WO2005113602A1 - Coiled-coil microbial antigens - Google Patents

Abstract

Numerous peptide antigen sequences are provided, which are derived from naturally occurring proteins and conformed to the coiled-coil structure. The peptides are immunogenic and can be used to elicit an immune response in an animal. Accordingly, they are useful as vaccines or to stimulate antibody production or cell-mediated immunity to the naturally occurring protein.

Description

COILED-COIL MICROBIAL ANTIGENS

FIELD OF THE INVENTION

This invention pertains to antigenic peptide sequences that are useful in eliciting immune responses.

REFERENCES

U.S. Patent Application No. 20030021795, published January 30, 2003.

U.S. Patent No. 6,075,181.

U.S. Patent No. 6,150,584.

Baquero, F., et al. (1991). A review of antibiotic resistance patterns of Streptococcus pneumoniae in Europe. J. Antimicrob. Chemother. 28(Supple. C):31-38.

Beuvery, E.G., et al. (1982). Comparison of the induction of immunoglobulin M and G antibodies in mice with purified pneumococcal type 3 and meningococcal group C polysaccharides and their protein conjugates. Infect. Immun. 37(1): 15- 22.

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Cowan, M.J., et al. (1978). Pneumococcal polysaccharide immunization in infants and children. Pediatrics 62(5):721-727.

Crain, M.J., et al. (1996). Evidence for the simultaneous expression of two PspAs by a clone of capsular serotype 6B Streptococcus pneumoniae. Microb. Pathog. 21(4):265-275.

Hennchsen, J. (1995). Six newly recognized types of Streptococcus pneumoniae. J. Clin. Microbiol. 33(10):2759-2762.

Hodges, et al. (1988).

Holmes, et al. (2001). Houston, M.E., et al. (1996). Lactam bridge stabilization of alpha-helices: the role of hydrophobicity in controlling dimeric versus monomeric alpha-helices. 5zocAeιw£sfry 35(31):10041-10050.

Lancet (1985). Acute respiratory infections in under-fives: 15 million deaths a year. Lancet 2(8457): 699-701. Lau, et al. (1984).

Remington 's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 18th Ed. (1990).

Robbins, J.B., et al. (1983). Considerations for formulating the second- generation pneumococcal capsular polysaccharide vaccine with emphasis on the cross-reactive types within groups. J. Infect. Dis. 148(6): 1136-1159.

Shapiro, E.D., et al. (1991). The protective efficacy of polyvalent pneumococcal polysaccharide vaccine. N. Engl. J. Med. 325(21): 1453-1460.

Shelly, M.A., et al. (1997). Comparison of pneumococcal polysaccharide and CRM197-conjugated pneumococcal oligosaccharide vaccines in young and elderly adults. Infect. Immun. 65(l):242-247.

Singh, D., and O'Hagan, D. (1999). Advances in vaccine adjuvants. Nat. Biotechnol. 17(11):1075-1081.

Spika, J.S., et al. (1991). Antimicrobial resistance of Streptococcus pneumoniae in the United States, 1979-1987. The Pneumococcal Surveillance Working Group. J. Infect. Dis. 163(6): 1273-1278.

Winter, G, and Milstein, C. (1991). Man-made antibodies. Nature 349(6307):293-299.

World Health Organization. WHO 9 (1995).

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All of the publications, patents and patent applications cited above or elsewhere in this application are herein incorporated by reference in their entirety to the same extent as if the disclosure of each individual publication, patent application or patent was specifically and individually indicated to be incorporated by reference in its entirety. BACKGROUND OF THE INVENTION

Streptococcus pneumoniae is an important pathogen causing life-threatening invasive diseases such as pneumonia, meningitis and bacteraemia. It is estimated that more than 1 million cases of pneumococcal pneumonia are diagnosed each year in the United States with 0.7% of these infections being fatal (Lancet, 1985). In developing countries, it is estimated that 4 million deaths are due to pneumonia, with S. pneumoniae infections accounting for 70% of the deaths (WHO, 1995). S. pneumoniae also causes less serious diseases such as otitis media and sinusitis which, due to their prevalence, are a significant burden on health care systems. The high morbidity and mortality rate associated with pneumococcal infections is exacerbated by the rate at which the organism acquires resistance to multiple antibiotics (Spika et al, 1991; Baquero et al, 1991). Thus, there is an unmet need for effective treatments for pneumococcal infections.

The current vaccine utilized for prevention of pneumococcal infection in humans is based on purified capsular polysaccharides. The design of a capsular polysaccharide vaccine is complicated by the fact that there are 90 different capsular types and the protection elicited by the capsule is type specific (Henrichsen, 1995). These problems are somewhat mitigated by the fact that certain capsules are more commonly associated with human disease than others and which ultimately led to their inclusion into the current 23 valent vaccine (Robbins et al., 1983). However, capsular polysaccharides, like most polymeric compounds possessing multiple repeating units, are inefficient in stimulating immune responses and subclass switching to IgG (Beuvery et al., 1982). Not surprisingly, the vaccine is only 60% effective in preventing fatal pneumococcal bacteraemia in the elderly (Shapiro et al., 1991) and is unable to elicit adequate antibody responses in children under the age of 2 (Cowan et al., 1978). The problem of poor immunogenicity in children is being addressed by conjugation of the polysaccharides to protein carriers such as diphtheria and tetanus toxoids (Shelly et al., 1997). h a recent clinical trial, a seven valent conjugate vaccine was shown to be immunogenic in children and elicited protection against invasive pneumococcal infection.

However, a separate vaccine will be required for adults because clinically important infections are caused by different capsular types than is the case for children. Coverage in the developing world is expected to be as low as 52% owing to geographical variation of pneumococcal strains, hi addition, it is anticipated that the conjugate vaccines may be too expensive for the developing world. Thus, there is an urgent need for alternative approaches to the development of pneumococcal vaccines.

U.S. Patent Application Publication No. 20030021795 describes vaccines, particularly pneumococcal vaccines, containing coiled-coil peptides that are derived from microbial proteins. The coiled-coil structure consists of two amphipathic alpha helices wrapped around each other with a left-handed supertwist. The coiled-coil is characterized by a heptad repeat (abcdefg), in which the a and d positions are typically occupied by hydrophobic amino acids. These residues are aligned such that they create a hydrophobic face that is responsible for the stability of this structure. It is disclosed in U.S. Patent Application Publication No. 20030021795 that by stabilizing the coiled-coil structure of microbial epitopes that have a coiled-coil propensity, the immunogenicity of the epitopes can be improved, resulting in better vaccines.

SUMMARY

The present invention provides various antigens which, particularly when conformed to a coiled-coil structure, effectively elicit immune responses. Exemplary peptide antigen sequences are listed in Table 1 as SEQ ID NO:l to SEQ ID NO: 144. Since these antigens are derived from pathogenic microorganisms, the antigens can serve as vaccines for diseases caused by the pathogens. The antigens can also be used to raise antibodies, which are useful in treating or preventing infections caused by the pathogens. Alternatively, the antigens and antibodies are also useful research tools. Accordingly, one aspect of the present invention provides an isolated peptide comprising at least one sequence selected from the group consisting of SEQ LO NO:l to SEQ ID NO: 144. The peptide may optionally comprise two or more sequences selected from the group consisting of SEQ ID NO:l to SEQ LD NO: 144. The two or more sequences are preferably connected by a spacer such that each two sequences and the spacer in between form a continuous heptad repeat structure. The two or more sequences may be from at least two different strains of the same species of microorganism, at least two different species of the same microbial genus, or microorganisms of different genuses. Alternatively, the two or more sequences maybe from at least two different regions of the same protein or different proteins of the same microorganism.

The peptide may contain additional sequences, particularly the sequences that facilitate coiled-coil formation, such as CGNle or CNleG. The peptide is preferably capable of forming a helical structure under physiological conditions, such as in an aqueous solution that contains no helical-promoting chemicals (e.g., trifluoroethanol). A typical aqueous solution is 50 mM KH₂PO₄, 50 mM KC1, pH 7.0.

The peptide is preferably less than about 250 amino acids long, more preferably less than about 200 amino acids long, yet more preferably less than about 150, 100, 75 or 50 amino acids long, and most preferably less than about 35 amino acids long.

The peptide may consist essentially of a sequence selected from the group consisting of SEQ ID NO:l to SEQ ID NO: 144, or a combination of at least two sequences selected from the group.

Another aspect of the present invention provides isolated nucleic acids that encode the peptides described herein, vectors that comprise the nucleic acids, as well as cells harboring the nucleic acids or vectors. Frequently used vectors include, without being limited to, plasmids, cosmids, phages, yeast artificial chromosomes, and viruses.

Another aspect of the present invention provides compositions that comprise the peptide described above. The peptide may be coupled to a carrier molecule, and the composition may further comprise an adjuvant and/or a pharmaceutically acceptable excipient or carrier. The composition is preferably useful for eliciting an immune response to multiple serotypes or strains of a microorganism, multiple species in a genus of microorganism, or multiple microorganisms of different genuses. This can be achieved, for example, if the coiled-coil peptide in the composition contains a common epitope that is shared among multiple serotypes, strains, species or genuses. It can also be achieved if the coiled-coil peptide contains at least two epitopes that are derived from different serotypes, strains, species or genuses. Furthermore, the composition may contain more than one coiled-coil peptides of the present invention, and the multiple peptides contains different epitopes that are derived from different serotypes, strains, species or genuses.

Further aspects of the present invention provide a method of eliciting an immune response in an animal, and a method of treating or preventing a microbial infection in an animal, comprising administering the composition described herein to the animal.

Also provided is a method for determining the presence in a biological sample of antibodies to a microorganism, comprising:

(a) contacting the biological sample with a peptide of the present invention; and

(b) determining whether antibodies in the biological sample bind to the peptide. The microorganism is preferably selected from the group consisting of Streptococcus pneumoniae, Staphylococcus aureus, Clostridium difficile, Haemophilus influenza, Pseudomonas aeruginosa, Neisseria meningitidis, Escherichia coli, Helicobacter pylori, andMoraxella catarrhalis.

Another aspect of the present invention provides a method of preparing an antibody using the composition described herein, as well as the antibodies so prepared, compositions comprising such antibodies, and methods of treating or preventing a microbial infection in an animal, comprising administering the antibody-containing composition to the animal. The antibody can also be used in a method of determining the presence of a microorganism in a sample, comprising contacting the sample with the antibody and determining if the antibody specifically recognizes the sample.

The animal is preferably a mammal or avian. The mammal is preferably selected from the group consisting of human, non-human primate, feline, canine, murine, rodent, equine, porcine, bovine and ovine, and most preferably a human.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. shows the CD spectrum of peptides PspA 41-48 and PavA 280-293 in benign conditions and in 50% TFE.

Figure 2. shows the antibody titer responses of PspA 41-48 and PavA 280-293 from two separate experiments.

Figure 3. shows the isotype responses of Pava 280-293 and PspA 41-48 antisera.

Figure 4. demonstrates the concentration dependent inhibition of free peptide antigens (PavA 280-293 and PspA 41-48) to inhibit binding of PavA 280-293 and PspA 41-48 antisera to the corresponding peptide BSA conjugates. Figure 5. Both PspA 41-48 and PavA 280-293 antisera demonstrated opsonic activity against S. pneumoniae serotypes 14 and 19F. Negative control sera was not opsonic and unable to assist in the phagocytosis process.

Figure 6. Whole cell bacterial lysates were separated electrophoretically and probed with PspA 41-48 antisera. The antisera bound to a particular band at 64 Kda. This band was sequenced by LC/MS/MS and shown to be PspA.

Figure 7. Mice immunized with 20 μg of PspA 41-48 or PavA 280-293 tetanus toxoid conjugates and subsequently challenged with a lethal dose of S. pneumoniae serotype 14 survived. In contrast, the mice that received a negative control (TT) did not. Figure 7A shows the number of surviving mice over time (hr), and Figure 7B shows the percentage of survival.

DETAILED DESCRIPTION

The present invention provides various antigens which, particularly when conformed to a coiled-coil structure, effectively elicit immune responses. Since these antigens are derived from pathogenic microorganisms, the antigens can serve as vaccines for diseases caused by the pathogens. The antigens can also be used to raise antibodies, which are useful in treating or preventing infections caused by the pathogens. Alternatively, the antigens and antibodies are also useful research tools.

Prior to describing the invention in further detail, the terms used in this application are defined as follows unless otherwise indicated.

Definitions

An "antigen" is a substance that is capable of eliciting an immune response in an animal. A "peptide antigen" is an antigen that comprises a peptide. A "composite antigen", as used herein, refers to an antigen that comprises at least two peptide antigens, particularly at least two peptide antigens that are covalently bonded.

A "native protein" is a protein which exists in nature.

A "coiled-coil protein" is a protein which, at least in part, forms a coiled-coil. A coiled-coil protein may have other conformational structures in addition to the coiled-coil. Encompassed within the term coiled-coil proteins are the proteins which have been shown to assume a coiled-coil structure and the proteins which are predicted to form coiled-coils by using a computer algorithm.

A "microbial protein" is a protein derived from a microorganism such as a bacterium, archaebacterium, fungus, virus, protozoan, parasite, alga, slime mold, or prion.

An "epitope" is a part of a protein or peptide which is an antigenic determinant.

A "derivative" of an amino acid is a non-naturally occurring amino acid residue or a chemically modified amino acid. Amino acid derivatives may be used to increase the half life of the peptide in serum or tissue, or to increase antigenicity of the peptide.

Non-naturally occurring amino acids include, but are not limited to, D-isoniers, norleucine, 4-amino butyric acid, aminoisobutyric acid, 4-amino-3-hydroxy-5- phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-rnethylheptanoic acid and 2-thienyl alanine. A chemically modified amino acid is an amino acid with a side chain modification. For example, the amino group of lysine may be modified by alkylation with an aldehyde followed by reduction with NaBH ; amidation with methylacetimidate; acylation with acetic anhydride; carbamoylation with cyanate; trinitrobenzylation with 2,4,6,-trinitrobenzene sulphonic acid (TNBS); acylation with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation with pridoxal-5'-ρhosphate followed by reduction with NaBH₄.

The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation via O- acylisourea formation followed by subsequent derivatization, for example, to a corresponding amide.

The sulfhydryl group may be modified by carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulfide with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4 chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; or carbamoylation with cyanate at an alkaline pH.

Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy- 5nitrobenzyl bromide or sulphenyl halides. Tyrosine residues may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or diethylpyrocarbonate. A "peptide" as used herein is a peptide or a protein. In other words, a peptide referred to in this application may contain any number of amino acids connected by peptide bonds. A peptide may contain naturally existing amino acids or amino acid derivatives. A peptide may also contain cross links between different portions of the peptide. For example, coiled-coil structures are often composed of two strands or three strands. It is preferable that the different strands of the coiled-coil are linked by disulfide bonds, which stabilize the coiled-coil structure. More preferably, the peptide contains a lactam bridge between the side chains of lysine and glutamic acid which are spaced 3 or 4 residues apart (Houston et al., 1996). These lactam bridges are incorporated preferably at the N- and C-termini of the peptide sequence and not in regions of the sequence where the epitope is being displayed.

A "solvent exposed" or "solvent accessible" amino acid residue is an amino acid residue of a protein or peptide which is exposed to the solvent when the protein or peptide exists in a solution. The solution is preferably an aqueous solution, more preferably a physiologically compatible solution, such as blood, lymphatic fluid or a benign buffer. Ixi particular, "solvent exposed" amino acid residues refer to the residues at the b, c, e, f or g positions of a native epitope which forms or is predicted to form a coiled-coil.

A benign buffer is a physiologically compatible buffer, such as a phosphate buffered solution. The "benign buffer" as used in this application is 50 mM KH₂PO₄, 50 mM KC1, pH 7.0.

An "antibody" is a protein molecule that reacts with a specific antigen and belongs to one of five distinct classes based on structural properties: IgA, IgD, IgE, IgG and IgM.

An "immune response" is the development in the host of a cellular and/or antibody-mediated immune response to a composition of interest. Such a response may consist of the production of one or more of the following: antibodies, B cells, helper T cells, suppressor T cells, and/or cytotoxic T cells directed specifically to an antigen or antigens included in the composition of interest.

A "vaccine" is a substance which is capable of eliciting in an animal an immune response which prevents, partially or completely, infection by a pathogen.

"Elicit" or "stimulate" an immune response is to cause an immune response by exposing an immune system to an immunogen.

An "immunogen" is a molecule which is capable of eliciting an immune response in an animal.

"Prevent a microbial infection" is to prevent, completely or partially, the development of a microbial infection.

"Treat a microbial infection" is to reduce, completely or partially, the symptoms of a microbial infection after the onset of the microbial infection.

A "sample" is an aliquot or a representative portion of a substance, material, or population. For example, a sample may be a sample of water, sewage, oil, sand, blood, biological tissue, urine or feces.

A "biological sample" is a sample collected from a biological subject, such as an animal, plant or microorganism.

An "effective amount" is an amount which is sufficient to achieve the intended purposes. For example, an effective amount of a vaccine is an amount of the vaccine sufficient to elicit an immune response in the recipient of the vaccine to protect the recipient from contracting the target disease or to prevent or alleviate medical conditions associated with the disease. Compositions and uses

Pneumococcal surface protein A has been found in all strains of S. pneumoniae studied to date and is required for full virulence (Crain et al., 1990). While PspA has a strain-dependent molecular weight ranging from 67 to 99 kDa, all proteins consist of four distinct domains: an N-terminal highly charged domain, a proline rich domain, a repeat domain comprising 10 highly conserved 20 amino acid repeats, and a short hydrophobic domain. The repeat region is responsible for attachment of the protein to the cell surface of S. pneumoniae by non-covalent binding to choline residues.

A preferred embodiment is the consensus amino acid sequence of residues 41-48 of PspA. To better conform this sequence to the coiled-coil structure, the residues at the a and expositions were changed to isolucine and leucine, respectively. Additional sequences were also added to the N-terminal and C- terminal end to further stabilize the coiled-coil structure. The resulting peptide is designated PspA 41-48 (SEQ ID NO:l).

PspA 41-48 is predominantly coiled-coil. As described in Example 1, PspA 41- 48 is 99% helical in a physiological aqueous solution (50 mM phosphate, 100 mM KCI, pH 7.0). In contrast, when the residues at the a and d positions were substituted with glycine (PspA 41-48 control), the helical content dropped to less than 5%. Although in a solution containing 50% TFE, which stabilizes alpha helices, both PspA 41-48 and PspA 41-48 control folded into an alpha-helix, this is not a physiological condition.

PspA 41-48 was used to raise antibodies as a conjugate with the carrier protein tetanus toxoid. The resulting antisera had high titer, specificity, and opsonic activities (Examples 3-6), indicating that PspA 41-48 is highly immunogenic.

Consistent with these results, in vivo administration of the PspA 41-48-tetanus toxoid conjugate provided animals protection against a subsequent challenge by

S. pneumoniae in various models, including systemic and intranasal infections

(Examples 9-11). Therefore, PspA 41-48 can be used as an effective vaccine. Antibodies raised using PspA 41-48 are also protective against S. pneumoniae, as demonstrated by the opsonic activities of the antibodies.

Significantly, the PspA 41-48 antisera recognize a number of serotypes of S. pneumoniae (Example 7). It has long been a problem that S. pneumoniae undergoes substantial antigenic variation. For example, at least 40 different sequences for PspA have been identified to date from various strains of S. pneumoniae, and the vaccines or antibodies prepared using one strain are often useless against another. PspA 41-48 thus provides a powerful, cross-reacting tool against various strains of S. pneumoniae.

Similarly, another preferred embodiment was derived from amino acids 280-293 of PavA. PavA is another surface protein that is conserved amongst numerous strains of S. pneumoniae. PavA is essential for virulence (Holmes et al. 2001). This protein is a putative adhesin and has been shown to bind to fibronectin. Analysis of the sequence by the protein multicoil program suggests that the protein has a coiled-coil region between residues 270 and 310. When the PavA 280-293 sequence was conformed to the coiled-coil structure, the resulting peptide (SEQ ID NO: 14) was strongly coiled-coil under physiological conditions (94% helical, see Example 2). This peptide is also highly immunogenic (Examples 3-6), provides protection against S. pneumoniae infections (Examples 9-13), and is capable of eliciting immune responses against multiple strains of S. pneumoniae (Example 7). Thus, like PspA 41-48, PavA 280-293 can be used as vaccines, as well as to raise antibodies, against various strains of S. pneumoniae.

In addition to PspA 41-48 and PavA 280-293, we have identified many other bacterial sequences that are useful in the same manner. These sequences are listed in Table 1 below. It is contemplated that the amino acids at the a and d positions in any peptide provided in this disclosure can be substituted with any hydrophobic amino acid, particularly isoleucine, leucine, valine, phenylalanine, tryptophan, methionine and tyrosine. Preferably, the a and d positions are occupied by a residue independently selected from the group consisting of isoleucine, leucine, and valine. More preferably, the residue at the a or d position is isoleucine or leucine. Most preferably, the residue at the a and d positions are isoleucine and leucine, respectively.

Table 1. Bacterial Protein Targets

* The first column shows the region in the native protein where the enumerated sequence is located. The italic letters in the top row, a to g, indicate the position of each amino acid in the coiled-coil heptad. _ _ ga b c Def ga b c def gΛb c def ga ID N__ S. pneumoniae Pneumococcal Surface Protein A

41^48 CG Nle El EALKKEI SKLEKDI DALKKEI EALKK ϊ S KLEKDI D 2

77-84 CG Nle El E ALKKAI KKLDDDI E ALKKEI E ALKK 3 KKLDDDI E 4

82-91 CG Nle El EALKKEI EALDDDI KKLEEKI EALKK 5 EALDDDI KKL 6

342-349 CG Nle El EALKKLI EELSDKI DELEKEI K 7 EELS DKI D 8

350-359 CG Nle ELEALKKEI DELDAEI AKLEKEI EALKK 9 DELDAEI AKL 10

386-395 CG Nle E I E AL KKE I E A L A AKI A E L E KE I E AL K K 11 EALAAKI AEL 12

S. pneumoniae PavA (AAF05332)

280-293 CG Nle El EALKKEI RRLENEI QKLRHEI EALKK 13 I RVENELQKNRHK 14

370-381 CG Nle El EALKKEI KYLTDLI EELKKEI EALKK 15 VKYLTDLI EET K 16 S. aureus PavA Homolog (NP 371732)

294-306 CNle G E I E AL KKE I RF L Q Q QI HKL QNE I E AL KK 17 VRF VQQQLHKYQN 18 SEQ ga b c Def ga b c def gAb c d ef ga ID NO:

307-320 CNle G E I E AL KKKI AKL I E E I E QL KNE I E L AKK 19 KL AKLI EE YE QS KN 20 S. aureus Hypothetical Protein Similar to AdcA Zn Binding Lipoprotein (NP 647145)

179-191 CNle G E I E AL KKHI DD L E KNI KKL NDE I E AL KK 21 HKDDYEKNYKKLN 22

193-206 CNle G E I E AL KKDI KKL DNDI KQL T KE I E AL KK 23 DLKKI DNDMKQVTK 24 S. aureus PsaA Homolog (GI.5923621)

160-172 CNle G E I E AL KKE I DNL KKHI ADL E KE I E AL KK 25 I DNDKKHKADYEK 26 C. difficile Fibronectin Binding Protein (GI-.21666416)

318-333 CNle G E I E AL KKKI DR L YNKI KKL E E E I E A L KK 27 KLDRL YNKLKKQEEEL 28

408-421 CNle G El EALSHQI SLLKEEI DYLKKEI EALKK 29 S HQI S LNKEEI DYL 30 H. influenzae Conserved Hypothetical Protein (GI.16272697)

59-67 CNle G E I E AL KKE I AKL Q ANI KKL E S E I E AL KK 31 QANLKKHES 32

68-80 CNle G E I E AL KKKI NS L E GE I L E L E I E I E AL KK 33 Kl NS VEGELLET E 34

82-94 CNle G E I E AL KKS I KE L RKQI ADL DKE I E AL KK 35 S LKEI RKQI ADAD 36

184-197 CNle G El EALKKQI S TLKKQI QAL QKEI EALKK 37 QLS TQKKQQQAL QK 38

198-211 CNle G E I E AL KK Al QE L QS T I NE L NKE I E AL KK 39 AQQEHQS TLNEL NK 40

212-225 CNle G El EALKKNI ALLQDKI NTLKAEI EALKK 41 NL ALDQDKLNTL KA 42

226-239 CNle G E I E AL KKNI Q AL R Q E I QRL E QE I E AL KK 43 NEQALRQEI QRAEQ 44_ SEQ b c Def ga b c def gAb c def ga ID NO-

242-252 CNle G E I E AL KK Al R E L E KRI R E L L AE I E AL KK 45 REQEKREREAL 46 H. influenzae HAP (Haemophilus Adherence and Penetration Protein) (GI:23506944)

984-996 CNle G E I E AL KKE I QE L R S DI VRL E Q Al E AL KK 47 QELRS DL VRAE QA 48

998-

1007 CNle G El EALKKEI RTLEAKI VEL KKEI EALKK 49 RTLEAKQVEQ 50 H. influenzae HIA (AAC43721)

71-81 CNle G EE Il EE AALL KKKKEE II EE AA LL NNNN SS II AADDLL EE KK QQ II EE AALL KK KK 51 NNS I ADAEKQV 52

87-94 CNle G EElI EEAALLKKKKEEII EEGGLLLLNNLLII EEKKLLKKKKEEII EEAALLKKKK 53 GLLNLNEK 54

607-618 CNle G E II EEAALLTTSSSSII DDNNLLTTKKQQII DDAALLKKKKEEII EEAALLKKKK 55 TS S ADNLTKQND 56

620-630 CNle G EElI EEAALLYYKKGGII TTNNLLDDEEKKIl EEAALLKKKKEEII EEAALLKKKK 57 AYKGLTNLDEK 58 P. aeruginosa PspA Like Protein (G 560Q326)

21-34 CNle G EElI EEAALLKKKKEEII AADDLLQ QRRQQII EE Q QLL Q QKKEEII EEAALLKKKK 59 ERADTQRQL E QT QK 60

35-48 CNle G EElI EEAALLKKKKDDII GGEELLKKKKLLII DDGGLL QQQQEEII EEAALLKKKK 61 DI GELKKLL DGI QQ 62

49-62 CNle G EElI EEAALLKKKKEEII SS GGLLQQKKQQII KKSSLL EETTEEII EEAALLKKKK 63 EKS GVQKQL KST ET 64

63-76 CNle G EElI EEAALLKKKKEEII GGDDLLEEKKQ QII KKAALL Q QDDEEII EEAALLKKKK 65 EMGDLEKQI KAL QD 66

77-90 CNle G EElI EEAALLKKKKEEII DDKKLLEEAAEEII KKRRLL DDGGEEII EEAALLKKKK 67 ELDKS E AEL KRL DG 68

239-252 CNle G EElI EEAALLKKKKQQII RREELLAAAAAAIl EERRLL RRQ QEEII EEAALLKKKK 69 QARE AAAAA ERE RQ ' 70 SEQ ga b c Def ga b c def gAb c def ga ID NO: N. meningitidis Aap (Adhesin and Penetration Protein) (GI:11071865)

1058-

1069 CNle G E I E AL KKE I QE L S DKI GKL E AKI E AL KK 71 QELSDKLGKAEA 72

1071-

1082 CNle G E I E AL KKE I Q AL KDNI Q S L D AE I E AL KK 73 KQAEKDNAQSLD 74

1113-

1125 CNle G GEI EALKKEI MQLEEEI KRLQADI EALKK 75 MQAEEEKKR VQAD 76 TV^", meningitidis Conserved Hypothetical Protein (GI.1567719)

180-192 CNle G E I E ALK KE I KNL KT D I DEL K A Al E AL KK 77 KNAKTDS DEL KAA 78

195-207 CNle G E I E ALK KE I E AL T NDI E NL K AL I E AL KK 79 AATNDVENKKALL 80

234-247 CNle G El EALKKEI RQLRGNI ASLNRKI EALKK 81 I RQARGNL AS VNRK 82

256-268 CNle G El EALKKEI QKLNTEI NRLKTEEI EALKK 83 QKLNTELNRL KTE 84

323-332 C Nle G El EALKKEI KDLEKQI KAL KKEI EALKK 85 KDLEKQQKAL 86

404-416 CNle G E I E ALK KE I KKL E HR I Q DL E AE I E AL KK 87 KKKAEHRI QDAEA 88

N. meningitidis Putative Adhesin/Advasin (G 21427120)

112-121 CNle G E I E AL KKE I E AL E S E I E KL T T Kl E AL KK 89 ES EI EKL TTK 90

180-191 CNle G E I E AL KKE I D S L DE T I T KL D E E I E AL KK 91 DSLDETNTKADE 92 JV. meningitidis PspA Like Protein (GI: 15676400) SEQ ga b c Def ga b c def gAb c def ga ID NO:

234-242 C Nle G El EALKKEI LELELQI TD EAEI AALKK 93 LE AELQNTD 94 E. coti yigN Protein (GI-.15804424)

54-67 CNle G E I E AL KKQI E HL R AE I E L L NNE I E AL KK 95 QSEHλV AECELLNN 96

61-74 CNle G E I E AL KKE I E L L NNE I R S L Q S E I E AL KK 97 ECELLNNEVRSL QS 98

68-81 CNle G El EALKKEI RS LQS I I TSL EAEI EALKK 99 EVRS LQS I NTSL EA 100

75-88 CNle G E I E AL KKI I T S L E AD I REL T T E I E AL KK 101 I NTSLEADLREVTT 102

82-95 CNle G E I E AL KKD I R E L T T R I E AL Q Q E I E AL KK 103 DLRE VTTRMEAAQQ 104 Kcoli YigP Protein (GI:15804157)

68-81 CNle G E I E AL KKQI A S L L AQ I KKL E E E I E AL K K 105 QRAS LL AQLKKQE A 106

75-88 CNle G E I E AL KKQI KKL E E Al S EL T R E I E AL KK 107 QLKKQEE Al S EATR 108

82-95 CNle G E I E AL KK Al S E L T R Kl R E L QNE I E AL KK 109 Al S E ATRKLRET QN 110

89-102 CNle G El EALKKKI RELQNTI NQLNKEI EALKK 111 KLRETQNTLNQL NK 112

96-109 CNle G E I E AL KKT I NQ L NKQ I D E L N A E I E AL KK 113 TLNQLNKQI DEMNA 114

103-116 CNle G E I E AL KK QI DE L N A S I AKL E Q E I E AL KK 115 QI DEMNAS I AKL E Q 116

110-123 CNle G E I E AL KK S I AKL E Q QI A AL E R E I E AL KK 117 S I AKLEQQKAAQER 118

170-183 CNle G El EALKKTI AQLKQTI EELAMEI EALKK 119 TI AQLKQTREEVAM 120 SEQ g a b c Def g a b c def gAb c d e f g a ID NO:

177-190 CNle G El EALKKTI EEL AMQI AEL EEEI EALKK 121 TREE VAMQRAEL EE 122

184-197 CNle G E I E AL KKQI AE L E E Kl S E L Q T E I E A L KK 123 QRAELEEKQS EQQT 124 E. coli STEC Autoagglutinating Adhesion (GI:16565696)

307-320 CNle G E I E AL KKQI N AL E QNI NQL L QE I E AL KK 125 QVNALEQNTNQQLQ 126 H. pylori PavA Homolog (NP_224153)

177-187 CNle G E I E AL KKQI KE L E HKI NQL KKE I E AL KK 127 QHKELEHKKNQ 128

184-197 CNle G E I E AL KKKI N Q L I KR I N AL KE E I E AL KK 129 KKNQI I KRLNAQKE 130

191-204, CNle G El EALKKRI NALKERI KELLEEI EALKK 131 RLNAQKERLKEKLE 132 H. pylori OMP 26 (NP 206880)

349-361 CNle G E I E AL KKE I NKL ME VI D KL DNE I E AL KK 133 VNKAMEVRDKL DN 134

355-368 CNle G E I E AL KK VI DKL D NNI NQL DNE I E AL K K 135 VRDKLDNNLNQL DN 136

362-374 C Nle G E I E AL KKNI NQ L D ND I KDL KKE I E AL KK 137 NLNQLDNDLKDQK 138 H. pylori Toxin Like OMP (GI; 15645538)

1390-

1403 CNle G El EALKKEI LQLLDKI Kl L QVEI EALKK 139 EVLQLLDKI KI T QV 140

1397-

1410 CNle G E I E AL KKKI Kl L Q V QI Q AL L E E I E AL KK 141 Kl Kl TQVQKQAL LE 142

1404-

1417 CNle G E I E AL KK QI Q AL L E T I NHL T D E I E A L KK 143 QKQALLETI NHL TD 144_ Of particular interest are peptide antigens that comprise at least two sequences disclosed herein. For example, PspA 41-48 and PavA 280-293 can be combined in the same antigen. For the purpose of forming a continuous coiled-coil structure, spacers can be inserted between the two sequences to maintain the continuity of heptad repeats. In the case of the composite antigen of PspA 41-48 and PavA 280-293, 5 or 1 residue can be inserted between PspA 41-48 and PavA 280-293, depending on the order of PspA 41-48 and PavA 280-293, as explained below.

The core sequence of PspA 41-48 (SKLEKDLD; SEQ LD NO:2) runs from a b position to a 5 position, and the core sequence of PavA 280-293 (IRRVENELQKNRHK; SEQ ID NO: 14) runs from an a position to a g position (see Table 1). A composite antigen in which SEQ ID NO:2 (b to b) precedes SEQ ID NO: 14 (a to g) needs 5 spacer residues to fill the c, d, e,/and g positions between SEQ ID NO:2 and SEQ LD NO: 14. In contrast, if SEQ ID NO: 14 (a to g) precedes SEQ ID NO:2 (b to b) in the composite antigen, only one spacer residue would be needed to occupy the a position immediately after SEQ ID NO: 14. In addition to the 5 or 1 spacer residue discussed above, extra spacer residues in multiples of 7 (such as 7, 14, 21, 28, etc.) can also be included. The use of spacer residues in multiples of 7 maintains the continuity of heptad repeats.

These spacer residues can be any amino acids, but preferably the a and d positions are occupied by hydrophobic amino acids, particularly isoleucine, leucine, valine, phenylalanine, tryptophan, methionine and tyrosine. Preferably, the a and d positions are occupied by a residue independently selected from the group consisting of isoleucine, leucine, and valine. More preferably, the residue at the a or d position is isoleucine or leucine. Most preferably, the residues at the a and ^positions are isoleucine and leucine, respectively. A composite antigen can also be prepared by covalently linking two peptides, each of which contains a different sequence of interest. In a preferred embodiment, the two peptides are linked by a disulfide bond. For example, the N-terminal cysteine residues of PspA 41-48 (SEQ ID NO:l) and PavA 280-293 (SEQ ID NO: 14) were reacted to form a disulfide bond. The resulting molecule formed an intra-molecular coiled-coil, with the two strands of the coiled-coil being PspA 41-48 and PavA 280-293, respectively. In this approach, if the two peptides have different lengths, it is preferable to add spacer residues to the shorter one in a manner that facilitates coiled-coil formation. For example, suppose SEQ ID NO:l and SEQ ED NO:7 are to be bonded by a disulfide bond via the N-terminal cysteine residues of the peptides. Upon aligning the heptad positions of the two sequences (i.e., a position with a position, b position with b position, and so on), SEQ ID NO:7 is shorter at the C-terminus by 4 residues, at the c, d, e, and/positions, respectively. It is preferable to add 4 residues that are suitable for these positions, e.g., a Leu at position d.

Peptides having the same sequences can also be covalently bonded to form intramolecular coiled-coils. In this case, the peptide may already contain more than one sequence of interest. For instance, a peptide having both SEQ LD NO:2 and SEQ ID NO: 14, as well as a CGNle at the N-terminus, can be covalently bonded to an identical peptide, and the resulting molecule will have two copies each of SEQ ID NO:2 and SEQ ID NO: 14.

By combining at least two sequences provided herein, it is possible to elicit not only an immune response against multiple serotypes or strains of a microorganism, but also an immune response against multiple microorganisms. For example, since SEQ ID NO:l is derived from S. pneumoniae and SEQ LD NO: 18 is derived from S. aureus, these two sequences can be included in a peptide antigen for the purpose of eliciting an immune response against both S. pneumoniae and S. aureus. The peptide antigens of the present invention, comprising at least one sequence selected from the group consisting of SEQ LD NO:l to SEQ ID NO: 144, are preferably less than about 250 amino acids long. The antigens are more preferably less than about 200, 150, 100, 75, or 50 amino acids long. Most preferably, the antigens are less than about 35 amino acids long. The peptides can be prepared by any methods established in the art, such as peptide synthesis or recombinant DNA techniques.

A composition comprising the peptide antigens of the present invention can be used to elicit an immune response in an animal for at least two purposes. Where the composition acts as a vaccine by eliciting an immune response in the animal, the resulting antibodies or T-cell mediated immunity can protect the animal from a subsequent attack involving a microorganism which comprises the same epitopes (active immunity). Alternatively, the composition can be used to produce antibodies which can be used as a research tool, or administered to a second animal to protect the second animal from a subsequent attack involving a microorganism which comprises the same epitopes (passive immunity).

To augment the immune response elicited, it may be preferable to couple the peptide antigen, especially the smaller peptides (e.g., those containing less than about 50 amino acids), to a carrier molecule. Carrier molecules and methods of coupling are known in the art. Typical carrier molecules include proteins and polysaccharides.

In addition, the peptide antigens or conjugates thereof may be further mixed with adjuvants to elicit an immune response, as adjuvants may increase i munoprotective antibody titers or cell mediated immunity response. Such adjuvants may include, but are not limited to, Freunds complete adjuvant, Freunds incomplete adjuvant, aluminum hydroxide, dimethyldioctadecyl- ammonium bromide, Adjuvax (Alpha-Beta Technology), Inject Alum (Pierce), Monophosphoryl Lipid A (Ribi Immunochem Research), MPL+TDM (Ribi Immunochem Research), Titermax (CytRx), QS21, the CpG sequences (Singh et al., 1999), CoVaccine HT (CoVaccine BV, Lelystad, Holland), toxins, toxoids, glycoproteins, lipids, glycolipids, bacterial cell walls, subunits (bacterial or viral), carbohydrate moieties (mono-, di-, tri-, tetra-, oligo- and polysaccharide), various liposome formulations or saponins. Combinations of various adjuvants may be used with the antigen to prepare the immunogen formulation.

The composition may be administered by various delivery methods including intravascularly, intraperitoneally , intramuscularly, intradermally, subcutaneously, orally, nasally or by inhalation. The composition may further comprise a pharmaceutically acceptable exicipient and/or carrier. Such compositions are useful for immunizing any animal which is capable of initiating an immune response, such as primate, rodent, bovine, ovine, caprine, equine, leporine, porcine, canine and avian species. Both domestic and wild animals may be immunized. The exact formulation of the compositions will depend on the particular peptide or peptide-carrier conjugate, the species to be immunized, and the route of administration.

The antibodies produced against the peptide antigens can be included in a pharmaceutical composition and administered to an animal. The pharmaceutical composition typically comprises a pharmaceutically acceptable carrier, and may include pharmaceutically acceptable excipients. The pharmaceutical composition can be administered, e.g., intravascularly, intraperitoneally, intramuscularly, intradermally, subcutaneously, orally, nasally or by aerosol inhalation. Preferably, the pharmaceutical composition is administered intravascularly, intramuscularly, nasally or by aerosol inhalation.

Also encompassed by the present invention are antibodies, particularly monoclonal antibodies, which are derived from the antibodies produced against the peptide antigens. In particular, hybridomas can be generated using the peptide antigens, and recombinant derivative antibodies can be made using these hybridomas according to well-known genetic engineering methods (for a review, see Winter et al., 1991). For example, the DNA fragment coding for the variable regions of the monoclonal antibodies can be obtained by polymerase chain reactions (PCR). The PCR primers can be oligonucleotides which are complementary to the constant regions of the heavy chain or light chain, and the PCR template can be the total cDNA or genomic DNA prepared from the hybridomas. Alternatively, a cDNA library can be prepared from the hybridomas and screened with probes which correspond to the constant regions of immunoglobulin heavy chain or light chain to obtain clones of the heavy chain or light chain produced by the particular hybridoma.

Subsequently, the DNA fragment for the variable regions can be inserted into an expression vector and joined in frame with the cDNA sequences of a selected constant region. The constant region can be the human constant sequences to make humanized antibodies, the goat constant sequences to make goat antibodies, the IgE constant sequences to make IgE which recognizes the peptide of formula I, and the like. Thus, antibodies with the same antigen recognition ability but different constant regions can be produced. Of particular interest are humanized antibodies, which can be used as therapeutic agents against a disease associated with the cognate antigen in humans without eliciting an undesired immune response against the humanized constant region.

Other methods known in the art to humanize antibodies or produce human antibodies can be utilized as well, including but not limited to the xenomouse technology developed by Abgenix Inc. (U.S. Patent Nos. 6,075,181 ; 6,150,584) and the methods developed by Biovation, Bioinvent International AB, Protein Design Labs., Applied Molecular Evolution, Inc., ImmGenics Pharmaceuticals Inc., Medarex, Inc., Cambridge Antibody Technology, Elan, Eos Biotechnology, Medlmmune, MorphoSys or UroGensys Inc. Likewise, other methods known in the art to screen human antibody secreting cells to coiled-coil peptide antigens can also be utilized.

The formulation for the composition, comprising either a coiled-coil peptide or an antibody against a coiled-coil peptide, will vary depending on factors such as the administration route, the size and species of the animal to be administered, and the purpose of the administration. Suitable formulations for use in the present invention can be found in Remington 's Pharmaceutical Sciences.

The following examples are offered to illustrate this invention and are not to be construed in any way as limiting the scope of the present invention.

EXAMPLES

In the examples below, the following abbreviations have the following meanings. Abbreviations not defined have their generally accepted meanings.

°C = Degree Celsius Hr — Hour min = Minute μM = Micromolar mM = Millimolar M = Molar ML = Milliliter μL = Microliter Mg = Milligram μg _= Microgram rpm = revolutions per minute ID = inner diameter TFE = Trifluoroethanol EDT = Ethanedithiol TFA = trifloroacetic acid PBS = phosphate buffered saline θ-ME = θ-mercaptoethanol DMSO = Dimethylsulfoxide ELISA = enzyme linked immunosorbent assay HRP = horse radish peroxidase TD = Thymus dependent TT — Tetanus toxoid KLH = keyhole limpet hemocyanin BSA = Bovine serum albumin i.n. = Intranasal i.p. = Intraperitoneal i.v. = Intravenous The one letter code and the three letter code for amino acids used throughout this application are listed below:

A = Ala = Alanine C -= Cys -= Cysteine D = Asp = Aspartic Acid E = Glu = Glutamic Acid F = Phe = Phenylalanine G = Gly = Glycine H = His = Histidine I = He = Isoleucine K = Lys = _. Lysine L = Leu = Leucine M = Me = Methionine N = Asn = Asparagine P = Pro = Proline Q = Gin = Glutamine R = Arg = Arginine S = Ser = Serine T = Thr = Threonine V -= Val = Valine w = Tip = Tryptophan Y = Tyr = Tyrosine Nle = Norleucine

Materials and Methods

Peptide synthesis and purification

The peptide analogs were prepared by t-Boc chemistry on an Applied Biosystem 431 A peptide synthesizer. The peptide resin (700 mg) was cleaved with 10 mL of HF containing 10% anisole and 2.5% EDT for 1 h at 0-4°C. After removal of the HF the peptide/resin was transferred to a sintered glass funnel and washed with diethyl ether (2 x 50 mL) followed by glacial acetic acid (2 x 50 mL). The acetic acid solution was then lyophilized. The crude peptide was taken up in 15 mL of water containing 0.05% TFA and 3 mL acetic acid. After stirring and sonication, the mixture was transferred to 1.5 mL Eppendorf tubes and centrifuged at 13000 rpm. The supernatant was collected and filtered through a Millex GV 0.22 μm syringe filter. This solution was loaded onto a Zorbax RX- C8 (22.1 mm TD x 250 mm, 5μ particle size) through a 5 mL injection loop at a flow rate of 3 mL/min. The purification was accomplished by running a linear AB gradient of 0.1% B/min where solvent A is 0.05% TFA in water and solvent B is 0.05% TFA in acetonitrile. μ

A linker and cysteine residue was incorporated to enable the strands of the coiled- coil to be linked together by a disulfide bond. The disulfide-bridged coiled-coils were formed by overnight air oxidation at room temperature of 1 mg/mL peptide in 100 mM NH₄HCO₃, pH 8.5. All peptides were characterized by analytical HPLC and electrospray mass spectrometry, and protein concentrations as well as amino acid composition were determined by amino acid analysis.

A Benzoylbenzoic acid group was attached to the N-terminus of the peptides to facilitate attachment of the peptide to carrier proteins. The peptide and tetanus toxoid were mixed together in phosphate buffer and irradiated with a long wavelength UV lamp for 2 hours at room temperature. The peptide conjugate was diafiltered through a 25 Kda membrane to remove unconjugated peptide. The conjugation ratio was determined by amino acid analysis and the amount of peptide attached was calculated based upon the number of nmoles of norleucine.

CD Spectroscopy

Circular dichroism (CD) spectroscopy was performed using a Jasco J-500C spectropolarimeter (Jasco, Easton, Maryland) equipped with a Jasco DP-500N data processor. A 10-fold dilution of an 500 M stock solution of the disulfide- - linked peptide was loaded into a 0.02 cm fused silica cell and ellipticity scanned from 190 to 250 nm. Each disulfide-bridged analog was analyzed by CD spectroscopy under benign conditions (50 mM phosphate, 100 mM KCI, pH 7.0) and also in the presence of 50% trifluoroethanol (TFE) in the same buffer. TFE is a solvent that induces I-helical structure in proteins that have a propensity to fold into an I-helical conformation. A Lauda water bath (model RMS, Brinkmann Instruments, Rexdale, Ont.) was used to control the temperature of the cell. CD spectra were the average of four scans obtained by collecting data at 0.1 nm intervals from 250 to 190 nm.

Direct ELISA

The following is the basic procedure to measure antibody levels by direct ELISA:

1. Coat EIA plates (COSTAR) with 1.0 μg/mL (0.1 μg per well) of antigen in 0.05 M carbonate-bicarbonate buffer (pH 9.6, 100 μL/well).

2. Incubate the plate(s) at 4°C overnight.

3. On the next day, wash plates 3X with washing buffer (PBS / 0.05% Tween). Flick off excess liquid by tapping the plates on the bench top.

4. Block plates with 100 μL blocking buffer per well (PBS/2% BSA). Incubate plates for 1 hour at 37°C.

5. Wash plates 5X with washing buffer (PBS/0.05% Tween). Flick off excess liquid by tapping the plates on the bench top.

6. Add 100 μL per well of test antibody appropriately diluted in dilution buffer (PBS/0.1% Tween). Incubate plates for 60 minutes at 37°C.

7. Wash plates 3X with washing buffer (PBS/0.05% Tween). Flick off excess liquid by tapping the plates on the bench top.

8. Dilute HRP anti-mouse IgG (Jackson Lab) in dilution buffer (PBS/0.1 % Tween) to a concentration of 1:5000. Add 100 μL per well and incubate at 37°C for 60 minutes.

9. Wash plates 3X with washing buffer (PBS/0.05% Tween). Flick off excess liquid by tapping the plates on the bench top.

10. Prepare HRP substrate; Prepare 0.01 M citrate buffer by dissolving 1.48 g of sodium citrate in 500 mL of distilled water. Prepare a 0.01 M citric acid solution by dissolving 1.58 g of citric acid in 750 mL distilled water. Prepare a solution of 0.3% hydrogen peroxide and 1 mM ABTS in the citate buffer. Pour citric acid solution into sodium citrate solution to get a pH 4.2. Add 100 μL to each well and develop in a dark place for 30 minutes.

11. Read the absorbance with an ELISA plate reader at 405 nm at 30 minutes.

Determination of antibody isotype levels

1. Coat EIA plates (COSTAR) with 1.0 μg/mL (0.1 μg per well) of antigen in 0.05 M carbonate-bicarbonate buffer pH 9.6, 100 μL/well.

2. Incubate the plate(s) at 4°C overnight.

3. On the next day, wash plates 3X with washing buffer (PBS/0.05% Tween). Flick off excess liquid by tapping the plates on the bench top.

4. Block plates with 100 μL blocking buffer per well (PBS/2% BSA). Incubate plates for 1 hour at 37°C.

5. Wash plates 5X with washing buffer (PBS/0.05% Tween). Flick off excess liquid by tapping the plates on the bench top.

6. Prepare 1:250 dilution mouse serum in the working buffer (PBS/0.1% Tween). Add 100 μL/well into the appropriate well. Incubate plates for 1 hour at 37°C.

7. Wash plates 3X with washing buffer (PBS/0.05% Tween). Flick off excess liquid by tapping the plates on the bench top.

8. Dilute HRP-labeled detection antibodies (Southern Biotechnology Associates Inc.). Add 100 μL/well into the appropriate well. Incubate plates for 1 hour at 37°C. 9. Wash plates 3X with washing buffer (PBS/0.05% Tween). Flick off excess liquid by tapping the plates on the bench top.

10. Prepare HRP substrate;

Prepare 0.01 M citrate buffer by dissolving 1.48 g of sodium citrate in 500 mL of distilled water. Prepare a 0.01 M citric acid solution by dissolving 1.58 g of citric acid in 750 mL distilled water. Prepare a solution of 0.3% hydrogen peroxide and 1 mM ABTS in the citate buffer. Pour citric acid solution into sodium citrate solution to get a pH 4.2. Add 100 μL to each well and develop in a dark place for 30 minutes.

11. Read the absorbance with an ELISA plate reader at 405 nm at 30 minutes.

Inhibition ELISA

1. Dilute the coating antigen to 1.0 μg/mL (0.1 μg per well) in carbonate- bicarbonate buffer. Use glass tubes.

2. Add 100 μL of the coating antigen to each well of the plate. Store the plate(s) overnight at 4°C.

3. Shake out the wells and tap upside-down onto Kimwipes. Wash plate three times with -200 μL wash buffer (PBS/0.05% Tween, v/v) per well.

4. Add 200 μL blocking buffer per well (PBS/2% BSA, w/v). Incubate plates for 60 minutes at 37°C.

5. Shake out the wells and tap upside-down onto Kimwipes. Wash plate five times with -200 μL wash buffer (PBS/0.05% Tween, v/v) per well.

6. Add 50 μL of selected competitive conjugates or free peptides appropriately diluted in dilution buffer (PBS/0.1 % Tween, v/v). (NOTE: the competitive peptide has a x 2 concentration in 50 μL). 7. Added 50 μL per well of test antibody appropriately diluted in dilution buffer (PBS/0.1% Tween, v/v). (NOTE: the test antibody has a x 2 concentration in 50 μL). The final volume in each well was 100 μL.

8. Incubate plates for 60 minutes at 37°C with slow shaking on an ELISA shaker.

9. Shake out the wells and tap upside-down onto Kimwipes. Wash plate three times with -200 μL wash buffer (PBS/0.05% Tween, v/v) per well. Dilute Peroxidase-conjugated anti-mouse IgG in dilution buffer (PBS/0.1% Tween, v/v) to a concentration of 1 :5000. Add 100 μL per well and incubate at 37°C for 60 minutes.

10. Shake out the wells and tap upside-down onto Kimwipes. Wash plate three times with ~200μL wash buffer (PBS/0.05% Tween, v/v) per well.

11. Prepare HRP substrate;

0.03 % Hydrogen peroxide and ImM ABTS dissolved in 0.01M Sodium citrate buffer (Sodium citrate (1.48 g) dissolve sodium citrate in 500 mL distilled H2O. Citric acid (1.58 g) dissolve citric acid in 750 mL distilled H2O. Pour citric acid solution into sodium citrate solution to get pH 4.2). Add 100 μL to each well and develop in a dark place for 30 minutes.

12. Read the absorbance with an ELISA plate reader at 405 nm at 30 minutes.

Measurement of Antisera Cross-Reactivity by Western Blot

Streptococcus pneumoniae serotypes were originally isolated from clinical patients. The organisms were stored at -80°C in double strength skim milk and passaged on fresh blood agar plates overnight at 35°C with 5% CO2. A scaping of S. pneumoniae serotype was added to 1 mL PBS until turbid, and the bacterial suspension was resolved using a 12% Tris-HCl precast polyacrylamide gel using standard SDS-PAGE techniques. The proteins in the gel were transferred to a nitrocellulose membrane. Western blot fiber pads, filter paper and nitrocellulose membrane were pre-soaked in transfer buffer. The gel was rinsed, placed against the nitrocellulose and sandwiched between blot paper and fiber pad. Gel/membrane sandwich was placed in a gel transfer cassette holder, inserted into a transfer apparatus, and run at 100 volts for 1 hour.

The membrane was removed and blocked in skim milk for 1 hour at 37°C. The membrane was then washed with Tris Buffered Saline (TBS) (0.035M Tris base, 0.22 M sodium hydroxide, pH 7.2) + 0.1% Tween (TTBS). For inhibition of anti-sera, a scraping of S. pneumoniae serotype of interest was added to 1 mL tryptic soy broth (Fisher Scientific, Cat# 211822) until turbid. Anti-sera / bacteria suspension was added to the membrane. The membrane was incubated at 37°C for 1 hour while shaking. Membrane was washed 3 times for 5 minutes with TTBS. The membrane was incubated with goat anti-mouse alkaline phosphatase antibody (1:10000 dilution) at 37°C for 1 hour while shaking, and washed 2 times for 5 minutes with TTBS and once with TBS. For development, NBT/NICP (1 tablet/10 mL distilled H2O) was added to the membrane for several minutes. Development was stopped by rinsing with distilled H2O.

Identification of Protein Bands from Western Blot by LC/MS/MS

Bands from the Western blot were excised from the gel using a razor blade and transferred to an Eppendorf tub containing distilled water. The band was subsequently treated with trypsin. After enzymatic digestion, the tryptic peptides were subjected to LC/MS/MS analysis on a Micromass Q-ToF-2 mass spectrometer (Micromass, Manchester, UK) coupled with a Waters CapLC capillary HPLC (Waters Corp., USA). This procedure involved separation on a PicoFrit capillary reversed-phase column (5 micron BioBasic C18, 300 Angstrom pore size, 75 micron LD x 10 cm, 15 micron tip; New Objectives, MA, USA), using a linear water/acetonitrile gradient (0.2% Formic acid), with a 300 micron x 5 mm PepMap C18 column (LC Packings, CA, USA) as a loading/desalting column. The eluent was introduced directly to the mass spectrometer by electrospray ionization at the tip of the capillary column. Data dependent MS/MS acquisitions were performed on detected peptides with a charge state of 2 or 3.

Protein identification from the MS/MS data thus generated was achieved by searching the NCBI non-redundant database using Mascot Daemon (Matrix Science, UK). Search parameters included carbamidomethylation of cysteine, possible oxidation of methionine and one missed cleavage per peptide.

Opsonization assay

1. Streak an agar plate with desired gram positive bacteria procured from various culture collections (ATCC). Incubate at 37°C overnight.

2. Next day, pick an isolated colony and inoculate it in 1.0 mL of Todd- Hewitt Broth (THB) + Yeast Extraction (YE) media in a sterile test tube. Incubate at 37°C overnight.

3. The next day prepare 100 U/mL of sterile heparin.

4. I. V. inject 100 μL of sterile heparin into tail vein of each mouse (5-10). After 10 minutes, cardiac bleed mice into a sterile tube.

5. Measure O.D. of inoculated bacteria at 420 nm wavelength. Use THB+YE media as blank.

6. To a sterile flat bottom 96-well plate, add a sterile 2.5 mm glass bead in each well. To each well, add: a. 50 TL of heparinized blood b. 10 TL of mouse serum to be tested c. 5 TL of bacteria incubate at 37°C for 1 hour.

This step in performed in triplicate.

7. After 1 hour incubation, prepare 1 :20 dilution exogenous complement (Low-Tox guinea pig Complement, Cedarlane) in distilled water. Add 50 TL/well. Incubate at 37°C for 1 hour on a shaker (slow motion).

8. After one hour, 100 TL aliquot is plated out on agar plates using glass spreader.

9. Wrap all agar plates in plastic bags and incubate at 37°C. overnight. 10. Next day, count colony- forming units (CFU).

Animal housing

Mice were housed in polycarbonate rodent cages (Nalgene, Cat.# 01 -288-1 C) and fed certified autoclavable mouse diet (Purina) and deionized autoclaved water. All animal studies complied with the guidelines established by the Canadian Council on Animal Care and the requirements of the Health Sciences Animal Policy and Welfare Committee at the University of Alberta. Environmental parameters of the animal room were monitored using a data logger. The light cycle was maintained at 12 hours light and 12 hours dark. Temperature was maintained at 22°C (± 2°C) and humidity was maintained between 40% and 70% relative humidity.

Preparation of Streptococcus pneumoniae challenge culture Streptococcus pneumoniae serotype 14 was used in the challenge studies. This serotype was originally isolated from clinical patients. For challenge, the organisms, stored at 80°C in double strength skim milk, were passaged on fresh blood agar (Dalynn labs Cat. # BP-75) plates overnight at 35°C in 5% CO2. A scraping of S. pneumoniae was used to inoculate 10 mL of Todd-Hewitt Broth (Difco, Cat.# 249240) + 1% yeast extract (Difco, Cat# 212750) + 10% horse serum (Pel-Freez, Cat# 39399-0) in a side-arm flask until an optical density of 0.15 (560 nm) was achieved (Biochrom Ultraspec 2100 pro spectrophotometer, Fisher Scientific, Cat.# BC80-2112-21). This culture was then incubated at 35°C in 5% CO2. When the appropriate O.D. value of the culture was obtained, the concentration was diluted to achieve the required challenge dose. Serial dilutions of the challenge culture were streaked onto blood agar plates to confirm the number of Colony Forming Units/mL (CFU/mL).

Preparation of bacterial laden agarose beads and subsequent immunizations of animals

1. A lOμl aliquot of 5. pneumoniae from glycerol stocks will be streaked onto blood agar and incubated at 35°C overnight. 2. One colony will be picked from the plate and used to inoculate 50 mL of Todd Hewitt broth (THB) containing 1% yeast extract and 10% horse serum in a sterile 125 mL Erlenmeyer flask.

3. Incubate this flask at 35°C overnight in 7% CO₂ without shaking.

4. Place a 0.5 to 1.0 mL aliquot of the bacterial suspension into a sterile sidearm of 50 mL THB.

5. Grow the sidearm flask of 50 mL THB to late log phase (approx. 6 hrs.) in a non-shaking incubator at 35°C. Measure the Optical Density (O.D.) to determine the CFU/mL.

6. Prepare 50 mL of a 2% agarose, low Electro Endo Osmosis, (EEO) in sterile phosphate buffered saline (PSB) pH 7.4.

7. Mix a 5 mL aliquot of the late log phase bacteria suspension in THB into the 50 mL 2% agarose solution. Warm this to 50-55°C.

8. Warm and equilibrate 150 mL heavy mineral oil to 50-55°C in a sterile 600 mL beaker.

9. Add the warm 50 mL agarose-broth mixture to the heavy mineral oil and stir rapidly on a stirrer/hot plate for 6 minutes at room temperature.

10. Cool this mixture adding ice around the flask slowly over 10 minutes.

11. Pour the oil agarose mixture into a sterile 500 mL separatory funnel containing 150 mL of a 0.5% deoxycholic acid, sodium salt (SDC) in sterile PBS solution.

12. Allow the agarose bead suspension to settle out.

13. Remove the oil/SDC layers by aspiration. 14. Wash the agarose beads suspension in the separatory flask once again with 150 mL of a 0.25% SDC in sterile PBS and allow the beads to settle.

15. Remove the remaining oil SDC layer by aspiration

16. Wash the remaining agarose beads 3 to 4 times with 200 mL sterile PBS.

17. Passively filter the last agarose bead/PBS wash through a 200 micron filter.

18. Aspirate the remaining PBS and allow the filtered bead slurry to settle.

19. Re-suspend the bead slurry with an equal volume of PBS into a sterile container. Maintain at 4°C overnight.

20. Sterile agarose beads are prepared using 5 mL of sterile THB.

21. Remove a 1 mL aliquot of the final agarose bead (bacteria laden & sterile preparations) suspensions for quantitative microbiology.

22. Add the 1 mL aliquot for microbiology to 49mL sterile PBS.

23. Homogenize this aliquot (polytron homogenizer) and plate out serial dilutions on blood agar to determine the colony forming units per mL (CFU/mL) of the bacteria laden agarose beads, and to confirm sterility of the control beads. Use the CFU/mL count to determine final dilution factors of the inoculums.

24. Remove an aliquot of the final agarose bead suspension for confirmation of bead size.

25. Use an inverted light microscope and a micrometer to count several fields and determine the final bead size range.

In order to determine the CFU per mL necessary to produce a chronic (14 to 21 day) lung infection, the following in vivo experiments will be conducted using various amounts of bacteria. This protocol should be performed for each separate mouse model and its wild type control.

1. Groups of mice will be inoculated with the following concentrations of S. pneumoniae laden agarose beads (van Heeckeren, Gosselin, McMoran). The concentration may vary depending on strains of mice, such as CF, C57 and BY mice. A: 5 X lO⁴ B: 1 X 10⁵ C: 5 X 10⁵

2. On the day of inoculation, mice will be intratracheally inoculated with 50μl of a 1:10 dilution of the concentration of bacteria laden agarose beads, (A, B, or C) or 50μl of a 1:10 dilution of the sterile agarose beads.

EXAMPLE 1 The PspA 41-48 Coiled-coil Peptide

To date 40 partial and full sequences of pneumococcal surface protein A from various strains have been released to a number of sequence databases. These proteins share a common architecture consisting of a leader sequence, a coiled- coil domain, a proline domain and a choline binding domain. Analysis of PspA sequences by the program MultiCoil (Wolf et al, 1997) indicates that the coiled- coil regions are broken up by sections that contain proline residues. Multiple sequence analysis using the software Biotools indicates that the published sequences vary in similarity from 24.1% to 97%. However, certain regions of the helical domain of these proteins are remarkably similar, suggesting that these conserved regions are important to the function of the protein.

A consensus sequence was deduced from a conserved region (amino acids 41-48 of PspA) which was shown to have antigenic activities. Complete and partial PspA sequences were downloaded from the National Library of Medicine PubMed web site into the bioinformatics software Peptools (Biotools, Edmonton, Alberta). A total of 40 complete and partial sequences were transferred to the alignment module of the software and a consensus threshold was set to 65%. The consensus threshold defines the minimum residue plurality amongst a group of aligned sequences. In effect, the consensus threshold acts to filter out insignificant matches and highlights conserved residues within a sequence. This allows for easy identification of similarities by visual inspection.

The resulting consensus sequence was further conformed to a coiled-coil structure by replacing the amino acid residues at the a and d positions with He and Leu, respectively. Residues that enhance coiled-coil formation were also added to both sides of the sequence to form SEQ LD NO:l (designated PspA 41- 48). As described below, this peptide has extremely high coiled-coil propensity, displaying almost identical coiled-coil confirmation both under physiological conditions and in the presence of a coiled-coil enhancing agent, TFE. The CD spectra were determined under benign conditions (50 mM KH₂P0₄, 100 mM KCI, pH 7.0) and in aqueous buffer containing 50% TFE. The CD spectrum of peptide PspA 41-48 under benign conditions at 20°C is shown in Figure 1. The CD spectra is typical of a helical peptide with minima at 222 nm and 208 nm and high positive ellipticity below 200 nm. Typically, the molar ellipticity at 222 nm ([ΘJ₂₂₂) has been used to measure helical content in peptide. For peptide PspA 41-48 this corresponds to a value of 33700 which indicates that this peptide is predominantly I-helical. Theoretically, the [θ]₂₂₂ value for a peptide of 27 residues is -33900 (Chen et al., 1974). Therefore peptide PspA 41-48 is 99% helical. It should be noted that the linker region (Cys Gly Nle) is designed not to be helical and therefore decreases the [ΘJ222 signal. The ratio of the molar ellipticity at 222 and 208 ([θ]₂₂₂/[θ] os) is greater than 1.02 and similar to that observed before for coiled coils (Hodges et al, 1988; Lau et al, 1984; Zhou et al, 1992) and distinctly different from non-interacting I-helices in which the [θ]208 is greater than the [θ]222- In the presence of 50% TFE, helical content increased slightly to -33900. The data indicate that peptide PspA 41-48 is highly helical and found predominantly in the coiled coil conformation under aqueous (normal physiological) conditions.

A control peptide was prepared in which only the hydrophobic residues at a and d positions (He and Leu) were replaced with Gly residues in peptide PspA 41-48. While the epitope is still present, the lack of hydrophobic residues prevent this peptide frpm forming a coiled-coil. The CD spectra of this peptide under buffered conditions is reminiscent of a random coil with a minima around 199 and a very small signal -1600 at 222 nm. Under these conditions the helical content is less than 5%. In the presence of 50% TFE, this peptide folds into an I- helix. This indicates the peptide can adopt a helical conformation but requires an appropriate hydrophobic heptad repeat at a and d positions to do so. EXAMPLE 2 The PavA 280-293 Coiled-coil Peptide

PavA is a streptococcal surface protein which appears to be involved in adherence and virulence of S. pneumoniae. Analysis of the native sequence by the program multicoil (http://nightingale.lcs.mit.edu/cgi-bin/multicoil) indicated potential coiled-coil regions between residues 270-310. Within this region, a potential B-cell epitope was identified (280-293) and inserted into the coiled-coil template to form PavA 280-293 (SEQ TD NO: 13).

The CD spectrum of peptide PavA 280-293 under benign conditions at 20°C is shown in Figure 1. The CD spectra is typical of a helical peptide with minima at 222 nm and 208 nm and high positive ellipticity below 200 nm. Typically, the molar ellipticity at 222 nm ([θ]₂₂ ) has been used to measure helical content in peptide. For peptide PavA 280-293 this corresponds to a value of 31950 which indicates that this peptide is predominantly I-helical. Theoretically, the [θ]₂₂₂ value for a peptide of 27 residues is 33900 (Chen et al, 1974). Therefore peptide PavA 280-293 is 94% helical. It should be noted that the linker region (Cys Gly Nle) is designed not to be helical and therefore decreases the [θ]₂₂₂ signal. The ratio of the molar ellipticity at 222 and 208 ([θ]222/[θj20_δ) is greater than 1.02 and similar to that observed before for coiled coils (Hodges et al., 1988; Lau et al, 1984; Zhou et al, 1992) and distinctly different from non interacting I-helices in which the [θ]₂₀₈ is greater than the [θ]22₂- hi the presence of 50% TFE, helical content increased slightly to -32800. The data indicate that peptide PavA 280-293 is highly helical and found predominantly in the coiled coil conformation under aqueous conditions.

A control peptide was prepared in which only the hydrophobic residues at a and cl positions (He and Leu) were replaced with Gly residues in peptide PavA 280-293. While the epitope is still present, the lack of hydrophobic residues prevent this peptide from forming a coiled-coil. The CD spectra of this peptide under buffered conditions is remiscent of a random coil with a minima around 199 and a very small signal -1400 at 222 nm. Under these conditions the helical content is less than 5%. In the presence of 50%) TFE, this peptide folds into an I-helix. This indicates the peptide can adopt a helical conformation but requires an appropriate hydrophobic heptad repeat at a and d positions to do so.

The CD data for PspA 41-48 and PavA 280-293 are summarized below.

Table 2. CD Spectroscopy Analysis of Coiled-coil Peptides

EXAMPLE 3 Immunogenicity of Coiled-Coil Proteins

Peptides PavA 280-293 (SEQ LD NO:13) and PspA 41-48 (SEQ LD NO:l) as described in Examples 1 and 2 were coupled to the protein carrier tetanus toxoid (TT) as described in Materials and Methods. The immunogenicity of these peptides was determined as follows.

BALB/c mice (6-8 weeks, female, Charles River) were immunized on day 0 (1° immunization), day 7 (2° secondary immunization), day 28 (3° tertiary immunization), and day 42 (boost) by intraperitoneal injection (100 μL total, 50% Alhydrogel 2% adjuvant, Cedarlane, Catalog # SF2000-250) with the conjugates of interest. These antigens were diluted to various doses in 0.9% NaCl and mice injected with 0.9% NaCl were used as negative controls. Mice were bled following the 1°, 2°, 3° and boost injections on days 6, 14, 35 and 49 to collect serum to assay for antibody titers (direct ELISA) and antibody isotype response (isotyping ELISA), as well as to determine antibody specificity (inhibition ELISA) and measure immunoprotective antibody responses (bactericidal/opsonization assays).

A typical immunization schedule is shown in Table 3. Various other immunization schedules and adjuvant formulations known in the art would also be effective. Similarly, various delivery methods including intravascular, intramuscular, intradermal, subcutaneous, oral, nasal and aerosol inhalation routes would also be effective.

Table 3: BALB/c Mouse Immunization Schedule

The animals were immunized with the following materials (10 A.BY/SnJ mice / group): , 1. Pava 280-293 TT (20 μg/mouse) + Alum 2. PspA 41-48 TT (20 μg/mouse) + Alum 3. Scrambled Sequence Peptide TT (20 μg/mouse) + Alum 4. Tetanus toxoid (20 μg/mouse) + Alum 5. PBS (without Alum)

Antibody levels were measured with direct ELISA as described in Materials and

Methods. The mean O.D. ELISA readings obtained with antisera to the PspA

41-48 and PavA 280-293 conjugates are shown in Table 4. The two conjugates elicited significant antibody titers to their corresponding peptide haptens. These antibodies were immunogen specific, as the negative control and unconjugated tetanus toxoid did not elicit any antibodies or cross-reactive antibodies to these antigens. End point titers for both peptide conjugates were high: 2,700,000 and 3,380,000 for PspA 41-48 and PavA 280-293, respectively.

Table 4: ELISA Results of Mouse Serum Antibodies to the Coiled-Coil Antigens Experiment 1 (CC0029)

¹ mice were i.p. injected on day 0, 7, 28 and 42 and sera were collected on day 49. Experiment 2 (CC0031)

¹ mice were i.p. injected on day 0, 7, 28 and 42 and sera were collected on day 49. Table 5 lists end point titers for a number of S. pneumoniae coiled-coil peptide antigens. As can be seen from the table, these peptides, when conjugated to tetanus toxoid, are highly immunogenic in mice.

Table 5. End Point Titer of S. pneumoniae Coiled-coil Peptides

¹ 2 x background

EXAMPLE 4 Determining Antibody Isotype Levels Elicited by Coiled-Coil Antigen Conjugates

To quantify the IgM, IgG, and IgA isotypes elicited by coil-coiled antigen conjugates, antibody isotype levels were determined as described in Materials and Methods.

The IgG antibody isotype response to the PavA 280-293 and PspA 41-48 coiled coil antigens is shown in Table 6. A significant IgG isotype switch from IgGl to IgG_2a and IgG2b was seen after the booster injection (Day 60 bleed). The observed IgG antibody maturation to both antigens is typical of a thymus- dependent response. Table 6. Isotyping ELISA of Mouse Serum Antibodies to the PavA 280-293 and PspA 41-48 Antigens

¹ Mouse sera (Day 49) were diluted 1:125000.

EXAMPLE 5 Determining Antibody Binding in Competition with Coiled-Coil Antigen Conjugates or Free Peptides

The specificity of the antisera was further examined by inhibition ELISA using peptides with or without a coiled-coil structure. Inhibition ELISA was performed as described in Materials and Methods. As shown in Table 7, the antibodies were specific to the coiled-coil antigens.

Table 7: Inhibition ELISA of Anti-Strep.(l :50,000 dilution, Day 49) Using Various Strep Antigen Inhibitors

EXAMPLE 6 Opsonization Assay to Measure Immunoprotective Antibodies Elicited by Coil-Coiled Antigen Conjugates.

Opsonization is the phenomenon where antibodies render bacteria susceptible to phagocytosis. PspA 41-49 antisera were incubated in whole mouse blood with an exogenous source of complement and S. pneumoniae bacteria, as described in Materials and Methpds. The opsonic activity of the antisera is demonstrated by a decrease in CFU counts of S. pneumoniae serotypes 14 and 19F (75%» and 70% respectively) relative to negative control antisera (Figure 5). Similarly, the PavA 280-293 antisera also exhibited significant opsonic activities against both serotypes (Figure 5). These results demonstrate that the antibodies elicited by the coiled-coil peptides can provide passive immunity and are thus therapeutically valuable.

EXAMPLE 7 Measurement of Antisera Cross-Reactivity by Western Blot

To determine the cross-reactivity of the antisera prepared using the coiled-coil antigens, various S. pneumoniae serotypes (6B, 14, 19 and 23F) were separated and transferred onto a Western blot as described in Materials and Methods. The antisera raised against the PspA 41-48 peptide antigen were used to probe the separated bacterial lysate. The protein band at 64 Kda (Figure 6) was cut out from the gel and subjected to tryptic digestion, followed by HPLCMS/MS sequencing. This band was identified as pneumococcal surface protein A. Thus, the antisera cross-reacted with all the serotypes tested here.

Similarly, S. pneumoniae serotypes 6B, 7F, 10A, 12F, 14 17F, 19F 22 and 23F were separated and transferred onto a Western blot. Antisera raised against the PavA 280-293 peptide antigen was used to probe the separated bacterial lysate. A protein band at 62 Kda was observed in all lanes. The calculated molecular weight of the PavA protein is 63290 Dalton, which suggests that the PavA antisera bound to PavA protein. Moreover, these results indicate that the antisera produced by using the coiled-coil PavA 280-293 peptide antigen can recognize numerous different serotypes of S. pneumoniae.

EXAMPLE 8 Identification of Protein Bands from Western Blot by LC/MS MS

PspA 41-48 antisera were used to probe whole cell lysates from S. pneumoniae serotypes 6 A, 6B 9, 14 and 23F. The bands that reacted with the antisera were digested and sequenced as described in Materials and Methods. The proteins recognized by the PspA 41-48 antisera are listed below.

EXAMPLE 9 Streptococcus pneumoniae Systemic Infection Model

The vaccine effect of the coiled-coil peptides was assessed using a Streptococcus pneumoniae systemic infection model. Briefly, mice were immunized with the peptides of interest and challenged with a lethal dose (LD₈₀) of Streptococcus pneumoniae. The effects of the peptides on survival of the mice were then determined.

A breeding colony of the BALB/c mice was established at the Provincial Lab Vivarium (University of Alberta Hospital). Male and female inbred mice, three weeks old, were weaned from the BALB/c breeding colony and housed as described in Materials and Methods. Groups of these BALB/c mice (10 per group) were immunized by intraperitoneal injection (i.p.) on days 0, 7, 28, and 42 with 20 Tg of the PavA 280-293 peptide tetanus toxoid conjugate (PavA 280- 293-TT) or PspA 41-48 peptide tetanus toxoid conjugate (PspA 41-48-TT) + 2% Alhydrogel (alum) (Superfos Biosector, Denmark, Cat.# SE2000-1), or with linear scrambled peptide tetanus toxoid conjugate (20 Tg) + alum as a negative control. These conjugates were administered using a sterile 0.5 mL tuberculin syringe with a 27 gauge needle (Becton Dickinson, Cat.# 305620). The mice were tail bled 20 days post-final immunization (day 62) to collect serum for ELISA antibody titer assays and other immunoassays.

Also on day 62, the mice (final weight between 20-27 g) were i.p. challenged , with a 100 μL lethal dose (-LD80) of S. pneumoniae (serotype 14, 1.0x107 CFU/mouse, prepared as described in Materials and Methods). Mice were monitored for relative degrees of morbidity and mortality associated with systematic infection. This was accomplished by observing the challenged mice once per hour for 48 hours. Symptom monitoring began at 14 hours after challenge for signs of infection or illness. The potential symptoms include hunched back, ruffled fur, lethargy, cyanosis, dehydration, conjunctivitis, mucous diarrhea, emaciation and loss of righting reflex. Once the righting reflex was lost, the animal was euthanized with the time of death recorded. The survival time for each mouse was calculated. The results are shown in Table 8 and Figure 7.

Table 8. Active Immunization Studies with Coiled-coil Peptide Tetanus Toxoid Conjugates Experiment 1

Experiment 2

¹ Experiments were terminated at 48 hours.

Thus, the mice immunized with S. pneumoniae PspA 41-48 and PavA 280-293 tetanus toxoid conjugates were protected from LD80 dose of serotype 14 S. pneumoniae bacteria. The mice immunized with the control conjugates were not protected from S. pneumoniae challenge.

EXAMPLE 10 Streptococcus pneumoniae Intranasal Challenge Model

A breeding colony of the A.BY/SnJ mice was established at the Provincial Lab Vivarium (University of Alberta Hospital). Male and female inbred A.BY/SnJ mice, three weeks old, were weaned from the A.BY/SnJ breeding colony and housed as described in Materials and Methods. Groups of these A.BY/SnJ mice (10 per group) were immunized by intranasal injection (i.n.) on days 0, 7, 28, and 42 with 20 μg of the PavA 280-293 peptide tetanus toxoid conjugate (PavA 280- 293-TT) or PspA 41-48 peptide tetanus toxoid conjugate (PspA 41-48-TT), + Lipopolysaccharide (LPS) (Sigma, Cat#L4391), or with linear scrambled peptide tetanus toxoid conjugate (20 μg) + LPS as a negative control. These conjugates were administered using a sterile pipette with disposable tip. Conjugate was administered by adding 5 μL onto each nares and allowing it to be inhaled. The mice were tail bled 20 days post-final immunization (day 62) to collect serum for ELISA antibody titer assays and for other immunoassays.

On day 62, the mice (final weight between 20-27 g) were i.n. challenged with a

10 μL lethal dose (~LD₈₀) of S. pneumoniae (serotype 14, 1.0x105 CFU/mouse). The mice were monitored for relative degrees of distress and illness by observing the challenged mice once per hour for 7 days. Symptom monitoring began at 14 hours after challenge for signs of infection or illness. After 7 days, nasal washes were conducted by flushing 250 μL of PBS through the trachea and collected as it exits the nares. The wash was collected in sterile tubes, diluted and plated on BAP plates and incubated at 35°C with 5% CO₂. The colony forming units were counted.

Table 9. Reduction in Nasal Wash Counts with Mice Immunized with Coiled-coil tetanus toxoid conjugates

• mice were i.n. immunized on day 0, 7, 28 and 42. i.n. challenged with S. pneumoniae serotype 14 on Day 60. Nasal wash was collected on day 67.

Therefore, these results show that the vaccination with PspA 41-48 or PavA 280- 293 effectively protected the animals from S. pneumoniae infection.

Example 11 Streptococcus pneumoniae Intratracheal Challenge Model The intratracheal challenge model will be prepared according to Materials and Methods. In this chronic infection model, BAL fluid, saliva, blood and lungs will be collected 10 days post challenge for histopathological analysis. The histopathological examination of the mouse lungs reveal that the lungs immunized with PspA 41-48 and PavA 280-293 show no signs of infection and the microscopic appearance is similar to a normal, healthy mouse lung. In contrast, lungs from mice immunized with the negative control display extensive active bronchopneumonia histopathology. Supportive mononuclear reaction is observed in the affected alveoli. Inflammation cells present in the negative control animals include neutrophils, macrophages and lymphocytes.

Plating of BAL fluid indicated that the non immunized lung had substantially higher CFU counts compared to the immunized mice.

Example 12 Streptococcus pneumoniae Passive Immunization Animal Model

To evaluate the protection provided by passively administering anti-PspA or anti- PavA antibodies, BALB/c mice will receive the antibodies and lethal doses of Streptococcus pneumoniae, and the effects of the antibodies will be determined.

A breeding colony of BALB/c mice at the Health Sciences Laboratory Animal Services (University of Alberta) can provide the mice used in this study. Female BALB/c mice are received at 6-8 weeks of age and housed as described in Materials and Methods. The mice (final weight between 20-27 g) are i.v. administered anti -PavA or anti-PspA monoclonal antibodies (10-20 mg/kg dose) at 2 hours before or after a bacterial challenge. To challenge, the mice receive i.p. lethal doses of Streptococcus pneumoniae serotypes, and are observed as described in Example 9 for degrees of morbidity and mortality associated with systematic infection.

The results will show that the mice passively immunized with anti PspA 41-48 or anti PavA280-293 monoclonal antibodies are protected from lethal challenges of various S. pneumoniae serotypes (9, 14, or 23F), whether the antibody is given 2 hours before or after the bacterial challenge. The mice passively immunized with the negative control mouse antibody MOPC are not protected. These results will thus demonstrate the in vivo efficacy of these antibodies.

Claims

We claim:

1. An isolated peptide comprising at least one sequence selected from the group consisting of SEQ ID NO:l to SEQ ED NO: 144.

2. The peptide of claim 1 comprising at least two sequences selected from the group consisting of SEQ ID NO:l to SEQ ED NO: 144.

3. The peptide of claim 2 wherein the two sequences are connected by a spacer such that the two sequences and the spacer form a continuous heptad repeat structure.

4. The peptide of claim 1 further comprising the amino acid sequences CGNle or CNleG.

5. The peptide of claim 1 comprising sequences derived from at least two different strains of microorganism.

6. The peptide of claim 1 that is capable of forming a coiled coil in 50 mM KH₂PO₄, 50 mM KCI, pH 7.0.

7. The peptide of claim 1 that consists of less than about 200 amino acids.

8. The peptide of claim 1 that consists of less than about 150 amino acids.

9. The peptide of claim 1 that consists of less than about 100 amino acids.

10. The peptide of claim 1 that consists of less than about 50 amino acids.

11. The peptide of claim 1 that consists of less than about 35 amino acids.

12. The peptide of claim 1 that consists essentially of at least one sequence selected from the group consisting of SEQ LD NO:l to SEQ ED NO: 144.

13. A composition comprising the peptide of claim 1.

14. The composition of claim 13 wherein the peptide is conjugated to a carrier molecule.

15. The composition of claim 13 further comprising a pharmaceutically acceptable excipient.

16. The composition of claim 13 further comprising an adjuvant.

17. The composition of claim 13 capable of eliciting an immune response to more than one strain and/or species of a microorganism.

18. The composition of claim 13 capable of eliciting an immune response to at least two different microorganisms.

19. A method of eliciting an immune response in an animal, comprising administering the composition of claim 13 to the animal.

20. The method of claim 19 wherein the composition further comprises an adjuvant.

21. The method of claim 19 wherein the peptide is conjugated to a carrier molecule.

22. The method of claim 19 wherein the immune response is against more than one strain and/or species of microorganism.

23. The method of claim 19 wherein the immune response is against at least two different microorganisms.

24. A method of treating or preventing a microbial infection in an animal, comprising administering to the animal the composition of claim 13.

25. A method for determining the presence in a biological sample of antibodies to a microorganism selected from the group of Streptococcus pneumoniae, Staphylococcus aureus, Clostridium difficile, Haemophilus influenza, Pseudomonas aeruginosa, Neisseria meningitidis, Escherichia coli, Helicobacter pylori, and Moraxella catarrhalis, comprising: (a) contacting the biological sample with the peptide of claim 1 ; and (b) determining whether antibodies in the biological sample bind to the peptide.

26. The method of claim 25 which is used to determine prior exposure of an animal to the microorganism.

27. A method of preparing an antibody, comprising administering the composition of claim 13 to an animal to elicit an antibody response.

28. An antibody that recognizes the peptide of claim 1.

29. The antibody of claim 28 that recognize more than one strain and/or species of microorganisms.

30. A composition comprising the antibody of claim 28.

31. A method of treating or preventing a microbial infection in an animal, comprising administering the composition of claim 30 to the animal.

32. A method of determining the presence of a microorganism in a sample, comprising contacting the sample with the antibody of claim 28 and determining if the antibody specifically recognizes the sample.