WO2008016984A2

WO2008016984A2 - Polysaccharide vaccine and uses thereof

Info

Publication number: WO2008016984A2
Application number: PCT/US2007/074985
Authority: WO
Inventors: Lawrence C. Madoff; Lawrence Paoletti; Dennis L. Kasper; Catherine Lachenauer
Original assignee: Brigham and Womens Hospital Inc
Current assignee: Brigham and Womens Hospital Inc
Priority date: 2006-08-01
Filing date: 2007-08-01
Publication date: 2008-02-07
Anticipated expiration: 2009-02-01
Also published as: WO2008016984A3

Abstract

This invention relates to antigenic compositions that include an ALP3 polypeptide in combination with a polysaccharide such as a group B streptococcus polysaccharide and to methods of using the compositions to elicit an immune response.

Description

POLYSACCHARIDE VACCINE AND USES THEREOF

GOVERNMENT SUPPORT

The work described herein was funded, in part, through grants from the National Institutes of Health (Grant Nos. AI07061, AI01388, AI3842402, AI60603 and A125152). The United States government may therefore have certain rights in the invention.

TECHNICAL FIELD

This invention relates to antigenic compositions that include a group B Streptococcus polypeptide and to methods of using the compositions to elicit an immune response.

BACKGROUND

Group B Streptococcus (GBS; Streptoccocus agalactiae) is a gram-positive coccus that frequently colonizes the human genital and gastrointestinal tracts. GBS is a common cause of invasive infection in neonates, pregnant women, and immunocompromised subjects. Prior to the 1990's, virtually all invasive GBS infections were caused by serotypes Ia, Ib, II, and III. Recently, serotype VIII group B streptococci have become increasingly prevalent in Japan and represent more than 20% of colonizing isolates.

SUMMARY

The invention is based, in part, on the discovery of an alpha-like protein 3 (ALP3) in group B Streptococcus that induces an immune response to polysaccharide antigens. The ALP3 polypeptide can be used alone or in combination with a polysaccharide antigen, e.g., a group B streptococcal antigen. The ALP3 polypeptide can also be used as a carrier for polysaccharides, e.g., GBS polysaccharides, which induce a reduced immune response as compared to the response induced when conjugated with an ALP3 polypeptide.

In one aspect, the invention features an antigenic composition that includes an ALP3 polypeptide in combination with, e.g., physically associated with, a polysaccharide, e.g., a GBS polysaccharide. The combination can enhance immunogenicity of the polysaccharide in a host organism and/or stimulate an immune response which is more effective for protecting a host organism than the polysaccharide in the absence of an ALP3 polypeptide. In one embodiment, the ALP3 polypeptide is covalently associated with the polysaccharide, e.g., the GBS polysaccharide. In one embodiment, the ALP3 polypeptide can be linked directly or via a linking agent to the polysaccharide, e.g., the GBS polysaccharide. In one embodiment, the ALP3 polypeptide is linked to the polysaccharide, e.g., the GBS polysaccharide, via an amine bond between an epsilon amino group and a reduced sialic acid residue. For example, the ALP3 polypeptide can be linked to the polysaccharide, e.g., the GBS polysaccharide, via reductive amination.

In one embodiment, the polysaccharide is a polysaccharide described herein.

In one embodiment, the polysaccharide is a GBS polysaccharide from a GBS type Ia, Ib, II, III, IV, V, VI, VII, or VlII strain or modified polysaccharides thereof. In one embodiment, the GBS polysaccharide is a GBS type V polysaccharide, a GBS type VIII polysaccharide, or modified polysaccharides thereof. Examples of GBS polysaccharides include, but are not limited to, those described in U.S. Publication No. 20060134142, the contents of which are incorporated herein by reference.

In various embodiments, the composition includes a pharmaceutically acceptable carrier, and/or an adjuvant, such as alum, or complete or incomplete Freund's adjuvant.

In one embodiment, the composition includes one or more additional polypeptides, one or more additional polysaccharides, or combinations thereof. Examples of additional polypeptides and polysaccharides include, but are not limited to, an alpha C protein polypeptide, an Rib polypeptide, a GBS beta C protein polypeptide, an epsilon polypeptide, an R protein (e.g., an Rl or R4 protein), an alpha-like protein, a surface protein of group b streptococcus 1 (spbl), an spb2, a laminin binding protein (Lmb), a C5a peptidase, a matrix adhesion (Ema) polypeptide, a capsular polysaccharide, or an antigenic fragment of any of these polypeptides. The additional polypeptide or polypeptides can be from a strain of GBS type Ia, Ib, II, III, IV, V, VI, VII, or VIII strain. In one embodiment, the composition includes two, three, four, five or more different polypeptides. In one embodiment, the capsular polysaccharide is a capsular polysaccharide from GBS type Ia, Ib, II, HI, IV, V, VI, VII, or VIII strain or modified versions of such polysaccharides. In one embodiment, the composition includes two, three, four, five or more different polysaccharides. In one embodiment, the composition can include an ALP3 polypeptide physically associated with more than one polysaccharide, e.g., more than one GBS polysaccharide. For example, the composition can include an ALP3 polypeptide physically associated with a GBS type III strain polysaccharide and an ALP3 polypeptide physically associated with a GBS type V strain polysaccharide. The composition can include an ALP3 polypeptide physically associated with any combination of a first polysaccharide from a first GBS strain and a second polysaccharide from a second GBS strain.

In another aspect, the invention features a method of eliciting an immune response in a mammal by administering to the mammal a composition including an ALP3 polypeptide in combination with, e.g., physically associated with, a polysaccharide, e.g., a GBS polysaccharide. In one embodiment, the ALP3 polypeptide is covalently associated with the GBS polysaccharide. In one embodiment, the ALP3 polypeptide can be linked directly or via a linking agent to the GBS polysaccharide. In one embodiment, the ALP3 polypeptide is linked to the polysaccharide, e.g., a GBS polysaccharide, via an amine bond between an epsilon amino group and a reduced sialic acid residue. For example, the ALP3 polypeptide can be linked to the polysaccharide via reductive amination. In one embodiment, the composition is administered to the mammal two, three, four, or more times.

In one embodiment, the polysaccharide is a polysaccharide described herein.

In one embodiment, the polysaccharide is a GBS polysaccharide from a GBS type Ia, Ib, II, III, IV, V, VI, VII, or VIII strain or modified polysaccharides thereof. In one embodiment, the GBS polysaccharide is a GBS type V strain polysaccharide, a GBS type VIIl strain polysaccharide, or modified polysaccharides thereof. Examples of GBS polysaccharides include, but are not limited to, those described in U.S. Publication No. 20060134142, the contents of which are incorporated herein by reference.

In one embodiment, the composition includes one or more additional polypeptides, one or more additional polysaccharides, or combinations thereof. Examples of additional polypeptides and polysaccharides include, but are not limited to, an alpha C protein polypeptide, an Rib polypeptide, a GBS beta C protein polypeptide, an epsilon polypeptide, an R protein (e.g., an Rl or R4 protein), an alpha-like protein, a surface protein of group b streptococcus 1 (spbl ), an spb2, a laminin binding protein (Lmb), a C5a peptidase, a matrix adhesion (Ema) polypeptide, a capsular polysaccharide, or an antigenic fragment of any of these polypeptides. The additional polypeptide or polypeptides can be a polypeptide of GBS type Ia, Ib, II, III, IV, V, VI, VII, or VIIl strain. In one embodiment, the composition includes two, three, four, five or more different polypeptides. In one embodiment, the capsular polysaccharide is a capsular polysaccharide from GBS type Ia, Ib, II, III, IV, V, VI, VII, or VIII strain or modified versions of such polysaccharides. In one embodiment, the composition includes two, three, four, five or more different polysaccharides.

In one embodiment, the composition can include an ALP3 polypeptide physically associated with more than one polysaccharide, e.g., more than one GBS polysaccharide. For example, the composition can include an ALP3 polypeptide physically associated with a GBS type III strain polysaccharide and an ALP3 polypeptide physically associated with a GBS type V strain polysaccharide. For example, the composition can include an ALP3 polypeptide physically associated with any combination of a first polysaccharide from a first GBS strain and a second polysaccharide from a second GBS strain.

In one embodiment, the method elicits the production of antibodies, e.g., IgG or IgM antibodies, in the mammal that recognize the ALP3 polypeptide and/or thepolysaccharide, e.g., the GBS polysaccharide. In another embodiment the method elicits a protective response in the mammal, where the mammal is less likely to develop an infection, e.g., a group B streptococcal infection. In one embodiment, the method can further include evaluating an immune response to bacteria, e.g., GBS bacteria in the mammal (e.g., evaluating humoral immune response and/or a cell-mediated immune response).

In one embodiment, the mammal (e.g., human or nonhuman primate) receiving the composition is at risk for infection with a bacteria, e.g., a GBS bacteria. For example, the mammal can be immunocompromised or at risk of becoming immunocompromised or is elderly. In one embodiment, the mammal is pregnant. In another embodiment, the individual is a neonate, a woman in a peripartum period, or a nonpregnant adult. In yet another embodiment, the individual has diabetes mellitus or a malignancy.

In another aspect, the invention features a method of preparing an immunogenic composition, including linking a polysaccharide with an ALP3 polypeptide. In one embodiment, the ALP3 polypeptide is covalently associated with the polysaccharide, e.g., a GBS polysaccharide. In one embodiment, the ALP3 polypeptide can be linked directly or via a linking agent to the polysaccharide, e.g., a GBS polysaccharide.

In another aspect, the invention features a method of preventing or treating an infection caused by a group B Streptococcus including administering to an individual an effective amount of an antigenic composition including an ALP3 polypeptide in combination with a GBS polysaccharide, e.g., a composition described herein. In one embodiment, administering the effective amount of the composition prevents one or more of sepsis, pneumonia, meningitis, chorioamnionitis, or postpartum endometritis. In another embodiment, the individual is a neonate, a woman in a peripartum period, or a nonpregnant adult. In yet another embodiment, the individual is immunocompromised, or has diabetes mellitus or a malignancy.

Another aspect of the invention features a method of preventing or treating a disorder associated with a GBS infection that includes administering to an individual an effective amount of an antigenic composition including an ALP3 polypeptide in combination with a GBS polysaccharide, e.g., a composition described herein, In one embodiment, the disorder is sepsis, pneumonia, meningitis, chorioamnionitis, or postpartum endometritis. In another embodiment, the individual is a neonate, a woman in a peripartum period, or a nonpregnant adult. In yet another embodiment, the individual is immunocompromised, or has diabetes mellitus or a malignancy.

The details of one or more embodiments are set forth in the accompanying drawing and the description below. All cited patents, patent applications, and references (including references to public sequence database entries) are incorporated by reference in their entirety for all purposes.

DESCRIPTION OF DRAWINGS

FlG. 1 is a schematic diagram illustrating the amino acid domains of a full-length ALP3 polypeptide.

DETAILED DESCRIPTION

The ALP3 polypeptide of group B Streptococcus can be useful to induce an immune response to an infection caused by a pathogen that can be viral, bacterial, or eukaryotic. An ALP3 polypeptide was originally isolated from a type VIII GBS strain (JM9-130013, a neonatal sepsis isolate), and has subsequently been identified in a type V strain. The full-length ALP3 polypeptide includes a 56 amino acid signal sequence, a 172 amino acid N-terminal sequence, followed by an "A" region containing 51 amino acids, and then a "U" region of 137 amino acids. Following the "U" region is a "B" repeat region containing five tandem repeals, each 79 amino acids long, and one partial repeat of 13 amino acids. After the B repeat region is a C-terminal region of 45 amino acids, including a cell wall-associating motif (LPXTGX) (SEQ ID N0:3).

As used herein, an "ALP3 polypeptide" can include a full-length ALP3 polypeptide that is a polypeptide having an N-terminal sequence, an A region, a U region, a B region and a C- terminal region, such as a polypeptide having the sequence of SEQ ID NO:1, and fragments thereof. An ALP3 polypeptide can also include more than one, e.g., two, three, four, five, six, seven, eight, nine, ten but not more than fifty, thirty, twenty, fifteen, of the regions discussed above. For example, an ALP3 polypeptide can include an N-terminal or C-terminal fragment of ALP3 but can have more than one of the "B" repeat regions or portions of the "B" region. Thus, an ALP3 polypeptide can include two, three, four, five, six, seven, eight, nine, ten but not more than fifty, thirty, twenty, fifteen "B" regions or portion or portions of "B" regions. An ALP3 polypeptide can be a functional fragment or derivative of a full length ALP3 polypeptide that does not essentially affect the immunogenicity or protective antibody-producing function of the polypeptide as compared to the activity of an ALP3 polypeptide having the amino acid sequence of SEQ ID NO: 1 , or a mature form of the ALP3 polypeptide of SEQ ID NO: 1. A functional derivative will have a sequence similar to a full length ALP3 polypeptide such as the polypeptide having the amino acid sequence of SEQ ID NO: 1 or mature form of the ALP3 polypeptide of SEQ ID NO:1, but will include at least some amino acid changes (deletion, insertions, inversions, substitutions, etc.). Amino acid substitutions can be conservative substitutions, which include exchanges of one amino acid for another with similar polarity, steric arrangement, or of the same class (e.g., hydrophobic, acidic or basic). An ALP3 polypeptide can be 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to a full length ALP3 polypeptide such as the ALP3 polypeptide of SEQ ID NO: 1.

Percent identity between two amino acid sequences is determined by standard alignment algorithms such as, for example, Basic Local Alignment Tool (BLAST) described in Altschul et al, J. MoI. Biol. 215:403-410, 1990; the algorithm of Needleman et ai, J. MoI. Biol. 48:444-453, 1970; or the algorithm of Meyers et al., Comput. Appl. Biosci. 4:11-17, 1988. A derivative of an ALP3 polypeptide can, e.g., differ from a full length ALP3 polypeptide by at least 1, 2, 3, 5, 10, 15 amino acids but not more than 100, 75, 50, 30, 20 amino acids, e.g., it can differ by at least 1 , 2, 3, 5, 10, 15 amino acids but not more than 100, 75, 50, 30, 20 amino acids from SEQ ID NO: 1. Methods of assaying immune response are known in the art and include, for example, measuring antibody titers in a subject (e.g., by ELlSA, including quantitative ELISA), or providing a challenge, such as a skin test, and assaying for a reaction. An immune response can also be assayed by opsonophagocytic assay where the serum from immunized individuals is mixed with human polymorphonuclear leukocytes, complement (e.g., from human serum or rabbit serum), and GBS expressing one or more of the relevant antigens. The ability of the serum to kill the GBS over a fixed time period is tested (Baltimore et al., J. Immunol, 118: 673-678, 1977). In another assay, passive mouse protection can be assayed, where the ability of serum from individuals immunized by the vaccine is administered to mice and the mice subsequently challenged with GBS. Survival of the mice is a measure of the protective capacity of the vaccine.

As used herein, "polysaccharide" refers to a polysaccharide from a pathogen that is not naturally associated with an ALP3 polypeptide sequence or fragment of an ALP3 polypeptide, and modified versions of such polysaccharides. Polysaccharides include monosacharides, disaccharides, and longer chains.

As used herein, a "GBS polysaccharide" is a GBS polysaccharide from GBS that is not naturally associated with an ALP3 polypeptide sequence or fragment of an ALP3 polypeptide, and modified versions of such polysaccharides. The GBS polysaccharide can be, e.g., a capsular polysaccharide from a type I (e.g., type Ia, or type Ib), type II, type III, type IV, type V, type VI, type VII or type VlII GBS strain. Examples of modified GBS polysaccharides include those described in U.S. Publication No. 20060134142. The GBS polysaccharide is typically a polysaccharide that, when introduced into a mammal, elicits antibodies that are capable of reacting with group B Streptococcus.

As used herein, an "antigenic composition" is a composition including at least one antigenic polysaccharide, antigenic polypeptide, or a DNA encoding an antigenic polypeptide, that will elicit an immune response when the composition is administered to a subject. An antigenic composition is also called a vaccine. As used herein, the term "treatment", "treat" or "treating" refers to administering a composition in an amount, manner, and/or mode effective to improve a condition, symptom, or parameter associated with a disorder (e.g., a disorder associated with a GBS infection) or to reduce onset, progression, or exacerbation of the disorder (including secondary damage caused by the disorder), to either a statistically significant degree or to a degree detectable to one skilled in the art. Accordingly, treating can achieve therapeutic and/or prophylactic benefits. An effective amount, manner, or mode can vary depending on the subject and may be tailored to the subject. As used herein, "treatment" also encompasses the prophylactic treatment of subjects with an elevated risk for infection, e.g., a GBS infection, such as a neonate, a pregnant woman or a woman in the peripartum period, or a nonpregnant adult. Other subjects having an elevated risk of infection, e.g., a GBS infection, include adults with diabetes mellitus, malignancy, or immunocompromised adults.

As used herein, "an amount effective to treat", or a "therapeutically effective amount", refers to an amount of an antigenic composition that is effective, upon single or multiple dose administrations to a subject, to improve or prophylactically treat a condition, symptom, or parameter associated with a disorder or to reduce onset, progression, or exacerbation of the disorder, to either a statistically significant degree or to a degree detectable to one skilled in the art.

As used herein, the terms "prevent", "preventing", and "prevention" mean that the treated subject either does not develop a clinically observable level of the condition at all, or develops it more slowly and/or to a lesser degree than he/she would have absent the treatment. For example, infection by GBS may not occur at all in an individual who receives the antigenic composition described herein, or symptoms of infection may develop, or manifest themselves more slowly and/or to a lesser degree than the individual would have absent administration of the composition. These terms are not limited solely to a situation in which the subject experiences no aspect of the condition whatsoever. For example, a treatment will be said to have "prevented" the condition (e.g., a GBS infection, or a symptom associated with a GBS infection) if it is given during exposure of a subject to a stimulus that would have been expected to produce a given manifestation of the condition, and results in the subject's experiencing fewer and/or milder symptoms of the condition than otherwise expected. A treatment can "prevent" infection by resulting in the subject's displaying only mild overt symptoms of the infection; it does not imply that there must have been no penetration of any cell by the infecting microorganism. An antigenic composition can be said to prevent a disease if its administration to an individual results either in the total or partial attenuation (i.e., suppression) of a symptom or condition of the disease, or in the total or partial immunity of the individual to the disease.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). The antigenic composition may be provided in a substance that protects it from the action of acids and other natural compounds that may inactivate it.

Compositions. An antigenic composition described herein can be, for example, a polysaccharide-based vaccine, a protein-based vaccine, or a DNA-based vaccine. A polysaccharide-based antigenic composition includes an ALP3 polypeptide unassociated with, or associated with, e.g., physically associated with a polysaccharide. For example, the ALP3 polypeptide may be covalently associated with a GBS polysaccharide. For example, the ALP3 polypeptide can be linked directly to the GBS polysaccharide or via a linking agent. In some embodiments, a protein linker region (e.g., of 1, 2, 3, 4, 5, 6, or more amino acids) can separate the ALP3 polypeptide from the polysaccharide. Alternatively, the ALP3 polypeptide and the polysaccharide are chemically linked, such as by reductive amination. An ALP3 polypeptide can be used as a carrier for a polysaccharide (e.g., a capsular polysaccharide). Polysaccharides by themselves are incompletely immunogenic. Carrier polypeptides or polypeptide fragments that are coupled to other antigenic polypeptides can enhance immunogenicity of an antigen, e.g., a polypeptide or a polysaccharide and result in antibody levels that are less variable (see, e.g., Kasper et al, J. Clin. Investig. 98:2308-2314, 1996, and Gravekamp et al., Infec. and Immun. 67:2491-2496, 1999).

Suitable polysaccharides for use in embodiments of this invention include, but are not limited to, polysaccharides from encapsulated bacteria. The polysaccharides can be from any source. For example, they can be derived from naturally-occurring bacteria, genetically engineered bacteria, or can be produced synthetically. The polysaccharides can be subjected to one or more processing steps. For example, the polysaccharides can be processed by purification, functionalization, depolymerization using mild oxidative conditions, deacelylation, and the like. Post processing steps can also be employed, if desired. Methods for synthesizing, preparing, and/or purifying suitable polysaccharides are known in the art and can be employed.

Non-limiting examples of polysaccharides for use in this invention include, but are not limited to, pneumococcal polysaccharides, for example, of serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 1OA, HA, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F; meningococcal polysaccharides, for example, of serotypes A, B, C, W 135, and Y, Haemophilus influenzae type b polysaccharide polyribosylribitol phosphate, and Salmonella typhi Vi polysaccharide. Polysaccharides of pneumococcal and group B streptococcal serotypes, and meningococcal serogroups are also suitable for use herein, as T-independent polysaccharide antigens, for example, polysaccharides derived from group A streptococcus, Staphylococci, Enterococci, Klebsiella pneumoniae, E. coli, Pseudomonas aeruginosa, and Bacillus anthracis. While bacterial polysaccharides are particularly preferred, gram (-) bacterial lipopolysaccharides and their polysaccharide derivatives, and viral polysaccharides can also be employed.

A polysaccharide, e.g., a GBS polysaccharide, can be conjugated to an ALP3 polypeptide using art-known methods. For example, methods are described in U.S. Patent Application No. 20070141084, herein incorporated by reference. For example, polysaccharides can be oxidized by treatment with periodate (or a related reagent, e.g., paraperiodic acid or potassium metaperiodate) to leave aldehydic termini at sites with vicinal dihydroxy groups. A polysaccharide can be reacted with the ALP3 polypeptide under conditions that conjugate the components, e.g., using a linking reagent or via direct coupling to the moiety. Conjugation to the ALP3 polypeptide may be carried out, e.g., by reductive amination (see, e.g., Schwartz and Gray, Arch. Biochem. Biophys. 181: 542-549, 1977). For example, a GBS polysaccharide and an ALP3 polypeptide are reacted in the presence of cyanoborohydride ions, or another reducing agent which will not reduce the reducing ends of interest nor adversely affect the polypeptide or polysaccharide. Methods for conjugating polysaccharides are also described, e.g., in Wessels et al., JClin Invest., 86: 1428-1433, 1990 and Wang et at., Vaccine, 21: 1 112-7, 2003. An antigenic composition can have other components, including other antigens, such as other GBS antigens for an enhanced immune response.

The antigenic compositions described herein can be formulated with salts, buffers or adjuvants or other substances which are desirable for improving the efficacy of the composition. For example, adjuvants may enhance a mammal's immune response to an ALP3 polypeptide, or an ALP3- polysaccharide combination, e.g., to enhance antibody production and/or to induce an immune response with lower doses of polysaccharide. Normally, the adjuvant and the composition are mixed prior to presentation to the immune system, or are presented separately, but into the same site of the animal being immunized. Adjuvants include water emulsions (e.g., complete and incomplete Freund's adjuvant); oil; iron oxide; sodium alginate; aluminum hydroxide, aluminum and calcium salts (i.e., alum); unmethylated CpG motifs; glucan; dextran sulfate; bacterial extracts (e.g., mycobacterial extracts); QS-21 (Aquila Biopharmaceuticals, Inc., Framingham, Mass.); MPL™ (3-0-deacylated monophosphoryl lipid A; Corixa, Hamilton, Mont.); 529 (an amino alkyl glucosamine phosphate compound, Corixa, Hamilton, Mont.), N-acetyl-muramyl-L-theronyl-D-isoglutamine (thr-MDP); N-acetyl- normuramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP); and N- acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(r-2'-dipalmitoyl-sn-glycero-3- hydroxyphosphoryloxy-ethylamine) (CGP 19835A, referred to as MTP-PE). Polypeptide adjuvants such as IL- 12 (Genetics Institute, Cambridge, Mass.) and GM-CSF (Immunex Corp., Seattle, Wash.) may also be used to enhance the immune response to polysaccharide compositions.

The antigenic compositions described herein can be formulated in a suitable carrier media for administration to a subject (e.g., a media including sodium phosphate-buffered saline at a neutral pH, and/or an aluminum phosphate solution, or another conventional media). Suitable pharmaceutical carriers are known in the art.

Dosages and routes of administration. Compositions for administration to a subject can contain from about 0.01 to 1000 μg/mLper dose of an antigenic composition described herein, e.g., about 0.1 to about 500 μg/mL per dose, about 5 to about 300 μg/mL per dose, or about 10 to about 50 μg/mLper dose. The compositions can elicit effective levels of antibody against the ALP3 polypeptide and GBS polysaccharide in a mammal. The exact dosage can be determined by routine dose/response experimentation. Polysaccharide conjugate compositions can be administered as a single immunization or in a series, e.g., two, three or four doses at 1 month, 2 month, 3 month, or longer intervals. A booster may be given one year, two years, or more after the initial immunization or primary series of immunizations. An exemplary immunization schedule for pregnant women is a single dose given in the second or early third trimester. For immunization of non-pregnant adults, a single dose can be used. The requirement for subsequent booster doses in adults can be based on the immunogenicity of the polysaccharide composition and ongoing surveillance of efficacy of the vaccine in eliciting an immune response.

In general, the "effective amount" of the antigenic composition is one which is sufficient to achieve the desired biological effect. The desired biological effect is the production of antibodies in the recipient in response to the antigenic composition, which subsequently protects the recipient from infection, e.g., infection by GBS. Infection can be prevented or treated, as described above. Generally, the dosage needed to provide an effective amount of the composition will vary depending upon such factors as the subject's age, condition, sex, and extent of disease, if any, and other variables which can be adjusted by one of ordinary skill in the art.

The antigenic compositions described herein can be administered parenterally by injection, rapid infusion, nasopharyngeal absorption (intranasopharangeally), dermoabsorption, or orally. The compositions may alternatively be administered intramuscularly, or intravenously. Compositions for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Carriers or occlusive dressings can be used to increase skin permeability and enhance antigen absorption. Liquid dosage forms for oral administration may generally comprise a liposome solution containing the liquid dosage form. Suitable forms for suspending liposomes include emulsions, suspensions, solutions, syrups, and elixirs containing inert diluents commonly used in the art, such as purified water. Besides the inert diluents, such compositions can also include adjuvants, wetting agents, emulsifying and suspending agents, or sweetening, flavoring, or perfuming agents.

The antigenic compositions can be administered to a mammal, such as a human. The compositions are also suitable for administration to a nonhuman mammal such as a nonhuman primate (such as a monkey or ape), a rabbit, mouse, rat, goat, cow, bull, pig, horse, sheep, wild boar, sea otter, cat, or dog.

Methods of treatment and prevention. The administration of an antigenic composition described herein (or the antisera which it elicits) may be for either a "prophylactic" or "therapeutic" purpose. When provided prophylactically, the composition is provided in advance of any symptom of infection, e.g., group B Streptococcus infection. The prophylactic administration of the composition serves to prevent or attenuate any subsequent infection. When provided therapeutically, the composition is provided upon the detection of a symptom of actual infection, including signs or symptoms of sepsis or meningitis. The therapeutic administration of the composition serves to attenuate any actual infection. The antigenic composition may therefore be provided either prior to the onset of infection (so as to prevent or attenuate an anticipated infection) or after the initiation of an actual infection.

The antigenic compositions described herein can be administered to pregnant women to prevent infection during the peripartum period. Such treatment can prevent, for example, GBS- related infections related to, for example, chorioamnionitis, postpartum endometritis, and sepsis.

The antigenic compositions can also be administered to prevent infection in a neonate. GBS infections in neonates often occur in utero or during passage through the vagina and can subsequently cause sepsis, pneumonia, and/or meningitis. Heavy colonization with GBS may also lead to premature birth. Administration of the antigenic compositions described herein to a pregnant woman can treat or prevent, for example, a GBS infection in the woman, and also prevent infection of the neonate while in utero or during delivery. Administration of the antigenic composition can thereby be used to prevent GBS-associated symptoms in a neonate, including the prevention of sepsis, pneumonia, and/or meningitis.

The antigenic compositions described herein can also be used to treat or prevent infection by a pathogen, e.g., GBS infection, and associated symptoms, in nonpregnant adults. For example, the antigenic compositions can be administered to nonpregnant adults to treat or prevent sepsis, soft tissue infections, and other focal infections. Nonpregnant adults that are particularly susceptible to infection, e.g., by GBS, include adults with diabetes mellitus, malignancy, or immunocompromised adults. For example, an immunocompromised adult may be undergoing or have undergone chemotherapy, or stem cell, bone marrow, or organ transplantation. Thus the antigenic compositions can be administered to these adults to prevent infection, e.g.. GBS infection.

The antigenic compositions may confer resistance to a pathogen, e.g., viral, bacterial, or fungal, by either passive immunization or active immunization. In one embodiment of passive immunization, the composition is provided to a host (e.g., to a human or mammal) volunteer, and the elicited antisera is recovered and directly provided to a recipient suspected of having an infection caused by a group B Streptococcus.

In another embodiment of passive administration, the antigenic composition comprising an ALP3 polypeptide and a GBS polysaccharide is provided to a female at or prior to pregnancy or parturition, under condition of time and amount sufficient to cause the production of antisera which serve to protect both the female and the fetus or newborn, such as by passive incorporation of the antibodies across the placenta.

EXAMPLE

Example 1

An antigenic composition can include an ALP3 polypeptide covalently linked to a GBS polysaccharide. For example, the ALP3 polypeptide can include the sequence of SEQ ID NO: 1 , below, or a fragment of SEQ ID NO: 1.

Sequences:

ALP3 polypeptide sequence ( SEO ID NO: 1) (GenPept ID No. AAGOl 394)

1 mfrrsknnsy dtsqtkqrfs ikkfkfgaas vliglsflsg vtqgnlnife esivaastip 61 gsaatlntsi tkniqngnay idlydvkngl idpqnlivln pssysanyyi kqgakyysnp 121 seitttgsat itfnildetg nphkkadgqi divsvnltiy dstalrnrid evinnandpk 181 wsdgsrdevl tglekikkdi dnnpktqidi dnkinevnei ekllwslpd kikyspeakh 241 rtveqhaeld akdsiantde lpsnstynwk nghkpdtsts gekdgivevh ypdgtvddvn 301 vkvtvtskkt dntaptltvt peqqtvkvde ditftvtved enevelgldd lkakyendii 361 garvkikylt kesnkkvmev timkatladk gaitftakdk agnqaepktv tinvlplkds 421 nepkgkdqtv kvgetpkaed signlsdlpk gttvafetpv dtatpgdkpa kwvtypdgs 481 kdtvdvtvkv vdprtdadkn dpagkdqqvk vgetpkaeds ignlpdlpkg ttvafetpvd 541 tatpgdkpak vwtypdgsk dtvdvtvkw dprtdadknd pagkdqqvkv getpkaedsi 601 gnlsdlpkgt tvafetpvdt atpgdkpakv wtypdgskd tvdvtvkwd prtdadkndp 661 agkdqqvkvg etpkaedsig nlpdlpkgtt vafetpvdta tpgdkpakvv vtypdgskdt 721 vdvtvkwdp rtdadkndpa gkdqgvkvge tpkaedsign lsdlpkgttv afetpvdtat 781 pgdkpakvw typdgskdtv dvtvkwdpr tdadkndpag kdqgvngkgn klpatgenat 841 pf fnvaalti issvgllsvs kkked

ALP3 DNA sequence (SEO ID NO:2) (GenBank Accession No. AF245663). TheALP3 cDNA sequence is at nucleotide positions 365-2962.

l ttgtaaaaaa gtgaaatagt tttatcggat ttatactctc tgataacaga aaaattttga

61 tcacaaacag taatagttag acctagaaga ctgtgtaatg atttcaacaa taaagaatca

121 atcatatgag ttaccacctt tgatttaata tgttatctat tttagagaag atttctttaa

181 agtcaacaga ataattaggt agtcggtatt ttaaccaaat atgatttaaa aaatcgaaaa

241 agtcaaatta tatataaaaa aaaaagcaga ttagattaga taaaaaagta tagatattct

301 aatatttatt gtttaagcct ataatttact ctgtatagag ttatacagag taaaggagaa

361 tattatgttt agaaggtcta aaaataacag ttatgatact tcacagacga aacaacggtt

421 ttcaattaag aagttcaagt ttggtgcagc ttctgtacta attggtctta gttttttgag

481 tggggttaca caaggtaatc ttaatatttt tgaagagtca atagttgctg catctacaat

541 tccagggagt gcagcgacct taaatacaag catcactaaa aatatacaaa acggaaatgc

601 ttacatagat ttatatgatg taaagaatgg attgatcgat cctcaaaacc tcattgtatt

661 aaatccatca agctattcag caaattatta tatcaaacaa ggtgctaaat attatagtaa

721 tccgagtgaa attacaacaa ctggttcagc aactattact tttaatatac ttgatgaaac

781 tggaaatcca cataaaaaag ctgatggaca aattgatata gttagtgtga atttaactat

841 atatgattct acagctttaa gaaataggat agatgaagta ataaataatg caaatgatcc

901 taagtggagt gatgggagtc gtgatgaagt cttaactgga ttagaaaaaa taaaaaaaga

961 tattgataat aatccaaaaa cacaaataga tattgataat aaaattaatg aagtcaatga

1021 aatagagaaa ttgttagttg tatcgctacc agataaaatt aagtattcgc cagaggctaa

1081 gcataggact gttgaacaac acgcggaatt agatgcaaaa gatagcattg caaatacaga

1141 tgaattgcca tcaaattcaa cgtataactg gaaaaatggt cataaaccag acaccLcaac

1201 atcaggtgaa aaagacggaa ttgttgaagt tcactatcca gatggtactg ttgatgatgt

1261 gaatgttaaa gtaaccgtta catcgaaaaa aactgataat acagctccaa cattaaccgt

1321 cactccagag caacagactg ttaaagtgga tgaagatatt acctttacgg ttacagttga

1381 agacgaaaat gaagttgaac taggtttaga tgatcttaaa gctaagtatg aaaatgatat

1441 cattggagct cgtgttaaaa ttaagtatct tactaaagaa tctaataaga aagtcatgga

1501 agtgacaatt atgaaagcta ctttagcaga taagggcgca attaccttta ctgcaaaaga

1561 taaagcaggt aatcaagcag aacctaagac agctaccatc aatgttcttc cgcttaagga

1621 tagcaacgaa ccaaaaggta aggaccaaac ggtcaaagta ggagaaacac cgaaggcaga

1681 agattctatt ggtaacttat cagatcttcc gaaaggtaca acagtagcct ttgaaactcc

1741 agttgatacg gcaacaccgg gagacaaacc agcaaaagtt gttgtgactt acccagatgg 1801 t tcaaaagat actgtagatg tgactgttaa ggttgtcgat ccacgtacag atgccgataa 1861 gaatgatcca gcaggtaaag atcagcaagt caaagtagga gaaacaccga aggcagaaga 1921 t tctattggt aacttaccag atcttccgaa aggtacaaca gtagcctttg aaactccagt 1981 tgatacggca acaccgggag acaaaccagc aaaagttgtt gt gacttacc cagatggttc 2041 aaaagatact gtagatgtga ctgttaaggt tgtcgatcca cgtacagatg ccgataagaa 2101 tgatccagca ggtaaagatc agcaagtcaa agtaggagaa acaccgaagg cagaagattc 2161 tattggtaac ttatcagatc ttccgaaagg tacaacagta gcctt tgaaa ctccagttga 2221 tacggcaaca ccgggagaca aaccagcaaa agttgttgtg acttacccag atggttcaaa 2281 agatactgta gatgtgactg ttaaggttgt cgatccacgt acagatgccg ataagaatga 2341 t ccagcaggt aaagatcagc aagtcaaagt aggagaaaca ccgaaggcag aagattctat 2401 tggtaactta ccagatct tc cgaaaggtac aacagtagcc tt tgaaactc cagttgatac 2461 ggcaacaccg ggagacaaac cagcaaaagt tgttgtgact tacccagatg gttcaaaaga 2521 tactgtagat gtgactgt ta aggttgtcga tccacgtaca gatgccgata agaatgatcc 2581 agcaggtaaa gatcagcaag tcaaagtagg agaaacaccg aaggcagaag attctattgg 2641 taacttatca gatcttccga aaggtacaac agtagccttt gaaactccag ttgatacggc 2701 aacaccggga gacaaaccag caaaagttgt tgtgacttac ccagatggtt caaaagatac 2761 tgtagatgtg actgttaagg ttgtcgatcc acgtacagat gccgataaga atgatccagc 2821 aggtaaagat cagcaagtca atggtaaagg aaataaacta ccagcaacag gtgagaatgc 2881 aactccattc tttaatgt tg cagctttgac aattatatca tcagttggtt tat tat ctgt 2941 ttctaagaaa aaagaggatt aatcttttga cctaaaatgt cactaaacat ttcaccattt 3001 at

Example 2

The immunogenicity and protective efficacy of purified recombinant Alp3 (rAlp3) was evaluated in outbred mice. Specific IgG was elicited in mice following vaccination with rAlp3 or rAlp3 covalently coupled to purified GBS type III capsular polysaccharide (Tables 1 and 2). Purified rAlp3 also induced a strong immune response in rabbits.

Most (94%) pups born to dams vaccinated with Ill-rAlp3 survived challenge with an ordinarily lethal dose of viable group B Streptococcus type III strain M781 bacteria (Table 3). Partial (44%) protection was afforded to pups born to dams given uncoupled rAlp3 (Table 3) likely due to the cross reactivity between rAlp3 and Rib, a protein found on the surface of the challenge strain M781. As expected, none of 43 pups born to saline vaccinated dams survived the challenge indicating the lethality of the challenge dose.

The data suggested that rAlp3 can serve as an effective carrier protein for group B streptococcal polysaccharides. Table 1. Group B Streptococcus type III capsular polysaccharide-specific IgG titer in pooled sera from adult female outbred mice (n = 3 per group) following vaccination with rAlp3 and group B Streptococcus type III CPS conjugated to rAlp3 (III-rAlp3) vaccines.

Mice were vaccinated on days 0, 21, and 42. All vaccines contained alum adjuvant.

Table 2. Group B Streptococcus rAlp3 -specific IgG titer in pooled sera from adult female outbred mice (n = 3 per group) following vaccination with rAlp3 and group B Streptococcus type III CPS conjugated to rAlp3 (III-rAlp3) vaccines.

Table 3. Protective of neonatal mice by maternal vaccination with rALP3 and group B Streptococcus type UI CPS conjugated to rAlp3 (III-rAlp3) vaccines.

Dams immunized No. No. pups survived/No, pups challenged with dams

(% survival)

Mouse pups were challenged with GBS type III strain M781 rAlp3 18/41 (44)^{a b}

III-rAlp3 3 34/36 (94)"

Saline 3 0/43 (O)'

Pups were challenged within 48 hours of birth and survival assessed 48 hours thereafter. aP <0.0001 compared with HI-rAlp3, "P <0.0001 compared with saline. A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention.

Claims

WHAT IS CLAIMED IS:

1. An antigenic composition comprising an alpha-like protein 3 (ALP3) polypeptide in combination with a polysaccharide antigen.

2. The antigenic composition of claim 1, wherein the polysaccharide antigen is Group B streptococcus (GBS) polysaccharide.

3. The antigenic composition of claim 1 or claim 2, wherein the ALP3 polypeptide is covalently associated with the polysaccharide.

4. The antigenic composition of claim 1 or claim 2, wherein the ALP3 polypeptide is directly linked to the polysaccharide.

5. The antigenic composition of claim 1 or claim 2, wherein the ALP3 polypeptide is associated with the polysaccharide via a linking agent.

6. The composition of claim 2, wherein the GBS polysaccharide is from a GBS type Ia, Ib, II, III, IV, V, VI, VII, or VIII strain.

7. The composition of claim 2, wherein the GBS polysaccharide is from a type III GBS strain.

8. The composition of claim 2, further comprising a GBS polypeptide or a second GBS polysaccharide, wherein the GBS polypeptide or polysaccharide is an alpha C protein polypeptide, an Rib polypeptide a GBS beta C protein polypeptide, an epsilon polypeptide, an R protein, an alpha-like protein, a surface protein of group b streptococcus 1 (spbl), an spb2, a laminin binding protein (Lmb), a C5a peptidase, a matrix adhesion (Ema) polypeptide, a capsular polysaccharide, or an antigenic fragment of any of these polypeptides.

9. The composition of claim 8, wherein the R protein is Rl or R4.

10. The composition of claim 1 or claim 2, further comprising a pharmaceutically acceptable carrier.

11. The composition of claim 1 or claim 2, further comprising an adjuvant.

12. A method of eliciting an immune response in a mammal, comprising administering to the mammal a composition comprising an alpha-like protein 3 (ALP3) polypeptide in combination with a polysaccharide antigen.

13. The method of claim 12, wherein the polysaccharide antigen is a Group B streptococcus (GBS) polysaccharide.

14. The method of claim 12 or claim 13, wherein the ALP3 polypeptide is covalently associated with the polysaccharide.

15. The method of claim 14, wherein the ALP3 polypeptide is directly linked to the polysaccharide.

16. The method of claim 14, wherein the ALP3 polypeptide is associated with the polysaccharide via a linking agent.

17. The method of claim 13, wherein the GBS polysaccharide is from a GBS type Ia, Ib, II, III, IV, V, Vl, VII, or VIII strain.

18. The method of claim 13, wherein the GBS polysaccharide is a polypeptide from a type III GBS strain.

19. The method of claim 13, further comprising a GBS polypeptide or a second GBS polysaccharide, wherein the GBS polypeptide or polysaccharide is an alpha C protein polypeptide, an Rib polypeptide, a GBS beta C protein polypeptide, an epsilon polypeptide, an

R protein, an alpha-like protein, a surface protein of group b streptococcus 1 (spbl), an spb2, a laminin binding protein (Lmb), a C5a peptidase, a matrix adhesion (Ema) polypeptide, a capsular polysaccharide, or an antigenic fragment of any of these polypeptides.

20. The method of claim 12 or claim 13, wherein the composition further comprises a pharmaceutically acceptable carrier.

21. The method of claim 12 or claim 13, wherein the composition further comprises an adjuvant.

22. The method of claim 13, where the method elicits the production of antibodies in the mammal that recognize the ALP3 polypeptide or the GBS polysaccharide.

23. The method of claim 12 or claim 13, wherein the method elicits a protective response in the mammal.

24. The method of claim 13, wherein the mammal is at risk for infection with a GBS bacteria.

25. The method of claim 12 or claim 13, wherein the mammal is a primate.

26. The method of claim 12 or claim 13, wherein the mammal is a human.

27. A method of preparing an immunogenic composition, comprising linking a polysaccharide antigen with an ALP3 polypeptide.

28. The method of claim 27, wherein the polysaccharide antigen is a GBS polysaccharide.

29. The method of claim 27 or claim 28, wherein the ALP3 polypeptide is covalcntly associated with the polysaccharide.

30. The method of claim 29, wherein the ALP3 polypeptide is directly linked to the polysaccharide.

31. The method of claim 29, wherein the ALP3 polypeptide is associated with the polysaccharide via a linking agent.

32. A method of preventing or treating an infection caused by a group B Streptococcus comprising administering to an individual an effective amount of the composition of claim 2.

33. The method of claim 32, wherein administering the effective amount of the composition prevents a disorder selected from the group consisting of sepsis, pneumonia, meningitis, chorioamnionitis, and postpartum endometritis.

34. The method of claim 32, wherein the individual is a neonate, a woman in a peripartum period, or a nonpregnant adult.

35. The method of claim 32, wherein the individual is immunocompromised, or has diabetes mellitus or a malignancy.

36. A method of preventing or treating a disorder associated with an infection comprising administering to an individual an effective amount of the composition of claim 1.

37. The method of claim 36, wherein the infection is a GBS infection and the composition is a composition according to claim 2.

38. The method of claim 37, wherein the disorder is sepsis, pneumonia, meningitis, chorioamnionitis, or postpartum endometritis.

39. The method of claim 37, wherein the individual is a neonate, a woman in a peripartum period, or a nonpregnant adult. The method of claim 37, wherein the individual is immunocompromised, or has diabetes mellitus or a malignancy.