found that such polypeptides are unexpectedly immunogenic and capable of eliciting a bactericidal immune response.

As used herein, the term non-pyruvylated means that the polypeptide does not contain pyruvate. Pyruvate-containing non-lipidated ORF2086 polypeptides typically exhibit a mass change of +70 compared to the corresponding wild-type polypeptide. In one embodiment, the polypeptide of the invention does not exhibit a mass change of +70 when measured by mass spectrometry compared to the corresponding wild-type non-lipidated polypeptide. See, e.g., example 10.

In another embodiment, the isolated non-pyruvylated non-lipidated ORF2086 polypeptide includes a deletion of an N-terminal cysteine residue, as compared to a corresponding wild-type non-lipidated ORF2086 polypeptide. The term "N-terminal cysteine" refers to a cysteine (Cys) at the N-terminus or N-terminal tail of a polypeptide. In more detail, "N-terminal cysteine" as used herein refers to the N-terminal cysteine at which LP2086 lipoproteins are lipidated to have a tripalmitoyl lipid tail, as known in the art. For example, when referring to any of SEQ ID NOs: 12-21 as reference sequence, the N-terminal cysteine is at position 1.

The term "wild-type non-lipidated ORF2086 polypeptide" refers to an ORF2086 polypeptide having an amino acid sequence identical to the amino acid sequence of a corresponding mature lipidated ORF2086 polypeptide found in nature. The only difference between the non-lipidated and lipidated molecules was that the wild-type non-lipidated ORF2086 polypeptide was not lipidated at the N-terminal cysteine to have a tripalmitoyl lipid tail.

As is also known in the art, the non-lipidated 2086 form is produced from a protein lacking the original leader sequence or from a leader sequence that has been replaced with a portion of a sequence that does not specify a site for fatty acid acylation in a host cell. See, for example, WO2003/063766, which is incorporated herein by reference in its entirety.

Examples of non-lipidated ORF2086 include not only the wild-type non-lipidated ORF2086 polypeptide just described but also polypeptides having an amino acid sequence corresponding to any of seq id nos. 12-21, wherein the N-terminal Cys is deleted, and polypeptides having an amino acid sequence corresponding to any of seq id nos. 12-21, wherein the N-terminal Cys is substituted. Other examples of non-lipidated ORF2086 polypeptides include SEQ ID NO:44, SEQ ID NO:49, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:62, and SEQ ID NO: 64.

Examples of wild-type non-lipidated ORF2086 polypeptides include polypeptides having amino acid sequences corresponding to any one of seq id nos. 12-21, 58, 59, and 60 shown in fig. 2. These exemplary wild-type non-lipidated ORF2086 polypeptides include an N-terminal Cys deletion.

As used herein, for example, a "non-lipidated" B44 polypeptide includes a polypeptide having an amino acid sequence selected from seq id No. 21, seq id No. 21 in which the N-terminal Cys at position 1 is deleted, and seq id No. 44. The "wild-type non-lipidated" B44 polypeptide includes a polypeptide having the amino acid sequence SEQ ID NO: 21. The "non-pyruvylated non-lipidated" B44 polypeptide includes a polypeptide having an amino acid sequence selected from seq id no:21 and seq id no:44 in which the N-terminal Cys at position 1 is deleted.

As another example, as used herein, a "non-lipidated" B09 polypeptide includes a polypeptide having an amino acid sequence selected from seq id No. 18, seq id No. 18 in which the N-terminal Cys at position 1 is deleted, seq id No. 49 and seq id No. 50. The "wild-type non-lipidated" B09 polypeptide includes a polypeptide having the amino acid sequence of seq id No. 18. The "non-pyruvylated non-lipidated" B44 polypeptide includes a polypeptide having an amino acid sequence selected from seq id no:18, seq id no:49 and seq id no:50 in which the N-terminal Cys at position 1 is deleted.

As another example, as used herein, a "non-lipidated" a05 polypeptide includes a polypeptide having an amino acid sequence selected from seq id No. 13, seq id No. 13 in which the N-terminal Cys at position 1 is deleted, and seq id No. 55. The "wild-type non-lipidated" A05 polypeptide includes a polypeptide having the amino acid sequence of SEQ ID NO. 13. The "non-pyruvylated non-lipidated" a05 polypeptide includes a polypeptide having an amino acid sequence selected from seq id no:13 and seq id no:55 in which the N-terminal Cys at position 1 is deleted.

The term "deletion" of an N-terminal Cys as used herein includes a mutation that deletes the N-terminal Cys compared to the wild-type non-lipidated polypeptide sequence. For example, a "deletion" of an N-terminal Cys refers to the removal of the amino acid Cys from a reference sequence, e.g., from the corresponding wild-type sequence, thereby resulting in a reduction of amino acid residues compared to the reference sequence.

In another embodiment, the N-terminal Cys is substituted with an amino acid that is not a Cys residue. For example, in exemplary embodiments, the N-terminal Cys at position 1 of SEQ ID NOs 12-21 includes a C to G substitution at position 1. See, for example, SEQ ID NO:62 as compared to SEQ ID NO:19(B22 wild-type); SEQ ID NO:64 compared to SEQ ID NO:15 (wild type A22). Exemplary amino acids for replacing the N-terminal Cys include any non-Cys amino acid, preferably a polar uncharged amino acid such as glycine. In a preferred embodiment, the substitution is with a non-conserved residue of Cys.

The inventors surprisingly found that expression of a non-lipidated ORF2086 polypeptide lacking an N-terminal Cys residue results in no detectable pyruvylation as measured by mass spectrometry compared to the corresponding wild-type non-lipidated ORF2086 polypeptide. Examples of non-pyruvylated non-lipidated ORF2086 polypeptides include polypeptides having an amino acid sequence selected from the group consisting of: SEQ ID NO. 12(A04), SEQ ID NO. 13(A05), SEQ ID NO. 14(A12), SEQ ID NO. 15(A22), SEQ ID NO. 16(B02), SEQ ID NO. 17(B03), SEQ ID NO. 18(B09), SEQ ID NO. 19(B22), SEQ ID NO. 20(B24) and SEQ ID NO. 21(B44), wherein cysteine at position 1 is deleted. Other examples of isolated non-pyruvylated non-lipidated ORF2086 polypeptides include polypeptides having an amino acid sequence selected from the group consisting of: SEQ ID NO. 44, SEQ ID NO. 49, SEQ ID NO. 50 and SEQ ID NO. 55. Preferably, the non-pyruvylated non-lipidated 2086 polypeptide includes at least about 250, 255, or 260 consecutive amino acids, and at most about 270, 269, 268, 267, 266, 265, 264, 263, 260, 259, 258, 257, 256, or 255 consecutive amino acids. Any minimum value may be combined with any maximum value to define a range. Preferably, the polypeptide has at least 254 or 262 contiguous amino acids.

In one embodiment, the isolated non-pyruvylated non-lipidated ORF2086 polypeptide is encoded by a nucleotide sequence operably linked to an expression system, wherein the expression system is capable of expression in a bacterial cell. In an exemplary embodiment, the nucleotide sequence is linked to a regulatory sequence that controls the expression of the nucleotide sequence.

Suitable expression systems, regulatory sequences and bacterial cells are known in the art. For example, any plasmid expression vector may be used, such as PET^TM(Novogen, Madison Wis.) or PMAL^TM(NewEngland Biolabs, Beverly, Mass.) as long as the polypeptide is expressed in bacterial cells. Preferably, PET^TMThe vector is used for cloning and expressing recombinant protein in Escherichia coli. In PET^TMIn the system, the cloned gene can be expressed under the control of the bacteriophage T7 promoter. Exemplary bacterial cells include Pseudomonas fluorescens (Pseudomonas fluorescens), preferably E.coli.

In one aspect, the invention relates to a non-pyruvylated non-lipidated ORF2086 polypeptide obtainable by a method. The polypeptide is preferably isolated. The invention further relates to compositions comprising a non-pyruvylated non-lipidated ORF2086 polypeptide obtainable by a method. The composition is preferably an immunogenic composition. The method comprises expressing a nucleotide sequence encoding a polypeptide having an amino acid sequence selected from the group consisting of: 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 and SEQ ID NO. 21, wherein the cysteine at position 1 is deleted. The nucleotide sequence is operably linked to an expression system capable of expression in a bacterial cell. In one embodiment, the method comprises expressing a nucleotide sequence encoding a polypeptide having an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO. 44, SEQ ID NO. 49, SEQ ID NO. 50 and SEQ ID NO. 55. In another embodiment, the nucleotide sequence is selected from the group consisting of seq id no:43, 51, 46, 47, 48, 45 and 54. Preferably, the bacterial cell is escherichia coli.

In one aspect, the invention relates to a composition comprising a first isolated polypeptide comprising the amino acid sequence set forth in seq id No. 49 and a second isolated polypeptide comprising the amino acid sequence set forth in seq id No. 44. In a preferred embodiment, the polypeptide is an immunogenic polypeptide. In another preferred embodiment, the composition further comprises an ORF2086 subfamily a polypeptide from serogroup B neisseria meningitidis. Preferably, the ORF2086 subfamily a polypeptide is a non-pyruvylated non-lipidated ORF2086 subfamily a polypeptide. In an exemplary embodiment, the ORF2086 subfamily a polypeptide is a05, examples of which include, for example, seq id no:13, wherein the N-terminal cysteine at position 1 is deleted; and SEQ ID NO: 55.

In another aspect, the invention relates to a method of producing an isolated polypeptide. The method comprises expressing in a bacterial cell a polypeptide comprising a sequence having greater than 90% identity to seq id No. 21, the sequence comprising at least one domain selected from the group consisting of: amino acids 13-18 of SEQ ID NO:21, amino acids 21-34 of SEQ ID NO:21 and amino acids 70-80 of SEQ ID NO:21, or a combination thereof, wherein the polypeptide lacks the N-terminal cysteine. The method further comprises purifying the polypeptide. Wherein the polypeptide produced comprises a non-pyruvylated non-lipidated ORF2086 polypeptide. Preferably, the polypeptide is an immunogenic polypeptide. In a preferred embodiment, the bacterial cell is E.coli.

Examples of polypeptides comprising at least one domain selected from the group consisting of amino acids 13-18 of SEQ ID NO:21, amino acids 21-34 of SEQ ID NO:21 and amino acids 70-80 of SEQ ID NO:21, or a combination thereof, include SEQ ID NO:12(A04), SEQ ID NO:13(A05), SEQ ID NO:14(A12), SEQ ID NO:15(A22), SEQ ID NO:16(B02), SEQ ID NO:17(B03), SEQ ID NO:18(B09), SEQ ID NO:19(B22), SEQ ID NO:20(B24), and SEQ ID NO:21 (B44). Preferably, wherein the cysteine at position 1 is deleted. Other exemplary polypeptides include SEQ ID NO 44, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 55, SEQ ID NO 62 and SEQ ID NO 64.

In an exemplary embodiment, the isolated polypeptide sequence further comprises at least one domain selected from the group consisting of: amino acids 96-116 of SEQ ID NO. 21, amino acids 158-170 of SEQ ID NO. 21, amino acids 172-185 of SEQ ID NO. 21, amino acids 187-199 of SEQ ID NO. 21, amino acids 213-224 of SEQ ID NO. 21, amino acids 226-237 of SEQ ID NO. 21, amino acids 239-248 of SEQ ID NO. 21, or combinations thereof. Examples of polypeptides comprising at least one domain selected from the group consisting of amino acids 13-18 of SEQ ID NO:21, amino acids 21-34 of SEQ ID NO:21 and amino acids 70-80 of SEQ ID NO:21 or combinations thereof, and further comprising at least one domain selected from the group consisting of amino acids 96-116 of SEQ ID NO:21, amino acids 158-170 of SEQ ID NO:21, amino acids 172-185 of SEQ ID NO:21, amino acids 187-199 of SEQ ID NO:21, amino acids 213-224 of SEQ ID NO:21, amino acids 226-237 of SEQ ID NO:21, amino acids 239-248 of SEQ ID NO:21 or combinations thereof, include SEQ ID NO:16(B02), SEQ ID NO:17(B03), SEQ ID NO:18(B09), SEQ ID NO:19(B22), SEQ ID NO:20(B24) and SEQ ID NO:21(B44), preferably wherein the cysteine at position is deleted. Other exemplary polypeptides include SEQ ID NO 44, SEQ ID NO 49, SEQ ID NO 50 and SEQ ID NO 55 and SEQ ID NO 62.

In one aspect, the invention relates to an isolated polypeptide produced by the methods described herein. In one embodiment, the isolated polypeptide is a non-pyruvylated non-lipidated polypeptide. In another aspect, the invention relates to an immunogenic composition produced by the methods described herein.

In one aspect, the invention relates to an isolated polypeptide comprising the amino acid sequence shown in SEQ ID NO:18 (wherein the N-terminal Cys at position 1 is deleted) or SEQ ID NO: 49. Exemplary nucleotide sequences encoding SEQ ID NO 49 include SEQ ID NO 46, SEQ ID NO 47, and SEQ ID NO 48. Preferably, the nucleotide sequence is SEQ ID NO. 46. In one aspect, the invention relates to an isolated nucleotide sequence comprising SEQ ID NO. 46. In one aspect, the invention relates to an isolated nucleotide sequence comprising SEQ ID NO. 47. In one aspect, the invention relates to an isolated nucleotide sequence comprising SEQ ID NO. 48.

In one aspect, the invention relates to a plasmid comprising a nucleotide sequence selected from the group consisting of seq id No. 46, seq id No. 47, seq id No. 48 and seq id No. 45, wherein the plasmid is capable of being expressed in a bacterial cell. Suitable expression systems, regulatory sequences and bacterial cells are known in the art, as described above. Preferably, the bacterial cell is escherichia coli.

In another aspect, the invention relates to an isolated polypeptide comprising the amino acid sequence set forth in SEQ ID NO. 50. In an exemplary embodiment, SEQ ID NO. 50 is encoded by SEQ ID NO. 45.

In another aspect, the invention relates to an isolated polypeptide comprising the amino acid sequence set forth in SEQ ID NO:21 (wherein the N-terminal Cys is deleted) or SEQ ID NO: 44. Exemplary nucleotide sequences encoding SEQ ID NO 44 include SEQ ID NO 43 and SEQ ID NO 51. Preferably, the nucleotide sequence is SEQ ID NO: 43. In one aspect, the invention relates to an isolated nucleotide sequence comprising SEQ ID NO. 43.

Immunogenic compositions

In a preferred embodiment, the composition described herein comprising an isolated non-pyruvylated non-lipidated ORF2086 polypeptide is an immunogenic composition. Immunogenic compositions comprising a protein encoded by a nucleotide sequence from neisseria meningitidis ORF2086 are known in the art. Exemplary immunogenic compositions include those described in WO2003/063766 and U.S. patent application publication nos. US20060257413 and US20090202593, which are incorporated herein by reference in their entirety. The immunogenic compositions described therein include a protein exhibiting bactericidal activity identified as ORF2086 protein, immunogenic portions thereof, and/or biological equivalents thereof. The ORF2086 protein refers to the protein encoded by the open reading frame 2086 of the neisseria species.

The protein may be a recombinant protein or an isolated protein from a native neisseria species. For example, the neisserial ORF2086 proteins, as well as immunogenic portions and/or biological equivalents of such proteins, can be isolated from bacterial strains, such as strains of neisseria species, including strains of neisseria meningitidis (serogroups A, B, C, D, W-135, X, Y, Z, and 29E), neisseria gonorrhoeae (neisserial gonorrhoeae), and neisseria lactis (neisserial lactamica).

ORF2086 proteins include 2086 subfamily a and B proteins, immunogenic portions thereof, and/or biological equivalents thereof. 2086 subfamily a proteins and 2086 subfamily B proteins are known in the art, see, e.g., Fletcher et al, 2004, cited above. See also WO2003/063766, disclosing amino acid sequences related to 2086 subfamily A proteins represented by SEQ ID NOs: 260 to 278. Furthermore, WO2003/063766 discloses a method wherein SEQ ID NOs.279 to 299 represent amino acid sequences related to proteins of 2086 subfamily B. WO2003/063766 is incorporated herein by reference in its entirety. The ORF2086 protein or its equivalent, etc., may or may not be lipidated. Preferably, the neisserial ORF2086 protein is a non-lipidated protein. Alternatively, the immunogenic composition can be a combination of a lipidated ORF2086 protein and a non-lipidated ORF2086 protein.

In one embodiment, the immunogenic composition comprises an isolated protein having at least 95% amino acid sequence identity to a protein encoded by a nucleotide sequence from neisseria ORF 2086.

In one embodiment, the immunogenic composition comprises an isolated protein having at least 95% amino acid sequence identity to a subfamily a protein encoded by a nucleotide sequence from neisseria ORF 2086. Preferably, the immunogenic composition comprises an isolated subfamily a protein encoded by a nucleotide sequence from neisseria ORF 2086. In some embodiments, the ORF2086 subfamily a polypeptide is an a05, an a04, an a12, or an a22 variant.

In some embodiments, the ORF2086 subfamily a polypeptide is an a05, an a12, or an a22 variant.

In another embodiment, the immunogenic composition comprises an isolated protein having at least 95% amino acid sequence identity to a subfamily B protein encoded by a nucleotide sequence from neisseria ORF 2086. Preferably, the immunogenic composition comprises an isolated subfamily B protein encoded by a nucleotide sequence from neisseria ORF 2086. In some embodiments, the ORF2086 subfamily B protein is a B44, B02, B03, B22, B24, or B09 variant. In some embodiments, the ORF2086 subfamily B protein is a B44, B22, or B09 variant.

In a preferred embodiment, the immunogenic composition comprises an isolated non-pyruvylated non-lipidated polypeptide having at least 95% amino acid sequence identity to a subfamily B protein encoded by a nucleotide sequence from neisseria ORF 2086. For example, in some embodiments, the ORF2086 subfamily B protein is selected from B44 having an amino acid sequence as set forth in seq id No. 21; b02 having the amino acid sequence shown in SEQ ID NO. 16; b03 having the amino acid sequence shown in SEQ ID NO. 17; b22 having the amino acid sequence shown in SEQ ID NO. 19; b24 having the amino acid sequence shown in SEQ ID NO. 20; or a B09 variant having the amino acid sequence shown in seq id No. 18, wherein the N-terminal Cys is deleted; or a combination thereof.

More preferably, the immunogenic composition comprises a non-pyruvylated non-lipidated B09 polypeptide, a non-pyruvylated non-lipidated B44 polypeptide, or a combination thereof. In one embodiment, the composition includes a non-pyruvylated non-lipidated B09 variant having an amino acid sequence as set forth in seq id No. 18, wherein the N-terminal Cys is deleted; non-pyruvylated non-lipidated B44 having the amino acid sequence shown in seq id No. 21, wherein the N-terminal Cys is deleted; or a combination thereof. In another embodiment, the immunogenic composition comprises non-pyruvylated non-lipidated B09 having seq id No. 49, non-pyruvylated non-lipidated B44 having seq id No. 44, or a combination thereof.

In one aspect, the invention relates to an immunogenic composition comprising an ORF2086 subfamily B polypeptide from serogroup B neisseria meningitidis, wherein the polypeptide is non-pyruvylated non-lipidated B44. B44 may include an amino acid sequence as shown in SEQ ID NO:21 (in which the N-terminal Cys is deleted) or SEQ ID NO: 44. In one embodiment, the composition further comprises a second ORF2086 subfamily B polypeptide from serogroup B neisseria meningitidis, wherein the second polypeptide is non-pyruvylated non-lipidated B09. B09 may include an amino acid sequence as shown in SEQ ID NO:18 (in which the N-terminal Cys is deleted) or SEQ ID NO: 49. In one embodiment, the immunogenic composition is a vaccine.

In another embodiment, the composition includes up to 3 ORF2086 subfamily B polypeptides. In another embodiment, the composition includes up to 2 ORF2086 subfamily B polypeptides.

In one embodiment, the composition further comprises one or more ORF2086 subfamily a polypeptides. In a preferred embodiment, the composition comprises subfamily a polypeptide a 05.

In another embodiment, the immunogenic composition comprises an isolated protein having at least 95% amino acid sequence identity to a subfamily a protein encoded by a nucleotide sequence from neisseria ORF2086, and an isolated protein having at least 95% amino acid sequence identity to a subfamily B protein encoded by a nucleotide sequence from neisseria ORF 2086.

Preferably, the immunogenic composition comprises an isolated subfamily a protein encoded by a nucleotide sequence from neisseria ORF2086 and an isolated subfamily B protein encoded by a nucleotide sequence from neisseria ORF 2086. More preferably, the immunogenic composition comprises an isolated non-pyruvylated non-lipidated subfamily AORF2086 polypeptide and an isolated non-pyruvylated non-lipidated subfamily BORF2086 polypeptide. In some embodiments, the ORF2086 subfamily a polypeptide is an a05, an a04, an a12, or an a22 variant. In a preferred embodiment, the ORF2086 subfamily a polypeptide is a05 having an amino acid sequence as set forth in seq id No. 13; a04 having the amino acid sequence shown in SEQ ID NO. 12; a12 having the amino acid sequence shown in SEQ ID NO. 14; or an A22 variant having the amino acid sequence shown in SEQ ID NO. 15, wherein the N-terminal Cys is deleted; or any combination thereof. In some embodiments, the ORF2086 subfamily B protein is a B44, B02, B03, B22, B24, or B09 variant. In a preferred embodiment, the ORF2086 subfamily B protein is B44 having an amino acid sequence as set forth in seq id No. 21; b02 having the amino acid sequence shown in SEQ ID NO. 16; b03 having the amino acid sequence shown in SEQ ID NO. 17; b22 having the amino acid sequence shown in SEQ ID NO. 19; b24 having the amino acid sequence shown in SEQ ID NO. 20; or a B09 variant having the amino acid sequence shown in seq id No. 18, wherein the N-terminal Cys is deleted; or a combination thereof.

In one embodiment, the immunogenic composition comprises a 1:1 ratio of subfamily a protein to subfamily B protein.

In another aspect, the isolated polypeptides and compositions described herein elicit a bactericidal immune response in a mammal against an ORF2086 polypeptide from serogroup B neisseria meningitidis. The compositions are capable of inducing bactericidal anti-neisseria meningitidis antibodies, and in preferred embodiments, bactericidal antibodies against strains of the respective subfamilies, following administration to a mammal. Additional information about the bactericidal response is given below. See, e.g., examples 6,11, 12 and 13. Bactericidal antibodies are indicators of protection in humans, and preclinical studies serve as surrogates, and any novel immunogenic composition candidate should elicit these functional antibodies.

In an exemplary embodiment, isolated non-pyruvylated non-lipidated B09 polypeptides having seq id no:18 (wherein the N-terminal Cys at position 1 is deleted) or seq id no:49 and immunogenic compositions thereof elicit bactericidal antibodies against (e.g., can bind to) subfamily a or preferably subfamily BORF2086 polypeptides from serogroup B neisseria meningitidis. Preferably, the non-pyruvylated non-lipidated B09 polypeptide and immunogenic compositions thereof elicit an immune response against the A05 variant (SEQ ID NO: 13); the B44 variant (SEQ ID NO: 21); the B16 variant (SEQ ID NO: 60); the B24 variant (SEQ ID NO: 20); a B09 variant (SEQ ID NO:18) or a combination thereof. In an exemplary embodiment, the non-pyruvylated non-lipidated B09 polypeptide and immunogenic compositions thereof elicit antibodies directed against the B44 variant (SEQ ID NO: 21); the B16 variant (SEQ ID NO: 60); the B24 variant (SEQ ID NO: 20); a B09 variant (SEQ ID NO:18) or a combination thereof. See, e.g., example 11, example 12, and example 13.

In another exemplary embodiment, the isolated non-pyruvylated non-lipidated B44 polypeptides having seq id no:21 (wherein the N-terminal Cys at position 1 is deleted) or seq id no:44 and immunogenic compositions thereof elicit bactericidal antibodies against (e.g., can bind to) subfamily BORF2086 polypeptides from serogroup B neisseria meningitidis. Preferably, the non-pyruvylated non-lipidated B44 polypeptide and immunogenic compositions thereof elicit antibodies directed against the B44 variant (SEQ ID NO: 21); the B16 variant (SEQ ID NO: 60); the B24 variant (SEQ ID NO: 20); a B09 variant (SEQ ID NO:18) or a combination thereof. See, e.g., example 11. In addition, the non-pyruvylated non-lipidated B44 polypeptide and immunogenic compositions thereof can also elicit bactericidal antibodies that bind to the B02 variant (SEQ ID NO: 16). See, e.g., example 12 and example 13.

In addition, the non-pyruvylated non-lipidated B44 polypeptide and immunogenic compositions thereof may also elicit bactericidal antibodies that bind to the B03 variant (SEQ ID NO:17) and the B15 variant (SEQ ID NO: 59). See, e.g., example 6.

In another exemplary embodiment, the isolated non-pyruvylated non-lipidated B22 polypeptide having seq id no:19 (wherein the N-terminal Cys at position 1 is deleted) and immunogenic compositions thereof elicit bactericidal antibodies against (e.g., can bind to) subfamily BORF2086 polypeptides from serogroup B neisseria meningitidis. Preferably, the non-pyruvylated non-lipidated B22 polypeptide elicits activity against the B44 variant (SEQ ID NO: 21); the B16 variant (SEQ ID NO: 60); the B24 variant (SEQ ID NO: 20); a B09 variant (SEQ ID NO:18) or a combination thereof. See, e.g., example 13.

In one embodiment, the isolated non-pyruvylated non-lipidated a05 polypeptides having seq id no:13 (wherein the N-terminal Cys is deleted) or seq id no:55 and immunogenic compositions thereof elicit bactericidal antibodies against (e.g., bind to) subfamily AORF2086 polypeptides from serogroup B neisseria meningitidis. Preferably, the non-pyruvylated non-lipidated A05 and immunogenic compositions thereof elicit bactericidal antibodies against the A05 variant (SEQ ID NO:13), the A22 variant (SEQ ID NO:15), the A12 variant (SEQ ID NO:14) or combinations thereof. See, e.g., examples 6 and 13.

In one aspect, the invention relates to a method of eliciting a bactericidal antibody in a mammal that is specific for serogroup B neisseria meningitidis. In one exemplary embodiment, the method comprises eliciting a bactericidal antibody specific for ORF2086 subfamily B serogroup B neisseria meningitidis, ORF2086 subfamily a serogroup B neisseria meningitidis, or a combination thereof. The method includes administering to the mammal an effective amount of an isolated non-pyruvylated non-lipidated 2086 polypeptide or immunogenic composition thereof as described above.

In a preferred embodiment, the method comprises eliciting bactericidal antibodies specific for ORF2086 subfamily B serogroup B neisseria meningitidis. An isolated polypeptide or immunogenic composition includes a non-pyruvylated non-lipidated B44 polypeptide. In another preferred embodiment, the composition further comprises a non-pyruvylated non-lipidated B09 polypeptide, and in an exemplary embodiment, the isolated polypeptide or immunogenic composition comprises seq id No. 49, seq id No. 44, or a combination thereof. In a preferred embodiment, the isolated polypeptide or immunogenic composition comprises seq id no:18 (wherein the N-terminal Cys at position 1 is deleted), seq id no:21 (wherein the N-terminal Cys at position 1 is deleted), or a combination thereof. In another preferred embodiment, the isolated polypeptide or immunogenic composition comprises seq id No. 19 wherein the N-terminal Cys at position 1 is deleted.

In a preferred embodiment, the method comprises eliciting bactericidal antibodies specific for ORF2086 subfamily a, serogroup B, neisseria meningitidis. An isolated polypeptide or immunogenic composition includes a non-pyruvylated non-lipidated a05 polypeptide. In a preferred embodiment, the isolated polypeptide or immunogenic composition comprises seq id No. 13, wherein the N-terminal Cys at position 1 is deleted. In another preferred embodiment, the composition further comprises a non-pyruvylated non-lipidated B44 polypeptide. In an exemplary embodiment, the isolated polypeptide or immunogenic composition comprises seq id No. 55, seq id No. 44, or a combination thereof. In a preferred embodiment, the isolated polypeptide or immunogenic composition comprises seq id no:13 (wherein the N-terminal Cys at position 1 is deleted), seq id no:21 (wherein the N-terminal Cys at position 1 is deleted), or a combination thereof.

The immunogenic composition can include a protein encoded by a nucleotide sequence from neisseria ORF2086, a polynucleotide thereof, or an equivalent, as the only active immunogen in the immunogenic composition. Alternatively, the immunogenic composition may additionally include an active immunogen, including other neisserial immunogenic polypeptides, or an immunologically active protein of one or more other microbial pathogens (such as, but not limited to, a virus, prion, bacillus, or fungus), or a capsular polysaccharide. The compositions may comprise one or more desired proteins, fragments or pharmaceutical compounds as required for the chosen indication.

The present invention encompasses any multi-antigen or multi-valent immunogenic composition. For example, the immunogenic composition can include a combination of two or more ORF2086 proteins, a combination of an ORF2086 protein with one or more PorA proteins, a combination of an ORF2086 protein with neisseria meningitidis serogroup A, C, Y and W135 polysaccharide and/or polysaccharide conjugate, a combination of an ORF2086 protein with neisseria meningitidis and pneumococcus (pneumococcus), or a combination of any of the foregoing, in a form suitable for the desired administration (e.g., transmucosal delivery). One skilled in the art will be able to readily formulate such multiple antigen or multivalent immunogenic compositions.

The invention also encompasses multiple immunization regimens in which any composition suitable for targeting a pathogen may be combined with or with the instant inventionThe composition of the invention is combined. For example, but not by way of limitation, an immunogenic composition of the invention and another immunological composition (such as an HPV vaccine) for immunizing Human Papilloma Virus (HPV) may be administered to a patient) As part of a multiple immunization protocol. One skilled in the art will be readily able to select immunogenic compositions for use in combination with the immunogenic compositions of the present invention for the purpose of developing and implementing multiple immunization protocols.

ORF2086 polypeptides, fragments, and equivalents can be used as part of a conjugate immunogenic composition; wherein one or more proteins or polypeptides are conjugated to a carrier to produce a composition having immunogenicity against several serotypes or more precisely serogroups and/or against several diseases. Alternatively, one ORF2086 polypeptide can be used as a carrier protein for other immunogenic polypeptides. The formulation of such immunogenic compositions is well known to those skilled in the art.

The immunogenic compositions of the invention preferably include a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers and/or diluents include any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Suitable pharmaceutically acceptable carriers include, for example, one or more of the following: water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof.

Pharmaceutically acceptable carriers may additionally include minor amounts of auxiliary substances which enhance the shelf life or effectiveness of the antibody, such as wetting or emulsifying agents, preservatives or buffers. The preparation and use of pharmaceutically acceptable carriers is well known in the art. Unless any conventional media or agent is incompatible with the active ingredient, its use in the immunogenic compositions of the invention is contemplated.

The immunogenic composition can be administered parenterally (e.g., by subcutaneous or intramuscular injection) as well as orally or intranasally. Methods for intramuscular immunization are described by Wolff et al Biotechniques:11(4):474-85, (1991) and by Sedegah et al PNAS Vol 91, pp 9866 and 9870 (1994). By way of example, but not limitation, other modes of administration employ oral formulations, pulmonary formulations, suppositories, and transdermal administration. For example, oral formulations include commonly employed excipients such as, but not limited to, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Preferably, the immunogenic composition is administered intramuscularly.

The immunogenic compositions of the invention may additionally comprise one or more other "immunomodulators", which are agents that interfere with or alter the immune system in order to observe up-or down-regulation of humoral and/or cell-mediated immunity. In a particular embodiment, humoral and/or cell-mediated upregulation of the immune system (arm) is preferred. Examples of certain immunomodulators include, for example, adjuvants or cytokines or ISCOMATRIX (csllimed, Parkville, Australia), described inter alia in U.S. patent No. 5,254,339.

Non-limiting examples of adjuvants that can be used in the vaccines of the present invention include RIBI adjuvant system (RibiInc., Hamilton, Mont.), alum, mineral gels (such as aluminum hydroxide gels), oil-in-water emulsions, water-in-oil emulsions (e.g., Freund's complete and incomplete adjuvants), block copolymers (CytRx, AtlantaGa.), QS-21(Cambridge Biotech Inc., Cambridge Mass.), SAF-M (Chiron, Emeryville Calif.)Saponin, QuilA or other saponin moieties, monophosphoryl lipid a, and an alfuzidine lipid-amine adjuvant. Non-limiting examples of oil-in-water emulsions suitable for use in the vaccines of the present invention include modified SEAM62 and SEAM1/2 formulations. The improvement SEAM62 is an oil-in-water emulsion containing 5% (v/v) squalene (squalene) (Sigma), 1% (v/v)Detergent (ICIsur)factants)、0.7%(v/v)Detergents (ICISurfactants), 2.5% (v/v) ethanol, 200. mu.g/ml QuilA, 100. mu.g/ml cholesterol and 0.5% (v/v) lecithin (lecithin). The improvement SEAM1/2 is an oil-in-water emulsion comprising 5% (v/v) squalene, 1% (v/v)Detergent, 0.7% (v/v) polysorbate 80 detergent, 2.5% (v/v) ethanol, 100 μ g/ml QuilA and 50 μ g/ml cholesterol.

Other "immunomodulators" which may be included in a vaccine include, for example, one or more interleukins, interferons or other known cytokines or chemokines. In one embodiment, the adjuvant may be a cyclodextrin derivative or a polyanionic polymer, such as the cyclodextrin derivatives or polyanionic polymers described in U.S. Pat. nos. 6,165,995 and 6,610,310, respectively. It will be appreciated that the immunomodulator and/or adjuvant to be used will depend on the subject to which the vaccine or immunogenic composition is to be administered, the route of injection and the number of injections to be given.

In some embodiments, the adjuvant is a saponin. In some embodiments, the saponin concentration is between 1 μ g/ml and 250 μ g/ml; between 5 and 150 μ g/ml; or between 10 and 100. mu.g/ml. In some embodiments, the saponin concentration is about 1 μ g/ml; about 5 μ g/ml; about 10 μ g/ml; about 20 μ g/ml; about 30 μ g/ml; about 40 μ g/ml; about 50 μ g/ml; about 60 μ g/ml; about 70 μ g/ml; about 80 μ g/ml; about 90 μ g/ml; about 100 μ g/ml; about 110. mu.g/ml; about 120. mu.g/ml; about 130 μ g/ml; about 140. mu.g/ml; about 150. mu.g/ml; about 160 μ g/ml; about 170 μ g/ml; about 180 μ g/ml; about 190 μ g/ml; about 200. mu.g/ml; about 210. mu.g/ml; about 220 μ g/ml; about 230 μ g/ml; about 240 μ g/ml; or about 250. mu.g/ml.

In certain preferred embodiments, the proteins of the invention are used in immunogenic compositions comprising a transmucosal adjuvant for oral administration and for the treatment or prevention of neisseria meningitidis infection in a human host. The transmucosal adjuvant may be cholera toxin (choleratoxin); preferably, however, mucosal adjuvants other than cholera toxin that can be used according to the present invention include non-toxic derivatives of cholera holotoxin (cholerhotoxin), wherein the a subunit is a mutagenized and chemically modified cholera toxin; or related proteins produced by modifying the amino acid sequence of cholera toxin. For specific cholera toxins that may be particularly suitable for use in preparing the immunogenic compositions of the invention, see mutant cholera holotoxin E29H as disclosed in published international application WO00/18434, which is hereby incorporated by reference in its entirety. These adjuvants may be added to or combined with the polypeptides of the present invention. The same technique can be applied to other molecules with transmucosal adjuvant or delivery properties, such as E.coli heat Labile Toxin (LT).

Other compounds having transmucosal adjuvant or delivery activity may be used, such as bile; polycations such as DEAE-dextran and polyornithine; detergents such as sodium dodecylbenzene sulfate; a lipid conjugate material; antibiotics, such as streptomycin; a vitamin A; and other compounds that alter the structural or functional integrity of mucosal surfaces. Other compounds with transmucosal activity include derivatives of microbial structures (such as MDP); acridine and cimetidine (cimetidine). STIMULON as described above may also be used^TMQS-21, MPL and IL-12.

The immunogenic compositions of the invention may be delivered in the form of ISCOMS (immune stimulating complexes) containing CTBs, liposomes or encapsulated in compounds such as acrylates or poly (DL-lactide-co-glycosides) to form microspheres of a size suitable for absorption. The proteins of the invention may also be incorporated into oily emulsions.

The amount (i.e., dose) of immunogenic composition administered to a patient can be determined according to standard techniques known to the ordinary skilled artisan, taking into account factors such as: a specific antigen; adjuvant (if present); the age, sex, weight, species, condition of the particular patient; and the route of administration.

For example, a dose for a juvenile human patient may include at least 0.1 μ g, 1 μ g, 10 μ g, or 50 μ g of neisserial ORF2086 protein, and at most 80 μ g, 100 μ g, 150 μ g, or 200 μ g of neisserial ORF2086 protein. Any minimum value and any maximum value may be combined to define a suitable range.

Adjuvant

In certain embodiments, an immunogenic composition as described herein also comprises one or more adjuvants. An adjuvant is a substance that enhances the immune response when administered with an immunogen or antigen. A number of cytokines or lymphokines have been shown to have immunomodulatory activity, and are therefore useful as adjuvants, including, but not limited to, interleukins 1- α, 1- β,2, 4,5, 6,7, 8, 10, 12 (see, e.g., U.S. patent No. 5,723,127), 13, 14, 15, 16, 17, and 18 (and mutated forms thereof); interferon- α, β and γ; granulocyte-macrophage colony stimulating factor (GM-CSF) (see, e.g., U.S. patent No. 5,078,996 and ATCC accession No. 39900); macrophage colony stimulating factor (M-CSF); granulocyte colony stimulating factor (G-CSF); and tumor necrosis factors alpha and beta.

Other adjuvants suitable for use with the immunogenic compositions described herein include chemokines, including but not limited to MCP-1, MIP-1 α, MIP-1 β, and RANTES; adhesion molecules such as selectins (selectins), e.g., L-selectin, P-selectin and E-selectin; mucin (mucin) -like molecules, such as CD34, GlyCAM-1, and MadCAM-1; integrin (integrin) family members such as LFA-1, VLA-1, Mac-1 and p 150.95; immunoglobulin superfamily members such as PECAM, ICAMs (e.g., ICAM-1, ICAM-2, and ICAM-3), CD2, and LFA-3; co-stimulatory molecules such as B7-1, B7-2, CD40, and CD 40L; growth factors including vascular growth factor, nerve growth factor, fibroblast growth factor, epidermal growth factor, PDGF, BL-1 and vascular endothelial growth factor; receptor molecules including Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, and DR 6; and Caspase (ICE).

Other exemplary adjuvants include, but are not limited to, aluminum hydroxide; aluminum phosphate; STIMULON^TMQS-21(AquilaBiopharmaceuticals,Inc.,Framingham,Mass.)；MPL^TM(3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, Mont.), 529 (an aminoalkyl glucosamine phosphate compound, Corixa, Hamilton, Mont), IL-12(genetics institute, Cambridge, Mass.); GM-CSF (ImmunexCorp., Seattle, Wash.); N-acetyl-muramyl-L-threonyl-D-isopropylamide (thr-MDP); N-acetyl-desmethyl-muramyl-L-alanyl-D-isoglutamine (CGP11637, known as desmethyl MDP); N-acetylmuramoyl-L-alanyl-D-isoglutamine-L-alanine-2- (1'-2' -dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy-ethylamine) (CGP19835A, known as MTP-PE); and cholera toxin. In certain preferred embodiments, the adjuvant is QS-21.

Other exemplary adjuvants include non-toxic derivatives of cholera toxin (including its a subunit), and/or conjugates or genetically engineered fusions of neisseria meningitidis polypeptides with cholera toxin or its B subunit ("CTB"), pre-cholera toxoid (procologenid), fungal polysaccharides (including schizophyllan), muramyl dipeptide ("MDP") derivatives, phorbol esters, thermolabile toxins of e.

Aluminum phosphate has been used as an adjuvant in phase 1 clinical trials at a concentration of 0.125mg per dose, well below the limit of 0.85mg per dose specified by U.S. federal regulations [610.15(a) ]. Aluminum-containing adjuvants are widely used in humans to enhance the immune response of antigens when administered intramuscularly or subcutaneously. In some embodiments, the concentration of aluminum in the immunogenic composition is between 0.125 μ g/ml and 0.5 μ g/ml; between 0.20 and 0.40. mu.g/ml; or between 0.20 and 0.30. mu.g/ml. In some embodiments, the concentration of aluminum in the immunogenic composition is about 0.125 μ g/ml; about 0.15 μ g/ml; about 0.175. mu.g/ml; about 0.20 μ g/ml; about 0.225 μ g/ml; about 0.25 μ g/ml; about 0.275 μ g/ml; about 0.30 μ g/ml; about 0.325 μ g/ml; about 0.35 μ g/ml; about 0.375 μ g/ml; about 0.40 μ g/ml; about 0.425 μ g/ml; about 0.45 μ g/ml; about 0.475. mu.g/ml; or about 0.50. mu.g/ml.

In a preferred embodiment, the concentration of aluminum in the immunogenic composition is between 0.125mg/ml and 0.5 mg/ml; between 0.20mg/ml and 0.40 mg/ml; or between 0.20mg/ml and 0.30 mg/ml. In some embodiments, the concentration of aluminum in the immunogenic composition is about 0.125 mg/ml; about 0.15 mg/ml; about 0.175 mg/ml; about 0.20 mg/ml; about 0.225 mg/ml; about 0.25 mg/ml; about 0.275 mg/ml; about 0.30 mg/ml; about 0.325 mg/ml; about 0.35 mg/ml; about 0.375 mg/ml; about 0.40 mg/ml; about 0.425 mg/ml; about 0.45 mg/ml; about 0.475 mg/ml; or about 0.50 mg/ml.

Adjuvants suitable for enhancing an immune response additionally include, but are not limited to, MPL^TM(3-O-deacylated monophosphoryl lipid A, Corixa, Hamilton, MT), which is described in U.S. Pat. No. 4,912,094. Synthetic lipid a analogs or aminoalkyl glucosamine phosphate compounds (AGPs) or derivatives or analogs thereof available from Corixa (Hamilton, MT) and described in U.S. patent No. 6,113,918 are also useful as adjuvants. One such AGP is 2- [ (R) -3-tetradecanoyloxytetradecanoylamino]Ethyl 2-deoxy-4-O-phosphono-3-O- [ (R) -3-tetradecanoyloxytetradecanoyl]-2- [ (R) -3-tetradecanoyloxytetradecanoyl-amino]-b-D-glucopyranoside, which is also known as 529 (previously known as RC 529). This 529 adjuvant is formulated as an Aqueous Form (AF) or as a Stable Emulsion (SE).

Other adjuvants include muramyl peptides such as N-acetyl-muramyl-L-threonyl-D-isoglutamyl-amino acid (thr-MDP), N-acetyl-desmethyl muramyl-L-alanine-2- (1'-2' dipalmitoyl-sn-glycero-3-hydroxyphosphoryl-oxy) -ethylamine (MTP-PE); oil-in-water emulsions, such as MF59 (U.S. patent No. 6,299,884) (containing 5% squalene, 0.5% polysorbate 80 and 0.5% Span85 (optionally with varying amounts of MTP-PE), formulated as submicron particles using a microfluidizer (such as 110Y microfluidizer (Microfluidics, Newton, MA)), and SAF (containing 10% squalene, 0.4% polysorbate 80, 5% pluronic block polymers L121 and thr-MDP), microfluidized as a sub-microemulsion or vortexed to produce larger particle size milkLiquid); incomplete Freund's Adjuvant (IFA); aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate; aifenugine (Amphigen); alfvudine; l121/squalene; d-lactide-polylactide/glucoside; a pluronic polyol; killed Bordetella (Bordetella); saponins, such as Stimulon described in U.S. Pat. No. 5,057,540^TMQS-21 (antibiotics, Framingham, MA.), ISCOMATRIX (CSLLImized, Parkville, Australia), and ISCOMATRIX (ISCOMATRIX), described in U.S. Pat. No. 5,254,339; mycobacterium tuberculosis (Mycobacterium tuberculosis); bacterial lipopolysaccharides; synthetic polynucleotides, such as oligonucleotides containing CpG motifs (e.g., U.S. Pat. No. 6,207,646); IC-31(IntercellAG, Vienna, Austria), described in European patent Nos. 1,296,713 and 1,326,634; pertussis Toxin (PT) or a mutant thereof, cholera toxin or a mutant thereof (e.g., U.S. patent nos. 7,285,281, 7,332,174, 7,361,355, and 7,384,640); or Escherichia coli heat-Labile Toxin (LT) or a mutant thereof, specifically LT-K63, LT-R72 (for example, U.S. Pat. Nos. 6,149,919, 7,115,730 and 7,291,588).

Methods of producing non-lipidated P2086 antigens

In one aspect, the invention relates to a method of producing a non-pyruvylated non-lipidated ORF2086 polypeptide. The method comprises expressing a nucleotide sequence encoding an ORF2086 polypeptide, wherein the N-terminal cysteine is deleted compared to the corresponding wild-type sequence, and wherein the nucleotide sequence is operably linked to an expression system capable of expression in a bacterial cell. For example, preferably, the polypeptide has the amino acid sequence of SEQ ID NO. 12; 13 under the condition of SEQ ID NO; 14 parts of SEQ ID NO; 15 parts of SEQ ID NO; 16 is shown in SEQ ID NO; SEQ ID NO. 17; 18 parts of SEQ ID NO; 19 under SEQ ID NO; 20 under the condition of SEQ ID NO; 21, wherein the cysteine at position 1 is deleted compared to the corresponding wild type sequence. Other exemplary polypeptides include polypeptides having an amino acid sequence selected from the group consisting of SEQ ID NO 44, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 55, SEQ ID NO 57, SEQ ID NO 62, and SEQ ID NO 64. The method further comprises purifying the polypeptide.

In some embodiments, the present invention provides a method of producing a soluble non-lipidated P2086 antigen, comprising the steps of: cloning the ORF2086 variant nucleic acid sequence into an Escherichia coli expression vector without a lipidization control sequence, transforming Escherichia coli bacteria by using the ORF2086 expression vector, and carrying out induction expression and separation on the expressed P2086 protein. In some embodiments, expression is induced with IPTG.

In some embodiments, the codon for the N-terminal Cys of the ORF2086 variant is deleted. Examples of such codons include TGC. In some embodiments, the codon for the N-terminal Cys of the ORF2086 variant is mutated by point mutagenesis to generate Ala, Gly, or Val codons. In some embodiments, Ser and Gly codons are added to the N-terminal tail of the ORF2086 variant to extend the Gly/Ser stalk immediately downstream of the N-terminal Cys. In some embodiments, the total number of Gly and Ser residues within the Gly/Ser stalk is at least 7, 8,9, 10, 11, or 12. In some embodiments, the codon for the N-terminal Cys is deleted. In some embodiments, the N-terminal 7, 8,9, 10, 11, or 12 residues are Gly or Ser.

In some embodiments, the codons of the N-terminal tail of the non-lipidated ORF2086 variant are optimized by point mutagenesis. In some embodiments, the N-terminal tail of the non-lipidated ORF2086 variant is optimized to match the N-terminal tail of the B09 variant. In some embodiments, the codon of the N-terminal tail of the ORF2086 variant is optimized by point mutagenesis such that the codon encoding the 5 th amino acid of the ORF2086 variant is 13-15100% identical to nucleotides of seq id No. 8 and the codon encoding the 13 th amino acid of the ORF2086 variant is 37-39100% identical to nucleotides of seq id No. 8. In some embodiments, the N-terminal tail of the non-lipidated ORF2086 variant is optimized to make the 5'45 nucleic acids 1-45100% identical to the nucleic acids of seq id No. 8. In some embodiments, the N-terminal tail of the non-lipidated ORF2086 variant is optimized to make the 5'42 nucleic acids 4-45100% identical to the nucleic acids of seq id No. 8. In some embodiments, the N-terminal tail of the non-lipidated ORF2086 variant is optimized such that the 5'39 nucleic acids are 4-42100% identical to the nucleic acids of seq id No. 8. In some embodiments, the N-terminal tail of the non-lipidated P2086 variant comprises at least one amino acid substitution compared to amino acids 1-15 of seq id No. 18. In some embodiments, the N-terminal tail of the non-lipidated P2086 variant comprises two amino acid substitutions as compared to amino acids 1-15 of seq id No. 18. In some embodiments, the N-terminal tail of the non-lipidated P2086 variant comprises at least one amino acid substitution compared to amino acids 2-15 of seq id No. 18. In some embodiments, the N-terminal tail of the non-lipidated P2086 variant comprises two amino acid substitutions as compared to amino acids 2-15 of seq id No. 18. In some embodiments, the amino acid substitution is a conservative amino acid substitution.

In some embodiments, the codons of the non-lipidated variants have been optimized for increased expression. Codon optimization is known in the art. See, e.g., Sastalla et al, applied and environmental microbiology, Vol.75 (7): 2099-. In some embodiments, codon optimization comprises matching the codon usage of an amino acid sequence to the codon frequency of a selected host organism, while including and/or excluding a particular DNA sequence. In some embodiments, codon optimization further comprises minimizing the corresponding secondary mRNA structure to reduce translational disorders. In some embodiments, the N-terminal tail has been codon optimized to include any one of seq id nos 28, 30, 32, and 34. In some embodiments, the Gly/Ser stem has been codon optimized to comprise any one of SEQ ID NOs 28, 30, 32, and 34.

For a better understanding of the present invention, the following examples are set forth. The examples are for illustrative purposes only and should not be construed as limiting the scope of the invention.

Immunogenic composition formulations

In certain embodiments, the immunogenic compositions of the invention further comprise at least one of an adjuvant, buffer, cryoprotectant, salt, divalent cation, non-ionic detergent, free radical oxidation inhibitor, diluent, or carrier.

In addition to the plurality of neisseria meningitidis protein antigens and the capsular polysaccharide-protein conjugate, the immunogenic compositions of the invention may additionally comprise one or more preservatives. The FDA requires that the bioproduct in multi-dose vials contain preservatives with few exceptions. Preservative containing vaccine products include vaccines comprising benzethonium chloride (anthrax), 2-phenoxyethanol (DTaP, HepA, Lyme, Polio (Polio) (parenteral)), phenol (pneumonia (Pneumo), Typhoid (Typhoid) (parenteral), vaccinia) and thimerosal (DTaP, DT, Td, HepB, Hib, influenza, JE, meningitis (Mening), pneumonia, Rabies). Preservatives approved for use in injectable pharmaceuticals include, for example, chlorobutanol, m-cresol, methyl paraben, propyl paraben, 2-phenoxyethanol, benzethonium chloride, benzalkonium chloride, benzoic acid, benzyl alcohol, phenol, thimerosal, and phenylmercuric nitrate.

The formulations of the invention may additionally comprise one or more of the following: buffers, salts, divalent cations, non-ionic detergents, cryoprotectants (such as sugars), and antioxidants (such as radical scavengers or chelating agents), or any multiple combination thereof. The choice of either component (e.g., chelating agent) can determine whether the other component (e.g., scavenger) is desirable. The final composition formulated for administration should be sterile and/or pyrogen-free. One skilled in the art can empirically determine which combinations of these and other components will be best included in the preservative-containing immunogenic compositions of the present invention, depending on a variety of factors, such as the particular storage and administration conditions desired.

In certain embodiments, formulations of the invention that are compatible with parenteral administration comprise one or more physiologically acceptable buffers selected from, but not limited to, Tris (tromethamine), phosphate, acetate, borate, citrate, glycine, histidine and succinate. In certain embodiments, the formulation is buffered to a pH value in the range of about 6.0 to about 9.0, preferably about 6.4 to about 7.4.

In certain embodiments, it may be desirable to adjust the pH of the immunogenic composition or formulation of the invention. The pH of the formulations of the present invention can be adjusted using standard techniques in the art. The pH of the formulation can be adjusted to between 3.0 and 8.0. In certain embodiments, the pH of the formulation may be or may be adjusted to be between 3.0 and 6.0, 4.0 and 6.0, or 5.0 and 8.0. In other embodiments, the pH of the formulation may be or may be adjusted to be about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 5.8, about 6.0, about 6.5, about 7.0, about 7.5, or about 8.0. In certain embodiments, the pH may be or may be adjusted to the following ranges: 4.5 to 7.5, or 4.5 to 6.5, 5.0 to 5.4, 5.4 to 5.5, 5.5 to 5.6, 5.6 to 5.7, 5.7 to 5.8, 5.8 to 5.9, 5.9 to 6.0, 6.0 to 6.1, 6.1 to 6.2, 6.2 to 6.3, 6.3 to 6.5, 6.5 to 7.0, 7.0 to 7.5, or 7.5 to 8.0. In a particular embodiment, the formulation has a pH of about 5.8.

In certain embodiments, formulations of the invention that are compatible with parenteral administration comprise one or more divalent cations, including but not limited to MgCl, at a concentration in the range of about 0.1mM to about 10mM, with up to about 5mM being preferred₂、CaCl₂And MnCl₂。

In certain embodiments, the formulations of the present invention that are compatible with parenteral administration comprise one or more salts, including but not limited to sodium chloride, potassium chloride, sodium sulfate, and potassium sulfate, present at physiologically acceptable ionic strengths to a subject following parenteral administration and included in the final formulation at final concentrations that yield the selected ionic strength or osmolarity. The final ionic strength or osmolality of the formulation will be determined by various components, such as ions from buffering compounds and other non-buffering salts. One preferred salt, NaCl, is present in a range up to about 250mM, with the salt concentration being selected to complement other components (e.g., sugars) such that the final total osmolarity of the formulation is compatible with parenteral administration (e.g., intramuscular or subcutaneous injection) and will promote long term stability of the immunogenic components of the immunogenic composition formulation at different temperature ranges. Salt-free formulations will allow for an increased range of one or more selected cryoprotectants to maintain the desired final osmolarity level.

In certain embodiments, formulations of the invention that are compatible with parenteral administration comprise one or more cryoprotectants selected from, but not limited to, disaccharides (such as lactose, maltose, sucrose, or trehalose) and polyhydroxy hydrocarbons (such as galactitol, glycerol, mannitol, and sorbitol).

In certain embodiments, the osmolality of the formulation is in the range of about 200mOs/L to about 800mOs/L, with a preferred range of about 250mOs/L to about 500mOs/L, or about 300mOs/L to about 400 mOs/L. The salt-free formulation may contain, for example, from about 5% to about 25% sucrose, and preferably from about 7% to about 15%, or from about 10% to about 12% sucrose. Alternatively, the salt-free formulation may contain, for example, from about 3% to about 12% sorbitol, and preferably from about 4% to 7%, or from about 5% to about 6% sorbitol. If a salt such as sodium chloride is added, the effective range of sucrose or sorbitol is relatively reduced. These and other such osmolality and osmolality considerations are well within the skill of the art.

In certain embodiments, formulations of the invention that are compatible with parenteral administration comprise one or more free radical oxidation inhibitors and/or chelating agents. A variety of free radical scavengers and chelating agents are known in the art and are suitable for use in the formulations and methods of use described herein. Examples include, but are not limited to, ethanol, EDTA/ethanol combinations, triethanolamine, mannitol, histidine, glycerol, sodium citrate, inositol hexaphosphate, tripolyphosphate, ascorbic acid/ascorbate, succinic acid/succinate, malic acid/maleate, desferonium iodonate (desferal), EDDHA and DTPA, and various combinations of two or more thereof. In certain embodiments, the at least one non-reducing free radical scavenger may be added at a concentration effective to enhance the long-term stability of the formulation. One or more free radical oxidation inhibitors/chelating agents can also be added in various combinations, such as a scavenger in combination with a divalent cation. The choice of chelating agent will determine whether or not the addition of a scavenger is required.

In certain embodiments, formulations of the invention that are compatible with parenteral administration comprise one or more nonionic surfactants including, but not limited to, polyoxyethylene sorbitan fatty acid esters, polysorbate-80 (Tween80)), polysorbate-60 (Tween 60), polysorbate-40 (Tween 40) and polysorbate-20 (Tween 20), polyoxyethylene alkyl ethers (including, but not limited to, Brij58, Brij35), and other nonionic surfactants such as Triton X-100(Triton X-100), Triton X-114, NP40, Span85 (Span85), and the pluronic series of nonionic surfactants (e.g., Pluronic 121), preferred components therein are polysorbate-80 at a concentration of about 0.001% to about 2% (wherein up to about 0.25% is preferred) or polysorbate-40 at a concentration of about 0.001% to 1% (wherein up to about 0.5% is preferred).

In certain embodiments, the formulations of the invention comprise one or more other stabilizing agents suitable for parenteral administration, such as a reducing agent comprising at least one thiol group (-SH) (e.g., cysteine, N-acetyl cysteine, reduced glutathione, sodium thioglycolate, thiosulfate, monothioglycerol, or mixtures thereof). Alternatively or optionally, the preservative containing immunogenic composition formulations of the invention may be further stabilized by removing oxygen from the storage container, protecting the formulation from light (e.g., by using an amber glass container).

The preservative-containing immunogenic composition formulations of the present invention may comprise one or more pharmaceutically acceptable carriers or excipients, including any excipient that does not itself induce an immune response. Suitable excipients include, but are not limited to, macromolecules such as proteins, sugars, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, sucrose (Paoletti et al, 2001, Vaccine,19:2118), trehalose, lactose, and lipid aggregates such as oil droplets or lipid particles. Such vectors are well known to those skilled in the art. Pharmaceutically acceptable excipients are discussed, for example, in Gennaro,2000, Remington, the science and practice of pharmacy, 20 th edition, ISBN: 0683306472.

The compositions of the invention may be lyophilized or in aqueous form, i.e., solutions or suspensions. Liquid formulations are preferably administered directly from their packaged form and are therefore ideal for injection without reconstitution in an aqueous medium as would otherwise be required for a lyophilized composition of the invention.

Can be administered parenterally (intramuscular, intraperitoneal, intradermal, subcutaneous, intravenous, or to the interstitial space); or by rectal, oral, vaginal, topical, transdermal, intranasal, ocular, otic, pulmonary or other transmucosal administration to a subject. In a preferred embodiment, parenteral administration is achieved by intramuscular injection, for example, to the thigh or upper arm of the subject. Injection may be achieved via a needle (e.g., a hypodermic needle), but alternatively a needle-free injection may be used. A typical intramuscular dose is 0.5 mL. The compositions of the present invention may be prepared in various forms, for example as liquid solutions or suspensions for injection. In certain embodiments, the compositions may be prepared as a powder or spray for pulmonary administration (e.g., in an inhaler). In other embodiments, the compositions may be prepared as suppositories or pessaries, or for nasal, otic or ocular administration, for example as sprays, drops, gels or powders.

The optimal amounts of the components of a particular immunogenic composition can be determined by standard studies involving observing the appropriate immune response in a subject. After the initial vaccination, the subject may receive one or several booster immunizations at appropriate intervals.

Packaging and dosage form

The immunogenic compositions of the invention may be packaged in unit-dose or multi-dose form (e.g., 2 doses, 4 doses, or more than 4 doses). For multi-dose forms, vials are often, but not necessarily, preferred over prefilled syringes. Suitable multi-dose forms include (but are not limited to): 2 to 10 doses per container, 0.1 to 2mL per dose. In certain embodiments, the dose is a 0.5mL dose. See, for example, international patent application WO2007/127668, which is incorporated herein by reference.

The compositions may be provided in vials or other suitable storage containers, or may be provided in pre-filled delivery devices, such as single or multi-component syringes, which may or may not be provided with a needle. The syringe typically, but not necessarily, contains a single dose of the preservative containing immunogenic composition of the invention, although multiple dose pre-filled syringes are also contemplated. Likewise, a vial may comprise a single dose but may alternatively comprise multiple doses.

Effective dose volumes can be routinely established, but a typical dose of injectable composition has a volume of 0.5 mL. In certain embodiments, the dose is formulated for administration to a human subject. In certain embodiments, the dose is formulated for administration to an adult, juvenile, adolescent, infant or young child (i.e., no more than 1 year old) human subject and in preferred embodiments may be administered by injection.

The liquid immunogenic compositions of the invention are also suitable for reconstitution of other immunogenic compositions provided in lyophilized form. When the immunogenic composition is to be used for such extemporaneous reconstitution, the invention provides a kit having two or more vials, two or more ready-to-fill syringes, or one or more vials and ready-to-fill syringes, wherein the contents of the syringe are used to reconstitute the contents of the vial prior to injection, or vice versa.

Alternatively, the immunogenic compositions of the invention may be lyophilized and reconstituted, for example, using one of the numerous methods well known in the art for lyophilization to form dry, regularly shaped (e.g., spherical) particles, such as microparticles or microspheres, having particle characteristics, such as average diameter size, that can be selected and controlled by varying the precise method used to prepare the particles. The immunogenic composition may further comprise adjuvants, optionally prepared with or contained in separate dry, regularly shaped (e.g. spherical) particles, such as microparticles or microspheres. In these embodiments, the invention further provides an immunogenic composition kit comprising a first component comprising a stable dry immunogenic composition, optionally further comprising one or more preservatives of the invention; and a second component comprising a sterile aqueous solution for reconstituting the first component. In certain embodiments, the aqueous solution comprises one or more preservatives, and may optionally comprise at least one adjuvant (see, e.g., WO2009/109550 (incorporated herein by reference)).

In another embodiment, the container in a multi-dose form is one or more selected from the group consisting of (but not limited to): common laboratory glassware, flasks, beakers, graduated cylinders, fermentors, bioreactors, tubing, catheters (pipe), bags, bottles, vials, vial closures (e.g., rubber stoppers, screw caps), ampoules, syringes, dual or multi-chamber syringes, syringe stoppers, syringe plungers, rubber closures, plastic closures, glass closures, cartridges, and disposable pens and the like. The container of the present invention is not limited by the material of manufacture and includes a variety of materials such as glass, metals (e.g., steel, stainless steel, aluminum, etc.), and polymers (e.g., thermoplastics, elastomers, thermoplastic elastomers). In a particular embodiment, the container in this form is a glass vial of type 5mLSchott1 with a butyl plug. Those skilled in the art will appreciate that the above-described forms are by no means exhaustive and serve merely as a guide to the skilled person with respect to the many forms that can be used with the present invention. Other forms contemplated for use in the present invention may be found in the public catalogues of laboratory equipment vendors and manufacturers, such as united states plastics corp. (Lima, OH), VWR.

Examples

Example 1: experimental procedures

Serum bactericidal assay

Cynomolgus macaques (cynomolgus) (n =5 per group) adsorbed to AlPO₄The rLP2086 or rP2086(a + B) protein of (a). Animals were vaccinated at weeks 0, 4 and 24 and ORF 2086-specific IgG and functional antibody titers were determined at weeks 0, 4,6 and 26. Serum ORF 2086-specific IgG titers were determined against rLP2086A and rLP 2086B.

Functional antibodies were checked for potency by Serum Bactericidal Assay (SBA) against neisseria meningitidis strains expressing LP2086 with sequences homologous or heterologous to those contained in the vaccine.

Serum bactericidal antibodies in macaques or rabbits immunized with the ORF2086 vaccine were determined using SBA with human complement. Rabbit immune serum or cynomolgus monkey immune serum was heat inactivated to remove intrinsic complement activity, followed by 1:2 serial dilutions in Dulbecco's PBS (D-PBS) containing Ca2+ and Mg2+ in 96-well microtiter plates to test serum bactericidal activity against neisseria meningitidis strains. The bacteria used in the assay were grown in GC medium (GCK) supplemented with Kellogg's supplements and monitored by optical density at 650 nm. Final OD at 0.50-0.55₆₅₀The bacteria were collected for use in the assay, diluted in D-PBS, and 1000-3000 CFU's were added to the assay mixture containing 20% human complement.

Human serum without detectable bactericidal activity was used as the exogenous source of complement. The source of the complement was tested for suitability for each individual test strain. The complement source was used only when the number of surviving bacteria in the control without added immune serum was > 75%. 10 unique complement sources were required to perform the SBA described in this study.

At 37 ℃ and 5% CO₂After 30 min of incubation, D-PBS was added to the reaction mixture and aliquots were transferred to a micro-filter plate filled with 50% GCK medium. The micro-filtration plate is filtered at 37 ℃ and 5% CO₂Incubate overnight, stain and quantify the micro-colonies. Serum bactericidal titer was defined as the interpolated reciprocal of the serum dilution that reduced CFU by 50% compared to CFU in control wells without immune serum. SBA titers were defined as the reciprocal of the interpolated dilution of test serum that reduced bacterial counts by 50% after incubation for 30 minutes at 37 ℃. Sensitivity to killing by ORF2086 immune sera was established if SBA titers in ORF2086 immune sera increased 4-fold or more than 4-fold compared to the corresponding preimmune sera. Sera negative for the assay strain at the initial dilution were assigned a titer of half the assay detection limit (i.e., 4).

Example 2: cloning and expression of non-lipidated ORF2086 variants

The mature P2086 amino acid sequence corresponding to residues 27-286 from Neisseria meningitidis strain M98250771(A05) was originally generated by PCR amplification from genomic DNA. The forward primer, sequence TGCCATATGAGCAGCGGAAGCGGAAG (SEQ ID NO:22), was ligated to the 5' sequence and contained an NdeI cloning site. The reverse primer, sequence CGGATCCCTACTGTTTGCCGGCGATGC (SEQ ID NO:23), attached to the 3' end of the gene and containing the stop codon TAG followed by the restriction site BamHI. The 799bp amplified fragment was first cloned into the intermediate vector PCR2.1(Invitrogen, Carlesbac, Calif.). This plasmid was cleaved with NdeI and BamHI and ligated into NdeI and BamHI cleaved expression vector pET9a (Novagen, Madison, Wis.). The resulting vector pLA100 (which includes SEQ ID NO:54) expresses the mature subfamily A05P2086 from strain M98250771 (see SEQ ID NO:13 (where the N-terminal Cys at position 1 is deleted) or SEQ ID NO:55) without the N-terminal cysteine that would be present in the lipidated protein. Expression of fHBP was obtained using the BLR (DE3) E.coli host strain [ F-ompTs hsdSB (rB-mB-) galdcm Δ (srl-recA)306:: Tn10(TetR) (DE3) ] (Novagen).

The same cloning procedure was used to prepare B02, B03, B09, B22, B24, B44, a04, a12, and a22N Cys-deleted variants. Variants containing an N-terminal Cys are also prepared by this same method using a forward primer that also includes a Cys codon (e.g., the first codon of SEQ ID NO: 1-11). Based on the sequences provided herein, one skilled in the art will be able to design respective forward and reverse primers for these variants. For example, the following primers were used to amplify the B44 non-lipidated variant, followed by cloning into pET9a using NdeI and BlpI.

TABLE 1

Results

The non-lipidated plasmid construct was strongly expressed, but the non-lipidated protein variant was acylated with acetone at the N-terminal Cys residue. See examples 8 and 9, which describe, for example, a method of expressing a construct. To overcome this pyruvylation, the N-terminal Cys codon was deleted. See, e.g., example 10. However, deletion of the N-terminal Cys abolished expression of the a22 and B22 variants. See, for example, fig. 4. However, despite deletion of the N-terminal Cys residue, the a05, B01, and B44 variants were still expressed. See, e.g., seq id No. 13(a05), wherein the N-terminal Cys at position 1 is deleted; SEQ ID NO. 35 (end B01N); and SEQ ID NO:21(B44) wherein the N-terminal Cys at position 1 is deleted. See, for example, fig. 5. Furthermore, expression of the non-lipidated B09 variant was not affected by deletion of the N-terminal Cys residue. See, e.g., example 4.

Example 3: effect of Gly/Ser Stem on expression of non-lipidated variants

To determine the reason why the a05, B01, and B44 variants were expressed in the absence of the N-terminal Cys, but not the a22 and B22 variants, the sequences of these variants were aligned. The a05, B01, and B44 variants all have a series of extended 10 or 11 Gly and Ser residues (i.e., Gly/Ser stalk) immediately following the N-terminal Cys. However, the a22 and B22 variants only have a Gly/Ser stem consisting of 6 Gly and Ser residues. Thus, the Gly/Ser stalk of the a22 and B22 variants is extended by the insertion of additional Gly and Ser residues.

Long Gly/Ser stem variants were prepared by the method described in example 2 using a forward primer encoding Gly/Ser stem with 10 or 11 Gly and Ser residues.

The N-terminal Cys deleted long Gly/Ser stem (10-11 Gly/Ser residues) a22 and B22 variants showed increased expression compared to the N-terminal Cys deleted a22 and B22 short Gly/Ser stem (6 Gly/Ser residues) variants. However, these expression levels were still low compared to the expression levels of the a05, B01, and B44 variants.

Example 4: codon optimization

Expression of the non-lipidated B09 variant was not affected by deletion of the N-terminal Cys residue (see SEQ ID NO:18 (where the cysteine at position 1 is deleted) or SEQ ID NO: 49). See, for example, fig. 6. Sequence evaluation of the B09 variant indicated that the B09 variant had a Gly/Ser stalk consisting of 6 Gly and Ser residues, similar to the Gly/Ser stalks of the a22 and B22 variants. In fact, the N-terminal tails of the B09 and a22 variants are identical at the amino acid level. However, the N-terminal tails of the B09 and A22 variants (SEQ ID NOs: 53 and 42, respectively) differ at the nucleic acid level by 2 nucleic acids: nucleic acids 15 and 39 of seq id No. 8. See, for example, fig. 6. The first 14 amino acids of the N-terminal tail of the B22 variant were identical to the B09 and a22 variants, and the N-terminal tail of the B22 variant differed only at the 15 th amino acid. The nucleic acids 1-42 of the B22 variant are identical to the nucleic acids 1-42 of the A22 variant. If there is a difference between nucleic acids 15 and 39 when optimally aligned, nucleic acids 1-42 of the B22 variant (see SEQ ID NO:52) are identical to nucleic acids 1-42 of B09 (see SEQ ID NO: 53). Thus, the B22 variant differs from the B09 variant at amino acids 15 and 39 of seq id No. 8. The last sentence was typed incorrectly and it should be stated that the B22 variant differs from the B09 variant at nucleic acids 15 and 39 of SEQ ID NO. 8.

To determine whether nucleic acid differences affect the amount of expression of the B09 variant compared to the a22 and B22 variants, the a22 and B22 variants were mutated by point mutations to incorporate nucleic acids 15 and 39 into the corresponding codons of Gly5 and Gly 13. Incorporation of these silent nucleic acid mutations significantly increased the expression of a22 and B22N terminal Cys deletion variants to a similar extent as the N-terminal Cys deletion B09 variant. See, for example, fig. 7. Therefore, codon optimization to match the B09 variant may increase expression of the N-terminal Cys deleted non-lipidated P2086 variant.

Further analysis of the non-lipidated variant sequences indicated additional codon optimization in the Gly/Ser stem to improve expression. Thus, other non-lipidated variants were constructed by the method of example 2 using a forward primer comprising these codon-optimized sequences. The forward primers used to generate optimized Gly/Ser stems include any of the following sequences:

ATGAGCTCTGGAGGTGGAGGAAGCGGGGGCGGTGGA(SEQIDNO:28)

MSSGGGGSGGGG(SEQIDNO:29)

ATGAGCTCTGGAAGCGGAAGCGGGGGCGGTGCA(SEQIDNO:30)

MSSGSGSGGGG(SEQIDNO:31)

ATGAGCTCTGGAGGTGGAGGA(SEQIDNO:32)

MSSGGGG(SEQIDNO:33)

ATGAGCAGCGGGGGCGGTGGA(SEQIDNO:34)

MSSGGGG(SEQIDNO:33)

example 5: formulation optimization of immunogenic compositions

The ISCOMATRIX formulated vaccine produced a rapid immune response, resulting in a reduction in the number of doses required to achieve a 4-fold greater response rate as measured in a serum bactericidal assay. Groups of 5 rhesus monkeys (rhesumacaques) were immunized with different formulations of the bivalent non-lipidated rP2086 vaccine. Vaccines include the non-pyruvylated non-lipidated A05 variant (SEQ ID NO:13 (wherein the N-terminal Cys at position 1 is deleted) or SEQ ID NO:55 encoded by SEQ ID NO:54) and the non-pyruvylated non-lipidated B44 variant (SEQ ID NO:21 (wherein the N-terminal Cys at position 1 is deleted) or SEQ ID NO:44 encoded by SEQ ID NO: 51). The adjuvant units are as follows: AlPO₄At 250mcg, ISCOMATRIX is between 10 and 100 mcg. AlPO shown in tables 2 to 5₄The adjuvant unit of (a) is shown in milligram units and thus in contrast to 250mcg, is shown to be 0.25 (milligrams).

Immunization schedule was 0, 4 and 24 weeks with blood collected at 0, 4,6 and 26 weeks. For any group, there was no increase in SBA titers after dose 1. After dose 2, for the formulation containing ISCOMATRIX adjuvant, an increase in SBA titers and number of responders as determined by a 4-fold increase in SBA titers compared to baseline values was observed. Tables 2 and 3 provide the observed SBAGMT against fHBP subfamily a and B strains, respectively. ISCOMATRIX formulations SBAGMT against subfamily A and B strains, respectively, was the observed AlPO₄Preparation3-19 and 4-24 times of SBAGMT. Enhanced titers of iscomapatrix formulations against fHBP subfamily a and B strains were also observed after dose 3, compared to AlPO₄The preparation is 13-95 times and 2-10 times. Analysis of the responder ratio as determined by SBA titers increased 4-fold or more than 4-fold over baseline values revealed similar trends (tables 4 and 5).

Example 6: immunoprotection conferred by lipidated and non-lipidated variants

Recombinantly expressed non-lipidated P2086 variants (B44) induced responses to the expression of different fHBP variants (about 85% to about 85% >) as measured by SBA<92% identity) wide protection of strains of LP2086 sequences. These reaction rates are for AlPO₄A non-lipidated vaccine formulated together. See table 6, which shows SBA response rates to subfamily BfHBPMnB strains produced by bivalent fHBP vaccines. Non-lipidated vaccines (indicated by "-" under the column "lipidation") include the non-pyruvylated non-lipidated a05 variant (seq id no:13 (wherein the N-terminal Cys at position 1 is deleted) or encoded by seq id no:54Seq id No. 55) and the non-pyruvylated non-lipidated B44 variant (seq id No. 21 (wherein the N-terminal Cys at position 1 is deleted) or seq id No. 44 encoded by seq id No. 51).

Alternatively, recombinantly expressed non-lipidated P2086 variants (B44) induced a greater immune response against strains with similar (>92% identity) and different (<92% identity) LP2086 sequences than lipidated variants (B01), as measured by SBA titers. It was observed that the vaccine containing non-lipidated rP2086B44 had a higher response rate (as determined by a 4-fold or more-fold increase in SBA titers relative to baseline) compared to lipidated rLP2086B01 vaccine (table 6).

According to table 6, non-lipidated B44 is a preferred subfamily B component of fHBP in compositions for providing a broad spectrum of action against a variety of LP2086 variant strains (e.g., eliciting bactericidal antibodies against a variety of LP2086 variant strains).

Surprisingly, the inventors noted that LP2086B09 variant strains are particularly unlikely to have good SBA response rates for heterologous (non-B09) ORF2086 polypeptides. In particular, the inventors found that LP2086B09 is the exception for the assay strain against which the a05/B44 immunogenic composition described in table 6 elicits bactericidal antibodies. Thus, in a preferred embodiment, the immunogenic compositions of the invention include a B09 polypeptide, particularly in the case of compositions that include more than one ORF2086 subfamily B polypeptide. In a preferred embodiment, an immunogenic composition comprising non-lipidated B44 comprises a non-lipidated B09 polypeptide.

Example 7: codon optimization of B44 and B09 variants

Although the amount of expression achieved in the previous examples is sufficient for many applications, further optimization is desirable, and therefore E.coli expression constructs containing other codon optimizations over the full length of the protein are prepared and tested. One such improved sequence found for expression of Cys-free B44 protein is the nucleic acid sequence set forth in SEQ ID NO: 43. As shown in example 9, the expression construct containing seq id no:43 showed increased expression compared to the expression of the non-optimized wild type sequence.

Expression of the N-terminal Cys deleted B09 protein was improved by applying the codon changes from the above optimized B44(SEQ ID NO:43) to B09(SEQ ID NO: 48). To generate the optimized B09 sequence, the B44 optimized DNA sequence (SEQ ID NO:43) was first aligned with the DNA sequence of the B09 allele (SEQ ID NO: 48). The entire non-lipidated coding sequence of the B09 allele (SEQ ID NO:48) was optimized to reflect the codon changes seen in the B44 optimized allele (SEQ ID NO:43), in whatever case the amino acids between B44(SEQ ID NO:44) and B09(SEQ ID NO:49) were identical. The codon sequence in the B09 allele that corresponds to the same amino acid between the B09 allele and the B44 allele was altered to reflect the codons used in the B44 optimized sequence (SEQ ID NO: 43). The codon sequence of the amino acids differing between B09(SEQ ID NO:49) and B44(SEQ ID NO:44) in the B09DNA sequence was not altered.

In addition, the non-lipidated B44 amino acid sequence (SEQ ID NO:44) contains 2 consecutive serine-glycine repeats (S-G-G-G-G) (SEQ ID NO:56) at its N-terminus (see also amino acids 2 to 6 of SEQ ID NO:44), whereas the B09 allele contains only one serine-glycine repeat at the N-terminus (see amino acids 2 to 6 and amino acids 7 to 11 of SEQ ID NO: 49). The 2 serine-glycine repeats at the N-terminus of B44 (amino acids 2 to 6 and amino acids 7 to 11 of seq id no:44) also used different codons (see nucleotides 4 to 18 and nucleotides 19 to 33 of seq id no:43), and different combinations of the optimized B44 serine-glycine repeats (e.g. nucleotides 4 to 18 of seq id no:43, or nucleotides 19 to 33 of seq id no:43, or combinations thereof) were applied to the B09DNA sequence (seq id no:48, e.g. nucleotides 4 to 18 of seq id no:48) to examine the effect on recombinant protein expression.

3 different optimized B09 forms were constructed: SEQ ID NO:45 contains 2 serine-glycine repeats (GS1 and GS2) from optimized B44 (nucleic acids 4 to 33 of SEQ ID NO:43), SEQ ID NO:46 contains GS1 (nucleic acids 4 to 18 of SEQ ID NO:43), and SEQ ID NO:47 contains GS2 (nucleic acids 19 to 33 of SEQ ID NO: 43). All of the above codon-optimized sequences of DNA were chemically synthesized using standard in chemical techniques. The resulting DNA was cloned into an appropriate plasmid expression vector and tested for expression in e.coli host cells as described in examples 8 and 9.

Example 8: methods of expressing ORF2086B09 variants

Cells of the E.coli K-12 strain (derivative of wild-type W3110(CGSC4474), deletion of recA, fhuA and araA) were transformed with plasmid pEB063 comprising SEQ ID NO:45, pEB064 comprising SEQ ID NO:46, pEB065 comprising SEQ ID NO:47 or pLA134 comprising SEQ ID NO: 48. Preferred modifications to the K-12 strain are useful for fermentation purposes but are not required for protein expression.

After 8 hours of incubation at 37 ℃, linear glucose feeding was applied and further incubation for 3 hours was continued isopropyl β -D-1-thiogalactopyranoside (IPTG) was added to the culture to reach a final concentration of 0.1mM followed by 12 hours of incubation at 37 ℃, cells were collected by centrifugation at 16,000 × g for 10 minutes and by addition of Easy-lysine from lienco technologies (st^TMLysis kit' and loading buffer. Expression of B09 in the clarified lysate was analyzed by Coomassie staining and/or Western blot analysis of SDS-PAGE gels, quantified using a scanning densitometer. The results of the scanning densitometry are in table 7 below:

example 9: methods of expressing ORF2086B44 variants

Coli B strain (BLR (DE3), Novagen) was transformed with plasmid pLN056 including SEQ ID NO:51 cells of E.coli K-12 strain (derivative of wild type W3110) were transformed with plasmid pDK087 including SEQ ID NO:43 cells were inoculated into glucose-salt determination medium after 8 hours of incubation at 37 ℃, linear glucose feed was applied and further incubation was continued for 3 hours, isopropyl β -D-1-thiogalactopyranoside (IPTG) was added to the culture to reach a final concentration of 0.1mM, followed by 12 hours of incubation at 37 ℃, cells were collected by centrifugation at 16,000 × g for 10 minutes and by addition of Easy-lysine from Lienco technologies (St. Louis, Mo)^TMLysis kit' and loading buffer. The expression of B09 in the supernatant was analyzed by coomassie staining and/or western blot analysis of SDS-PAGE gels, and quantification was performed with a scanning densitometer. The results of the scanning densitometry are in table 8 below:

example 10: acylation of acetone

This example illustrates that the N-terminal Cys residue of the non-lipidated ORF2086 protein can be pyruvylated when expressed in e.g. e.

Reverse phase high performance liquid chromatography (RP-HPLC) was used to monitor the accumulation of heterologous protein during the production of variants A05(SEQ ID NO:13) and B44(SEQ ID NO: 21). This separation is combined with a quadrupole time-of-flight mass spectrometer (QTOF-MS) to provide a means of monitoring the formation of product-related variants.

After expression in E.coli B and/or K-12 host cells, the products produced from these fermentations were subjected to a purification procedure during which product modification was observed. Mass spectrometric deconvolution characterized variants exhibited mass changes of +70Da compared to the natural products of a05 and B44 (27640 and 27572Da, respectively).

Published literature indicates that mass changes of +70Da have been previously observed in proteins and have been attributed to acetonylation of amino-terminal residues.

Mass spectrometry data (MS/MS) was used to confirm the presence and position of pyruvate groups. The data indicate that the modification was at the amino-terminal cysteine residues of a05 and B44, i.e., at the amino acid at position 1. For A05, the percentage of acetone-acylated polypeptides was about 30% compared to the total number of A05 polypeptides (SEQ ID NO: 13). For B44, the percentage of pyruvylated polypeptides was about 25% compared to the total number of B44 polypeptides (SEQ ID NO: 21).

When A05(SEQ ID NO:13 (wherein the N-terminal Cys at position 1 is deleted) or SEQ ID NO:55) and the B44 variant (SEQ ID NO:21 (wherein the N-terminal Cys at position 1 is deleted) or SEQ ID NO:44) which do not contain an amino-terminal cysteine were purified, no acetoylation (+70Da) was detectable.

Example 11: immunogenicity of B09 and B44 alone and in combination

5-10 groups of rhesus monkeys were dosed with 250mcgAlPO per dose₄The B09 variant (SEQ ID NO:49 encoded by SEQ ID NO:48) or the B44 variant (SEQ ID NO:44 encoded by SEQ ID NO:43), or A05, B09 and B44(SEQ ID NO:55, SEQ ID NO:49 encoded by SEQ ID NO:48, and SEQ ID NO:44 encoded by SEQ ID NO:43, respectively) formulated together. Monkeys were vaccinated via intramuscular route at weeks 0, 4 and 8 with 10mcg of each non-lipidated fHBP, alone or in combination, as listed in tables 9 and 10. Both week 0 serum samples and week 12 serum samples were analyzed in SBA for MnB strains with subfamily a or subfamily BfHBP variants. Animals with a 4-fold increase in titer were recorded as responders. The B44 variant tested was the optimized construct (seq id no:43) and the broad response rate of this optimized construct B44 vaccine (alone or in combination with B09) observed in previous studies (table 9) was maintained (above table). The B09 vaccine alone (Table 10) also produced widespread cross-breedingCross-reactive immune responses (table 10).

Table 9: response rates of non-lipidated fHBP vaccines obtained in rhesus monkeys

Rhesus monkeys (n =10) were used at weeks 0, 4 and 8 with 250mcgAlPO alone or in combination as listed in the vaccine bar₄10mcg of each non-lipidated fHBP formulated together were immunized intramuscularly. Both week 0 serum samples and week 10 serum samples were analyzed in SBA for the MnB strains listed in the table. Animals with a 4-fold increase in titer were recorded as responders.

Table 9 indicates that compositions comprising combinations of non-pyruvylated non-lipidated B44, B09 and a05 show higher cross-reactivity rates against test variants, e.g. compared to cross-reactivity rates (cross-coverage) of compositions comprising only B44. In view of the results shown in the present application (including specifically table 6 together with table 9), compositions comprising B44, B09, and a05, alone or in combination, are preferred embodiments of the present invention. Specifically, compositions comprising both B44 and B09 are disclosed. Preferably, the composition additionally comprises a subfamily a polypeptide, such as in particular a 05.

Table 10: response rates of non-lipidated fHBPB09 vaccine obtained in rhesus monkeys

Rhesus monkeys (n =5) were used at weeks 0, 4 and 8 with 250mcgAlPO alone or in combination as listed in the vaccine bar₄10mcg of each non-lipidated fHBP formulated together were immunized intramuscularly. Both week 0 serum samples and week 10 serum samples were analyzed in SBA for the MnB strains listed in the table. Animals with a 4-fold increase in titer were recorded as responders.

Example 12: immunoprotection conferred by lipidated and non-lipidated variant constructs

20 female New Zealand (New Zealand) white rabbits, 2.5-3.5kg, obtained from Charles river Canada, were pre-screened by whole cell ELISA and 10 animals were selected for this study based on their low background titers against the test strains representing fHBP variants B02(SEQ ID NO:16) and B44(SEQ ID NO:21) (Table 11). Each group of 3 animals was immunized intramuscularly with 100 μ g of each protein formulated with 50 μ g ISCOMATRIX per 0.5ml dose at weeks 0, 4 and 9 (Table 12). Group 1 was inoculated with non-lipidated B44(SEQ ID NO: 44). Comprising with AlPO₄(250mcg) lipidated B01 inoculated control group formulated together. Rabbits were bled at weeks 0, 4,9 and 10. Individual sera from week 10 were prepared and analyzed by serobactericidal assay against a variety of serogroup B neisseria meningitidis strains from the fHBPB subfamily.

Table 11: rabbits used in the study

TABLE 12

Immunization protocol: weeks 0, 4, 9; the blood sampling scheme is as follows: week 0, 4,9, 10

Serum Bactericidal Assay (SBA): the micro-population based Serum Bactericidal Assay (SBA) was performed on individual serum samples against a variety of serogroup B neisseria meningitidis strains (table 13). Human serum from donors is suitable as a source of complement for the strains tested in the assay. Complement-mediated antibody-dependent bactericidal titers were interpolated and expressed as the reciprocal of the dilution of test serum that killed 50% of neisseria meningitidis cells in the assay. The detection limit determined was an SBA titer equal to 4. SBA titers of <4 were assigned a value of 2. And calculating and comparing the titer of the serum before blood collection, wherein the SBA titer in the serum at the 10 th week is increased by more than or equal to 4 times.

Serum bactericidal antibody activity as measured in SBA is an immune surrogate for protection against neisseria meningitidis disease. Immunization with non-lipidated rfHBP was assayed by SBA for the ability to elicit bactericidal antibodies in rabbits. SBA measures antibody levels in serum samples by mimicking naturally occurring complement-mediated bacterial lysis. Rabbit serum samples collected from week 10 were analyzed by SBA against strains with fHBP homologous to the B44 vaccine, and strains with fHBP closely related to the B01 vaccine (B02). As shown in table 14, at 1 week (week 10) after the third immunization, all serum samples showed bactericidal activity against both test strains. (Table 14). In New Zealand rabbits, it appears that non-lipidated B44(SEQ ID NO:44) is more immunogenic than non-lipidated B01. B44 is in the same subgroup as B01 (N4/N5). Non-lipidated B44(seq id no:44) formulated with iscomatrix adjuvant gave titers similar to lipidated B01 formulated with aluminum phosphate. The pre-blood serum of the rabbits showed substantially no pre-existing bactericidal activity against the test strain.

Table 13: serum bactericidal activity against fHBP subfamily B strains in new zealand white rabbits vaccinated with recombinant non-lipidated fHBP

Example 13: immunogenicity of 6 non-lipidated factor H binding proteins in New Zealand white rabbits

Groups of 5 rabbits were immunized with the non-lipidated fHBP variants as described in table 15. The vaccine was administered at weeks 0, 4 and 9. Rabbit serum samples collected from weeks 0 and 10 were analyzed by SBA against strains with homologous and heterologous fHBP sequences. Table 15 shows the percentage of responders after the third immunization. At 1 week (week 10) after the third immunization, all serum samples showed bactericidal activity against the homologous strain as well as other test strains from the same fHBP subfamily. The pre-blood serum of the rabbits showed substantially no pre-existing bactericidal activity against the test strain.

Table 14: percent responders after dose 3 in New Zealand white rabbits vaccinated with recombinant non-lipidated fHBP

MnBfHBP protein used

Test variants in table 14:

the present invention also provides the following embodiments as defined in the following clauses:

C1. an immunogenic composition comprising a P2086 polypeptide, wherein the P2086 is a B44, B02, B03, B22, B24, B09, a05, a04, a12, or a22 variant.

C2. An immunogenic composition comprising a P2086 subfamily B polypeptide, wherein the P2086 subfamily B polypeptide is a B44, B02, B03, B22, B24, or B09 variant.

C3. The immunogenic composition of C2, further comprising a P2086 subfamily a polypeptide.

C4. The immunogenic composition of C3, wherein the P2086 subfamily a polypeptide is an a05, a04, a12, or a22 variant.

C5. The immunogenic composition according to any one of C1 to 4, wherein the composition further comprises an adjuvant.

C6. The immunogenic composition of C5, wherein the adjuvant is selected from the group consisting of:

a) an aluminum adjuvant;

b) saponin

c) A CpG nucleotide sequence; and

d) any combination of a), b), and c).

C7. The immunogenic composition of C6, wherein the aluminum adjuvant is selected from the group consisting of: AlPO₄、Al(OH)₃、Al₂(SO₄)₃And alum.

C8. An immunogenic composition such as C6 or C7, wherein the aluminum concentration is between 0.125 μ g/ml and 0.5 μ g/ml.

C9. The immunogenic composition according to C8, wherein the aluminum concentration is 0.25 μ g/ml.

C10. The immunogenic composition according to any of C6 to 9, wherein the saponin concentration is between 1 μ g/ml and 250 μ g/ml.

C11. The immunogenic composition according to C10, wherein the saponin concentration is between 10 μ g/ml and 100 μ g/ml.

C12. The immunogenic composition according to C10, wherein the saponin concentration is 10 μ g/ml.

C13. The immunogenic composition according to C10, wherein the saponin concentration is 100 μ g/ml.

C14. The immunogenic composition of any of C6-13, wherein the saponin is QS-21 or ISCOMATRIX.

C15. The immunogenic composition according to any one of C1 to 14, wherein the composition confers the ability to elicit an immunogenic response to neisseria meningitidis bacteria after multiple doses have been administered to a subject.

C16. The immunogenic composition of C15, wherein the immunogenic response to the neisseria meningitidis bacterium is conferred after 2 doses have been administered to the subject.

C17. The immunogenic composition of C15, wherein the immunogenic response to the neisseria meningitidis bacterium is conferred after 3 doses have been administered to the subject.

C18. A composition that confers increased immunogenicity to a non-lipidated P2086 antigen, wherein the composition comprises a saponin and at least one non-lipidated P2086 antigen.

C19. The immunogenic composition according to C18, wherein the saponin concentration is between 1 μ g/ml and 250 μ g/ml.

C20. The immunogenic composition according to C19, wherein the saponin concentration is between 10 μ g/ml and 100 μ g/ml.

C21. The immunogenic composition according to C19, wherein the saponin concentration is 10 μ g/ml.

C22. The immunogenic composition according to C19, wherein the saponin concentration is 100 μ g/ml.

C23. The immunogenic composition of any of C18-22, wherein the saponin is QS-21 or ISCOMATRIX.

C24. The immunogenic composition of any one of C18-23, further comprising aluminum.

C25. The immunogenic composition of C24, wherein the aluminum concentration is between 0.125 μ g/ml and 0.5 μ g/ml.

C26. The immunogenic composition according to C25, wherein the aluminum concentration is 0.25 μ g/ml.

C27. The immunogenic composition according to any one of C18 to 26, wherein the composition confers an immunogenic response to neisseria meningitidis bacteria after multiple doses have been administered to a subject.

C28. The immunogenic composition of C27, wherein the immunogenic response to the neisseria meningitidis bacterium is conferred after 2 doses have been administered to the subject.

C29. The immunogenic composition of C27, wherein the immunogenic response to the neisseria meningitidis bacterium is conferred after 3 doses have been administered to the subject.

C30. The immunogenic composition of any one of C18-29, wherein the non-lipidated P2086 antigen is a non-lipidated P2086 subfamily B polypeptide.

C31. The immunogenic composition of C30, wherein the non-lipidated P2086 subfamily B polypeptide is a B44, B02, B03, B22, B24, or B09 variant.

C32. The immunogenic composition of any one of C18-29, wherein the non-lipidated P2086 antigen is a non-lipidated P2086 subfamily a polypeptide.

C33. The immunogenic composition of C32, wherein the non-lipidated P2086 subfamily a polypeptide is an a05, a04, a12, or a22 variant.

C34. The immunogenic composition according to any one of C18-33, wherein the composition comprises at least two non-lipidated P2086 antigens, wherein the two non-lipidated P2086 antigens are at least one non-lipidated P2086 subfamily a polypeptide and at least one non-lipidated P2086 subfamily B polypeptide.

C35. The immunogenic composition of C34, wherein the non-lipidated P2086 subfamily a polypeptide is an a05 variant and the non-lipidated P2086 subfamily B polypeptide is a B44 variant.

C36. The immunogenic composition of C34, wherein the non-lipidated P2086 subfamily a polypeptide is an a05 variant and the non-lipidated P2086 subfamily B polypeptide is a B22 variant.

C37. The immunogenic composition of C34, wherein the non-lipidated P2086 subfamily a polypeptide is an a05 variant and the non-lipidated P2086 subfamily B polypeptide is a B09 variant.

C38. A method of conferring immunity to a neisseria meningitidis bacterium in a subject, wherein the method comprises the step of administering to the subject an immunogenic composition comprising a P2086 subfamily B polypeptide, wherein the P2086 subfamily B polypeptide is a B44, B02, B03, B22, B24, or B09 variant.

C39. The method of C38, wherein the immunogenic composition further comprises a P2086 subfamily a polypeptide.

C40. The method of C39, wherein the P2086 subfamily a polypeptide is an a05, a04, a12, or a22 variant.

C41. The method of any one of C38-40, wherein the immunogenic composition further comprises an adjuvant.

C42. The method of C41, wherein the adjuvant is selected from the group consisting of:

a) an aluminum adjuvant;

b) saponin

c) A CpG nucleotide sequence; and

d) any combination of a), b), and c).

C43. The method of C42, wherein the aluminum adjuvant is selected from the group consisting of: AlPO₄、Al(OH)₃、Al₂(SO₄)₃And alum.

C44. The method of C42 or 43, wherein the aluminum concentration is between 0.125 μ g/ml and 0.5 μ g/ml.

C45. The method of C44, wherein the aluminum concentration is 0.25. mu.g/ml.

C46. The method of any one of C42-45, wherein the saponin concentration is between 1 μ g/ml and 250 μ g/ml.

C47. The method of C46, wherein the saponin concentration is between 10 μ g/ml and 100 μ g/ml.

C48. The method according to C47, wherein the saponin concentration is 10 μ g/ml.

C49. The method according to C48, wherein the saponin concentration is 100 μ g/ml.

C50. The method of any one of C42 to 49, wherein the saponin is QS-21 or ISCOMATRIX.

C51. The method of any of claims C38 to 50, wherein the immunogenic composition is administered to the subject in multiple doses in a dosing regimen.

C52. The method of C51, wherein the immunogenic composition is administered to the subject in 2 doses in a dosing regimen.

C53. The method of C51, wherein the immunogenic composition is administered to the subject in 3 doses in a dosing regimen.

C54. A method of producing a non-lipidated P2086 variant polypeptide, comprising the steps of:

a) cloning the ORF2086 variant nucleic acid sequence into an escherichia coli expression vector;

b) transforming a bacterium with the ORF2086 expression vector;

c) inducing expression; and

d) isolating the expressed P2086 protein;

wherein the ORF2086 expression vector does not comprise lipidation control sequences.

C55. The method of C54, wherein the codon encoding the N-terminal Cys of the ORF2086 variant is deleted.

C56. Such as C54, wherein the codon encoding the N-terminal Cys of the ORF2086 variant is mutated to generate an Ala, Gly, or Val codon.

C57. The method of C55 or 56, wherein the ORF2086 variant is an a05, B01, or B44 variant.

C58. The method of any one of C54-57, wherein the N-terminal tail is mutated to add Ser and Gly residues to extend the Gly/Ser stalk immediately downstream of the N-terminal Cys.

C59. The method of C58, wherein the total number of Gly and Ser residues in the Gly/Ser stem is at least 7.

C60. The method of C58, wherein the total number of Gly and Ser residues in the Gly/Ser stem is at least 8.

C61. The method of C58, wherein the total number of Gly and Ser residues in the Gly/Ser stem is at least 9.

C62. The method of C58, wherein the total number of Gly and Ser residues in the Gly/Ser stem is at least 10.

C63. The method of C58, wherein the total number of Gly and Ser residues in the Gly/Ser stem is at least 11.

C64. The method of C58, wherein the total number of Gly and Ser residues in the Gly/Ser stem is at least 12.

C65. The method according to any one of claims C54-57, wherein the codons for the N-terminal tail of the ORF2086 variant are optimized by point mutagenesis such that the codons encoding the 5 th amino acid of the ORF2086 variant are 13-15100% identical to nucleotides of seq id No. 8 and the codons encoding the 13 th amino acid of the ORF2086 variant are 37-39100% identical to nucleotides of seq id No. 8.

C66. The method of C65, wherein the codon of the N-terminal tail of the ORF2086 variant is 100% identical to nucleotides 1-458 of seq id No. 8.

C67. The method of C65, wherein the codon of the N-terminal tail of the ORF2086 variant is 100% identical to nucleotides 4-458 of seq id No. 8.

C68. The method of C65, wherein the codon at the N-terminal tail of the ORF2086 variant is 100% identical to nucleotides 4-42100 of seq id No. 8.

C69. Such as C65, wherein the N-terminal tail of the protein encoded by the ORF2086 variant comprises at least one amino acid substitution as compared to amino acids 1-15 of SEQ ID NO: 18.

C70. Such as C65, wherein the N-terminal tail of the protein encoded by the ORF2086 variant comprises at least one amino acid substitution as compared to amino acids 2-15 of SEQ ID NO: 18.

C71. Such as C65, wherein the N-terminal tail of the protein encoded by the ORF2086 variant comprises two amino acid substitutions as compared to amino acids 1-15 of SEQ ID NO: 18.

C72. Such as C65, wherein the N-terminal tail of the protein encoded by the ORF2086 variant comprises two amino acid substitutions as compared to amino acids 2-15 of SEQ ID NO: 18.

C73. The method of any one of C69-72, wherein the amino acid substitution is a conservative amino acid substitution.

C74. The method of any one of C65-73, wherein the ORF2086 variant is an a22 or B22 variant.

C75. The method of any one of C55 to 74, wherein expression is induced by addition of IPTG.

C76. The method of any one of C55-75, wherein the bacterium is e.

Claims (36)

1. A composition comprising an isolated non-pyruvylated non-lipidated ORF2086 polypeptide, wherein the polypeptide does not exhibit a mass change of +70Da compared to a corresponding wild-type non-lipidated polypeptide when measured by mass spectrometry, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO 13, SEQ ID NO 18, SEQ ID NO 58, and SEQ ID NO 21, wherein the cysteine at position 1 is absent.

2. The composition of claim 1, wherein the composition is an immunogenic composition.

3. The composition of claim 1, wherein the polypeptide comprises a deletion of an N-terminal Cys compared to a corresponding wild-type, non-lipidated ORF2086 polypeptide.

4. The composition of claim 1, wherein the cysteine at position 1 is deleted.

5. The composition of claim 4, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: SEQ ID NO. 44, SEQ ID NO. 49, SEQ ID NO. 50 and SEQ ID NO. 55.

6. The composition of claim 1, wherein the polypeptide is encoded by a nucleotide sequence operably linked to an expression system, wherein the expression system is capable of expression in a bacterial cell.

7. The composition of claim 6, wherein the expression system is a plasmid expression system.

8. The composition of claim 6, wherein the bacterial cell is an E.

9. The composition of claim 6, wherein said nucleotide sequence is linked to a regulatory sequence that controls expression of said nucleotide sequence.

10. The composition of claim 1, wherein the polypeptide comprises an N-terminal Cys substitution compared to a corresponding wild-type, non-lipidated ORF2086 polypeptide.

11. A composition comprising a non-pyruvylated non-lipidated ORF2086 polypeptide obtainable by a method comprising expressing a nucleotide sequence encoding a polypeptide having an amino acid sequence selected from the group consisting of seq id no:13, 18, 58 and 21, wherein the cysteine at position 1 is deleted, wherein said nucleotide sequence is operably linked to an expression system capable of expression in a bacterial cell.

12. The composition of claim 11, wherein the bacterial cell is escherichia coli (e.

13. A composition comprising an isolated polypeptide having an amino acid sequence shown in seq id No. 49 and an isolated polypeptide having an amino acid sequence shown in seq id No. 44.

14. The composition of any one of claims 1 to 13, wherein the composition is an immunogenic composition.

15. The composition of claim 13, further comprising an ORF2086 subfamily a polypeptide from serogroup B neisseria meningitidis (neuroseriameningitis).

16. The composition according to any one of claims 1 to 13, wherein the composition elicits a bactericidal immune response in a mammal against an ORF2086 subfamily B polypeptide of serogroup B neisseria meningitidis.

17. An isolated polypeptide has an amino acid sequence of SEQ ID NO. 49.

18. An isolated nucleotide sequence consisting of SEQ ID NO. 46.

19. An isolated nucleotide sequence consisting of SEQ ID NO: 47.

20. An isolated nucleotide sequence consisting of SEQ ID NO 48.

21. An isolated polypeptide has an amino acid sequence of SEQ ID NO 50.

22. An isolated nucleotide sequence consisting of SEQ ID NO: 45.

23. An isolated polypeptide has an amino acid sequence of SEQ ID NO: 44.

24. A plasmid comprising a nucleotide sequence selected from the group consisting of seq id No. 46, seq id No. 47, seq id No. 48 and seq id No. 45, wherein said plasmid is capable of being expressed in a bacterial cell.

25. The plasmid of claim 24, wherein the bacterial cell is e.

26. Use of an isolated polypeptide comprising an amino acid sequence selected from the group consisting of seq id no:44 and seq id no:49, or a combination thereof, for eliciting a bactericidal antibody in a mammal that is specific for ORF2086 subfamily B of serogroup B neisseria meningitidis.

27. An immunogenic composition comprising an ORF2086 subfamily B polypeptide from serogroup B neisseria meningitidis, wherein the polypeptide is non-pyruvylated non-lipidated B44 having the amino acid sequence of seq id no: 44.

28. The composition of claim 27, further comprising a second ORF2086 subfamily B polypeptide from serogroup B neisseria meningitidis, wherein the second polypeptide is non-pyruvylated non-lipidated B09 having the amino acid sequence of seq id no: 49.

29. The composition of claim 27, wherein the composition comprises up to 3 ORF2086 subfamily B polypeptides.

30. The composition of claim 27, wherein the composition includes up to two ORF2086 subfamily B polypeptides.

31. The composition of claim 27, wherein the composition further comprises one or more ORF2086 subfamily a polypeptides.

32. The composition of claim 31, wherein the composition comprises subfamily a polypeptide a05 having the amino acid sequence of seq id No. 55.

33. A composition comprising an isolated non-pyruvylated non-lipidated ORF2086 polypeptide having an amino acid sequence shown in seq id no:13, wherein the cysteine at position 1 is deleted.

34. A composition comprising an isolated non-pyruvylated non-lipidated ORF2086 polypeptide having an amino acid sequence shown in seq id no:18, wherein the cysteine at position 1 is deleted.

35. A composition comprising an isolated non-pyruvylated non-lipidated ORF2086 polypeptide having an amino acid sequence shown in seq id no:21, wherein the cysteine at position 1 is deleted.

36. A composition comprising an isolated non-pyruvylated non-lipidated ORF2086 polypeptide having an amino acid sequence shown in seq id no:58, wherein the cysteine at position 1 is deleted.