CA2144882A1 - Synthetic peptide vaccine for chlamydia trachomatis - Google Patents

Abstract

A synthetic peptide capable of producing an immunological response to C. trachomatis in a vertebrate is disclosed. The synthetic peptide comprises at least one conserved T-helper cell stimulating epitope located within the peptide sequence AL-NIWDRFDVFCTLGATTGYLKGNS and at least one B-cell neutralizing antibody stimulating epitope located within the pep-tide sequence FDVTTLNPTIAGAGDVK. In a preferred embodiment, the epitopes are colinear and in a particularly preferred embodiment the T-helper cell stimulating epitope is located closer to the N-terminus of the peptide than the B-cell neutralizing antibody stimulating epitope.

Description

21.q4882 ~ W O 94/06827 ~.~ P(~r/US93/08739 SYNTHETIC ~ L)E VACCINE FOR CHLAMYI)IA TRACHOM~TIS
FIELD OF THE lNVENTION
This invention relates to the development and testing of vaccines for Chlamydia frachomatis. In particular, this invention is directed to synthetic peptide vaccines against C.
S trachomatis containing conserved B cell and T cell epitopes derived from the same protein.
BACKGROUND OF THE INVEN'IION
- C trachomatis is a causative agent of sexually tr~n~mitted ~i~e~C~os (STDs) which afflict an estimated 3 million people a year in the United States alone (Washington, et al., MA~4 257:2070, 1987). In women, C trachomatis infection of the lower genital tract can ascend to the fallopian tubes causing salpingitis. Chlamydial salpingitis can lead to tubal blockage and cause infertility or ectopic pregnancy. It is estimated that in the United States 200,000 women per year become infertile as a result of chlamydial salpingitis. Measures to control or even prevent chlamydial SlL)s are badly needed.
Se~oly~ing of C. trachomatis isolates separates them into 15 distinct serovars and three selog,oul~s (Wang, et al., Infect. Immun. 7:356, (1973) and Wang, et al., J. Infect. Dzs.
152:791, 1985): the B-serogroup consists of serovars B, Ba, D, E, L1, and L2; the intermediate serogroup, serovars, F, G, K, and L3; and the C-serogroup, serovars A, C, H, I, and J. Serovars D, E, F, G, H, I, J, and K are most commonly associated with chlamydial STDs. Greater than 80% of C trachomatis caused STDs are due to infections caused by serovars D, E, F, or G (Kuo, et al., Infect. Immun. 41:865, 1983). The proportion of C:
trachomatis serovars isolated in the Seattle metropolitan area between 1965 and 1982 was 46.5% (se.ovar~. D and E), 24.6% (serovars G and F) and 5-7% (serovars H, I, J and K) (4).
This distribution of serovars has not changed over the past decade and is also representative of isolates obtained in other urban areas of the United States (Batteiger, et al., J. Infect. Dis.
159:661, 1989). Thus, a successful vaccine against chlamydial STDs must protect against multiple C trachomatis serovars with coverage against serovars D, E, F, and G being vital.
The most promising antigen for the development of a vaccine against chlamydial STDs is the C. trachomatis circa 40 kDa major outer membrane protein (MOMP). It is the principle C. trachomatis serotyping antigen (Caldwel~, H.D. and R.C. Judd., Infect. Immun.
38:960 (1982); Caldwell. H.D et al., Infect. Imn2U)2. 31:1161 (1981); Caldwell, H.D.~ndJ.
Schachter. Infect. Inv7tun. 35:1024, 19~2) and is the only surface component to which chlamydial neutralizing antibodies have been described (Zhang et al., J. Immunol. 138:575 (1987) and Zhang et al., Infect. Ir7tf~tun. 57:636, 1989). The MOMP genes of several C.
tracho~natis serovars have been sequenced (Pickett, et al., FEllqS Microbiol. Lett. 42:185 WO 94/06827 PCI'/US93/08739 ~_ (1987), Zhang, Y.-X., et al., NucleicAcids Res. 18:1061 (1990), and Hamilton, et al., Nucleic Acids Res. 17:8366, 1989) and are characterized by four symmetrically spaced hypervariable domains (VDs) that are flanked by regions of amino acid homology.
The MOMP VDs are the targets of species-specific chlamydial neutralizing S monoclonal antibodies (mAbs) and Fab fragments prepared from neutralizing mAbs inhibit chlamydial infectivity by blocking their attarl~ment to host cells (Su, H. and H.D. Caldwell.
Infect. Immun. 59(8):2843, 1991). Furthermore, proteolysis of chlamydiae with trypsin leads to a loss in their ability to attach to host cells and is associated with cleavage within the surface exposed VDS of the MOMP. These findings strongly support a role for the MOMP
VDs as surface structures which are important in the binding of chlamydiae to host cells.
Thus, epitopes located within surface exposed MOMP VDs are rational targets for the dcvclopll.ent of a synthetic chlamydial vaccine.
Synthetic peptide vaccines incorporate T-helper (TH) cell epitopes to enhance the immunogenicity of haptenic neutralizing B-cell epitopes and to evoke specific T-cell immunologic memory. By using overlapping synthetic peptides corresponding to the entire MOMP sequence in T-cell proliferation assays and as in vivo priming immunogens for the production of an ~n~mn~tic IgG antibody response, we showed that one peptide, termed A8, pocs~ ed functional TH activity. We directly demonstrated that peptide A8 possessed functional TH-cell activity by colinearly synthesizing it with the VDI sequence of serovar A, which contains a B-cell epitope, and showing that the production of IgG antibodies specific to B-cell epitopes within the VDI sequence was dependent on the A8 portion of the chimeric immunogen (Su, et al., J. Exp. Me~ 172:203, 1990). Peptide A8 corresponds to MOMP amino acid residues 106-130. This region of the MOMP is largely sequence invariant among the different C. trachomatis MOMPs suggesting that the TH-cell epitope contained within its sequence is antigenically conserved across serovars. While this synthetic peptide is useful for generating responses to serovar A, it does not generate protective responses to the other serovars.
Therefore, it is an object of this invention to prepare a synthetic peptide vaccine to a variety of C. trachomatis serovars that is suitable for generating protective immune responses in humans.
SUMMARY OF THE lNVENTION
The present invention provides a synthetic peptide capable of producing an immunological response to C. trac/lomatis in a vertebrate comprising a conserved T-helper stimulating epitope from the major outer membrane protein of C. tracllomat~s and a serovar ~_ WO 94/06827 2 1 ~ 4 8 8 2 PCI/US93/08739 conserved B-cell neutralizing antibody stimulating epitope from the major outer membrane protein of C. trachomatis. In a preferred embodiment of this invention, the T-helper cell stimulating epitope and the B-cell neutralizing antibody stimulating epitope are colinear.
Preferably the peptide contains the sequence identified as SEQ ID NO:3. In one embodiment the synthetic peptide T-helper cell stimulating epitope is located within SEQ
ID NO: 1 and in another embodiment the synthetic peptide is located within SEQ ID NO:2.
In yet another preferred embodiment of this invention, the T-helper stimulating epitope is located within SEQ ID NO:1 and the B cell neutralizing antibody stimulating epitope is located within SEQ ID NO:2. Preferably the T-helper stimulating epitope located within SEQ ID NO:1 is on the peptide N-terminus side of the B cell neutralizing antibody stimulating epitope located within SEQ ID NO:2. In another preferred embodiment of this invention the peptide additionally comprises at least one species specific B-cell neutralizing antibody stimulating epitope. It is also contemplated that the synthetic peptide of this invention additional~y comprises a known T-helper stimulating epitope from a protein other 1~ than the major outer membrane protein of C. trachomatis.
This invention also provides a method for inducing a protective immune response to C trachomatis in a vertebrate col,.p.i~ g introducing a synthetic peptide Colllplisi..g at least one conserved T-helper stimulating epitope located within SEQ ID NO: 1 and at least one B-cell neutralizing antibody stimulating epitope located within SEQ ID NO:2 n a pharm~ceutif~lly acceptable buffer into a vertebrate and testing for neutralizing antibody to C. trachomatis in the vertebrate. In a preferred embodiment of this invention the introducing step comprises injecting the synthetic peptide intramuscularly.
Further, this invention additionally relates to a peptide for use in the preparation of a vaccine for C. trachomat~s, comprising at least one conserved T-helper cell stimulating epitope located within SEQ ID NO: 1 and at least one B-cell neutralizing antibody stimulating epitope located within SEQ ID NO:2, wherein said peptide is formulated in a pharm~ceuti~lly acceptable buffer for use as a vaccine.
Finally this invention also relates to an immunoassay for detecting the presence of antibody to C. trachomatis in a sample, comprising a synthetic peptide and means for detecting antibody bound to said peptide, wherein the amino acid sequence of said peptide comprises a conserved T-helper cell stimulating epitope located in SEQ ID NO: 1 and a B-cell neutralizing antibody stimulating epitope located in SEQ ID NO:2.

W O 94/06827 '--~ PC~r/US93/08739 `_~

2 1 g ~ ~ 8 2 4 BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is a graphic summary of studies assessing the immunogenicity of peptideA8-VDIV in B10 H-2 congenic mouse strains. Graph (A) details the serum IgG antibody response of mice immunized with peptide A8-VDIV. Graph (B) details the serum IgGantibody response of mice immunized with peptide VDIV alone. The mouse sera weretested against peptide A8-VDIV (dark block), serovar D MOMP (striped block), andpeptide A8 (white block).
Figure 2 illustrates the results of the Pepscan ELISA analysis of the IgG antibody response of H-2 congenic mouse strains immunized with peptide A8-VDIV. All six mouse strains produced IgG antibodies reactive with octapeptides containing the septmeric species common LNPTIAG neutralizing B-cell epitope (Identified by the stippled pattern) contained within the A8-VDIV sequence.
Figure 3 is a comparison of the C. tracho~natis serovar specificity of the antibody response of different H-2 congenic mouse strains immunized with peptide A8-VDIV. Sera from five mice were pooled, diluted 1:100, and tested by ELISA against formalin fixed elementary bodies (EBs). Results are expressed as optical density values at A405.
Figure 4 is a comparison of the serum neutralizing activity of H-2 congenic mouse strains immunized with peptide A8-VDIV for three representative C. trachomatis serovars.
Serum dilutions were incubated with chlamydia and inoculated onto monolayers of HaK
cells to assay for chlamydial infectivity. The results are expressed as percent reduction in chlamydial infectivity.
Figure S illustrates the results of a Pepscan ELISA analysis of the lgG antibodyresponse of monkeys immunized with peptide A8-VDIV.
Figure 6 is a comparison of the serum neutralizing activity of three cynomolgus monkeys immunized with peptide A8-VDIV for three representative C. trachomatis serovars following the procedures described for Figure 4.
Figure 7 is a comparison of the immunogenicity of synthetic peptides A8-VI and A8-VDIV in primates. Solid bars represent absorbance values (A,l05) of pre-immune sera. The stippled bars are absorbance values of sera after immunization with the synthetic peptides.
DETAILED DESCRIPrlON OF THE INVENTION
As used herein the term "synthetic peptide" is used to describe a linear se4uence ot amino acids produced by laboratory chemical synthesis schemes that is preferably less than ~_ WO 94/06827 2 1 4 4 8 8 2 PCI'/US93/08739 100 amino acids in length however it is additionally contemplated that these peptides could similarly be created by recombinant DNA technology.
We have previously described a neutralizing mAB, designated Dlll-A3, that reactsby western blotting with the MOMPs of all C. trachomatis serovars except serovar K
Epitope mapping studies localized the DIII-A3 epitope to VDIV (Baehr et al., Proc. NatL
Acad. Sci USA 85:4000, 1988) and fine mapping of the mAb identified the epitope as the septapeptide sequence 298LNPTIAG304 within VDIV (Morrison, et al., 1992. "lmmunology of Chlamydia trachomatis infections: Immunoprotective and immunopathogenetic rei,~ollses." In: Sexually Transmitted Diseases. T.C. Quinn, ed. Raven Press, Ltd., New York, p. 57). The LNPTIAG sequence is invariant among C. trachomatis serovars except for serovar K where threonine replaces alanine at position 303. Although mAb DIII-A3 is broadly cross reactive with denatured MOMP by western blots, its immunoreactivity with intact C. trachomatis elementary bodies (EBs) by dot-immunoblot and its in vitro neutralizing activity is restricted to serovars within the B and intermediate serogroups indicating that this 1~ highly conserved epitope does not exhibit uniform surface accessibility among all C.
trachomatis serovars. While the LNPTIAG neutralizing site is ~cc~.s~ihle on the surfaces of B and intermediate complex serovars, it is not se~.min~y accessible on the surface of all serovars and would thus, not be expected to be a suitable epitope for a widely protective C.
trachomatis synthetic peptide vaccine.
As d;crlosed below, we found the chimeric peptide of this invention to be a goodimmunogen in both mice and primates, in that it: (i) targeted the production of neutralizing antibodies against the LNPTIAG B-cell epitope, (ii) was immunogenic in many mice strains disparate at H-2, and (iii) was a very effective priming immunogen for the production of an augmented IgG neutralizing response following secondary challenge with whole C.
trachomatis orgAni~m~
CONSTRUCTION OF THE SYI~ C PEPTIDE VACCINE
ln a preferred embodiment of this invention, we have linked together the A8 sequence with the MOMP VDIV sequence which contains the anti~enically common LNPTIAG neutralizing epitope, designated A8-VDIV, and have studied its immunogenic properties in both mice and primates. Methods for preparing the synthetic peptide are provided in Example 1. Peptide A8-VDIV corresponds to MOMP amino acids 106- 130 (A8) and 293-309 (VD]V). Residues 106-130 (ALNIWDRFDVFCTLGATTGYLKGNS) contain a functional Th-cell epitope. Peptide VDIV corresponds to MOMP residues 293-309 (FDVTTLNPTlAGAGDVK) and contains the sequence invariant LNPTL~G septmeric B-W0 94/06827 s PCI`/US93/08739 2:~g~81~2 -6-cell epitope recognized by the C. trachon~atis sub-species specilïc neutralizing monoclonal antibody DIII-A3. The chimeric peptide A8-VDIV was co-linearly synthesized with the A8 (T-cell site) sequence at its N-terminus and the VDIV sequence (B-cell site) at its carboxyl terminus. The A8-VDIV peptide was found to be a good immunogen in both species and preferentially targeted the production of antibodies to the LNPTLAG B-cell epitope.
Importantly, the anti-peptide antibodies neutralized the in vitro infectivity of those C.
trachomatis serovars that are epidemiologically important as causative agents of chlamydial STDs, suggesting that the oligopeptide may have considerable vaccine potential.
While the results described herein are as~ori~ted with the B cell and T cell combination provided in Example 1, it is contemplated that the T-helper epitope and the B cell epitope could be combined in either order with the T-helper epitope placed either before or after the B-cell neutralizing epitope. Thus, in one preferred embodiment of this invention, the synthetic vaccine contains, begins at the amino end of the peptide with the T-helper epitope linked to the B-cell neutralizing epitope and in another preferred embodiment of this invention, the synthetic vaccine contains, beginning at the amino end of the peptide, the B-cell neutralizing epitope followed by the T-helper epitope. ln addition, it is also contçmrl~ted within the scope of this invention, that the linking region sp~nning between the T-helper epitope and the B cell neutralizing epitope can incorporate any number of modifications known in the art. Thus, the peptide sequences incorporated as linking sequences between the epitopes of the vaccine could generate a flexible linking region, a rigid region, a hydrophobic region. a hydrophilic region or the like. Such modifications in the linking region between the conserved T-helper cell epitope and the conserved B-cell neutralizing epitope could be used to generate modified peptides suitable for testing as i,--prov~d peptide vaccines for C. trachomatis using the testing strategy outlined below.
Further, other T-helper epitopes and B cell neutralizing epitopes could be combined to further advance the efficacy of the synthetic peptide vaccine. For example, it is contemplated that serovar specific neutralizing epitopes could be incorporated into the vaccine. The addition of a second C. trachon~atis neutralizing epitope to this particular T-helper cell, B-cell neutralizing epitope combination could serve a number of functions. As one example, the additional neutralizing epitope could be incorporated into the vaccine to boost the efficacy of the vaccine for a particular serovar.
It is also contemplated within the scope of this invention that other T-helper epitopes could similarly be incorporated into the preferred combination of the T-helper and W O 94/06827 ~ 1 4 ~ 8 8 2 ~ PC~r/US93/08739 B-cell neutralizing epitope of this invention. Other synthetic vaccines for other adventitious agents have incorporated T-helper epitopes from proteins from unrelated agents. For example, U.S. Patent No. 4,882,145 to Thornton, et al. disclose the incorporation of T cell stimulating regions of the Hepatitis B virus nucleocapsid protein as a method for enhancing the immunogenicity of a polypeptide immunogen.
It is further contemplated that the preferred synthetic vaccine of this invention, peptide A8-VDIV, can be incorporated in biodegradable microspheres such as thosedescribed by Eldridge, et al., ( Mol. Immunol. 28:287, 1991 and Infect. Immw~. 59:2978, 1991). Alternatively, it is additionally contemplated within the scope of this invention that the protective immunity of the A8-VDIV vaccine could be bolstered by the construction of recombinant cholera toxin B subunit A8-VDIV gene fusions using the methods of Sanchez et al., Schodel, et al., or Lipscombe et al. (Proc. Natl. Acad. Sci USA 86:481, 1989; Gene 99:255, 1991; and Mol. Microbiol. 5:1385, 1991, respectively).
TESTING IMMUNOLOGIC ACT~VITY OF THE A8-VDIV CONTAINING PEPTIDE IN
V~VO
Once the preparation of the synthetic peptide is generated, it is introduced into test animals such as mice, primates, or the like, to study the efficacy of the protective immune response of the vaccine. Vaccination schemes for mice and primates are provided in Example 2 and Example 3. Preferably the peptide is capable of inducing mucosal immunity, therefore it is contemplated that the peptide may be introduced through any oral or parenteral route known in the art. It is anticipated that following vaccine trials in experimental animals such as mice and primates, the vaccine will he introduced into humans to begin efficacy trials for the protection of C. tracho~lalis associated STDs. Those with skill in the art of vaccine development will be readily able to adapt the primate vaccine strategy into a strategy suitable for human vaccination.
There are a variety of methods known in the art for studying the B-cell neutralizing activity and T-helper cell induced antibody responses using in vitro tests. As one example of a method to study the helper function of the A8 portion of peptide A8-VDIV and to ascertain the MHC class II restriction in the recognition of the TH-cell epitope(s) contained within the A8 sequence, we evaluated peptide A~-VDIV for its ability to elicit an IgG
antibody response to the B-cell VDIV portion of the peptide in Bl()H-2 congenic mouse strains. Eight B10 congenic strains of mice disparate at H-2 were immunized with peptide A-8 VDIV or peptide VDIV alone. Following the secondary immunization, the mouse sera were tested hy ELISA (as provided in Example 4) l`or l lG antibodies reactive to the free W O 94/06827 2 1 ~ 4 8 8 2 PC~r/US93/08739 _ peptides A8, VDIV, and whole MOMP (Fig. l). Six of the eight mouse strains, (C57BL/1() (H-2b), B10.A (H-2~), B10.M (H-2f), B10.WB (H-~!a), Bl0.BR (H-2~). and B10.SM (H-2U), immunized with peptide A8-VDIV produced high titer IgG antibodies reactive with the VDIV peptide and whole MOMP (Fig. lA). None of the mice produced IgG antibodies against the peptide A8 which contains the TH-cell site of the oligopeptide immunogen. Two strains, B10.D2 (H-2d) and B10.PL (H-2n), failed to produce IgG antibodies reactive with peptide VDIV or MOMP folIowing immunization with peptide A8-VDIV indicating thatmice having these H-2 haplotypes were incapable of recognizing the TH-cell epitope contained in the A8 portion of oligopeptide. Two of the eight strains of mice (C57BL/10 and B10.M) produced IgG antibodies reactive with peptide VDIV and MOMP followingimmunization with peptide VDIV alone (Fig. lB), demonstrating that in addition to B cell epitopes the VDIV sequence also contains functional TH cell epitope(s) whose recognition is restricted to the H-2b and H-2f haplotypes. Results are summarized in Table l below.
Collectively, these findings showed that the TH-cell epitopes contained within the A8-VDIV
peptide are recognized by multiple MHC class Il haplotypes and that these T-celldeterminants provide cognate help for antibody production to B-cell epitopes located in the VDIV sequenre 21~488~
~_ WO 94/06827 - PCr/US93/08739 Immunogenicity of Peptide A8-VDIV in B10 Congenic Mice Differing at H-~
.

Peptide pinNeutra~izing Mappingantibody titer ELISA IgG antibody titer (ND50) C. trachomatis C. trachomatis serovar serovar Mouse H-2 Peptide response to Strainhaplotype VDIV D E F G H (LNPTIAG) D G H
0C57BL/10(H 2b) 1024 81924096512102464 ~256128 '16 BlO.A ~H-2a) 256 4096204864 128 <16 ~25664 16 BlO.D2 (H-2d) <16 16 16 <16'16 16 - '16'16 '16 810.M (H-2~) 256 40964096512102432 ~256128 <16 BlO.UB(H-2ia) 1024 4096409664 256 '16 l12832 '16 15BlO.BR (H-2k) 1024 819281921024 2048 16 ~ 256 128 <16 BlO.PL (H-2U) ~16 64 16 <16<16 <16 - <16'16 '16 B10.SM (H-2V) 120 1024102464 256 C16 164 32 '16 The sera of the six responding strains of mice were tested by pepscan ELISA (seeExample 5) to determine if the anti-VDIV antibodies produced were reactive with the targeted LNPTIAG B-cell epitope contained in the VDIV sequence. Sequential and overlapping octapeptides corresponding to the MOMP VDIV sequence (residues 288-314) were synthesized on prederivated polypropylene pins and tested individually against a 1:200 dilution of pooled mouse sera for IgG antibody reactivity. The octapeptides tested are provided at the bottom of Figure 2. Each of the mouse strains produced IgG antibodies reactive with VDIV octapeptides containing the LNPTL~G B-cell epitope. Interestingly~ five of the six strains immunized with peptide A8-VDIV produced antibodies with enhanced immunoreactivity primarily to those VDIV octapeptides containing the targeted LNPTIAG
B-cell epitope.
The immunogenic properties of peptide A8-VDIV in mice were very encouraging and led us to further evaluate the peptides immunogenicity in sub-human primates. Table 2 shows the serum IgG antibody response of three cynomolgus monkeys (705, 752 and 907) after tertiary immunization with peptide A8-VDIV and two control monkeys (842 and 880) that received adjuvant alone. All three immunized monkeys produced significant lvG
antibodies to peptide VDlV and C. trachomatis serovars D, E, F~ G and K. The monkey sera were non-reactive or reacted weakly with the C-complex serovars H, 1 or J. None of WO 94/06827 ~ 8 8 2 PCr/US93/08739 the immunized monkeys produced IgG antibodies reactive with peptide A-X. The twocontrol monkeys that were immunized with adjuvant only, did not produce IgG antibodies reactive with peptide VDIV or EBs of the different C. trac~20matis serovars tested.

Immunogenicity of Peptide A8-VDIV in Cynomolgus Monkeys ELISA IgG antibody titerPeptide pin Neutralizing antibody Mappingtiter (ND50) C. trachomatis serovar C. trachomatis serovar 0 Monkey Peptide response to nurber VDIV D E F G H(LNPTIAG) D G H
705 4096409620482048 2048 32 ~ 256 128 c16 752 10242048 5122048 2048 64 + 128 64 ~16 907 5124096 10244096 4096 128 ~ 256 128 '16 842 ~16 ~16 ~16 ~16 ~16 ~16 - <16 ~16 ~16 880 ~16 16 <16 16 16 16 - <16 <16 <16 Monkey sera were further analyzed by pepscan ELISA against sequential and ove-la~pi,lg octapeptides corresponding to the VDTV sequence (Figure 5). All three immunized monkeys produced IgG antibodies that showed stron~ immunoreactivity against those VDIV octapeptides that contained the targeted LI~PTlAG B-cell epitope. These findings clearly showed that immunization with the A8-VDIV peptide was capable of preferentially directing antibody responsiveness to the LI~PTlAG epitope contained in the VDIV portion of the oligopeptide immunogen, and this targeted B-cell responsiveness was consistently observed in both mice and primates.
DETERMINATION OF SEROVAR SPECIFICITY ANI) CROSS-REACTIVITY
To ascertain whether the mouse anti-peptide antibodies reacted with intact chlamydiae, and to determine the serovar specificity of the anti-peptide response, mouse sera were tested by ELISA against formalin-fixed C. tracl7017tatis serovars D, E, F, G, H, I.
J and K EBs (Fig. 4). The anti-peptide antibodies produced hy each mouse strain reacted by ELISA with B-complex (serovars D and E) and lntermediate complex (serovars F, G and K) EBs. These same sera were weakly reactive or non-reactive with the C-~:omplex serovars H, I and J. Results are summarized in Table 3 below. Thus, the C. trac~tontati* serovar specificity of the polyclonal antibodies produced followinS~ immunization with peptide AX-VDIV was very similar to that of mAb DIII-A3. The exception was that the sera reacted with serovar K which is non-reactive with mAb DIII-A?,.

~_ WO W/06827 2 1 4 4 8 8.2~ .. PCI/U593/08739 The Ability of Peptide A8-VDIV to Prime Mice for an Augmented leG Response Following Secondary Challenge with Whole C. trachomatis EBs .
ELISA IgG antibody titer Primary immunization Secondary immunization Immunogen C. trachoratis serovar C. trachomatis serovar Primary 5e_u,~r~ Peptide D G H Peptide D G H
VDIV VDIV
0 PBS D EB <16 ~16 <16 <16 128 409616 <16 A8-VDlV PBS 10244096 1024 16 4096 81922048 32 A8-VDlV D EB 20484096 1024 16 16384 32768 16384 512 A8-VDlV G EB 10244096 512 ~16 16384 65536 16384 256 A8-VDlV H EB 10244096 512 16 16384 65536 16384 512 TESTING NEUTRALIZING ACT~VITY OF ANTl-A8-VDIV ANTIBODY
To ascertain whether the anti-peptide antibodies generated in the test animal orhuman patient, in response to the synthetic peptide, were functional neutralizing antibodies, the sera was tested for neutralizing activity using a chlamydial inclusion forming unit (IFU) reduction assay. In this example, the sera from vaccinated mice was tested for neutralizing activity (See Example 6). Neutralization assays were performed with C. trachomatis serovars D, G and H, since these three serovars are representative of the three chlamydial serogroups. All six responsive mouse strains had si~nifi-~nt serum neutralizing activity against C. trachomatis serovars D and G but failed to neutralize the infectivity of serovar H for HaK cells (Fig. 4). The exception was strain B 1 0.A which produced a low neutralizing titer (1:16) against serovar H. Thus, similar to the results found by ELISA using intact C.
trachomatis 2EBs, the serum neutralizing activity of mouse anti-A8-VDIV was also sub-species specific with only B and Intermediate complete serovars (D and G, respectively) being neutralized. Therefore, it is contemplated that the preferred peptide vaccine of this invention can additionally incorporate the B-cell neutralizing epitope of strains with low neutralizing activity such as strain B10.A.
Sera from monkeys immunized with peptide A8-VDIV (705~ 752 and 907) were assayed for their ability to neutralize C. trachomatis infectivity il? vitro. The sera from each of the immunized monkeys had significant neutralizing activity against C. trachomatis serovars D and G but were incapable of neutralizing the infectivity of serovar H (Figure 6).
Thus, primates immunized with peptide A8-VDIV produced neutralizing antibodies having a C tracho~natis sub-species specificity. These findings were consistent with those found in WO 94/06827 2 1 ~ 4 8 8 2 ~ PCI`/US93/08739 _~

the mouse and clearly demonstrated the ability of peptide A8-VDIV to evoke broadly cross-reactive neutralizing antibodies in both mice and primates.
Summaries of the neutralization studies are provided for mice and monkeys in Tables 1 and 2 respectively. These findings demonstrated as a whole that mouse and S monkey anti-A8-VDIV antibodies are capable of neutralizing the infectivity of chlamydiae in vitro.
COMPARISON OF A8-VOIV l'~;~lll)E VACCINE WITH A8-VDI(A) VACCINE
Previously a peptide vaccine A8-VDI(A) was prepared that combined the conserved T-helper cell epitope of this invention with another B cell neutralizing epitope. The following study compared the two vaccines for its ability to induce a protective response against a number of C trachomatis serovars in primates. Advantageously the combination of the conserved T-helper epitope with the VDIV-derived neutralizing epitope provided a broad protective response to a number of serovars as compared to A8-VDI(A) and as co"",ared with other combinations of epitopes.
Monkeys were immunized three times intramuscularly with 1 mg of peptide.
Animals were bled 14 days following the third immunization and their sera was tested by ELISA for IgG antibodies reactive against multiple C. trachomatis serovars. Results are provided in Figure 7. Solid bars r~resent absorbance values (A405) of pre-immune sera.
The stippled bars are absorbance values of sera after immunization with the synthetic peptides. Monkeys immunized with peptide A8-VDI produce antibodies against only serovar A. ln contrast monkeys immunized with peptide A8-VDIV produced IgG
antibodies reactive with the majority of the serovars. Most ~ignifir~ntly monkey anti-A8-VDIV antibodies reacted strongly against C. trachomatis serovars D E F and G; the most i"~po~lant serovars in terms of C. trachornatis caused sexually transmitted diseases (STD).
The anti-A8-VDIV sera neutralized chlamydial infectivity i)l vitro with a similar specificity as that shown by ELISA. Thus the A8-VDIV peptide has considerable potential as avaccine to prevent infection in humans by C. trachomatis because it is capable of evoking broadly cross reactive neutralizing antibodies against multiple serovars.
As provided in the invention disclosed above this invention has particular utility against the major serovars associated with STDs in this country. Advantas~eously~ and unlike other C. trachomatis vaccines disclosed thus far the synthetic peptide vaccine ot this invention has proved useful against challenges of C. tracltomatis in mice and primates. The results of studies with this vaccine indicated that immunization with the peptide effectively targeted the production of high titer antibodies against the B-cell portion of the peptide.

~_ WO 94/06827 2 1 4 4 8 8 2~ PCI/US93/1~8739 These anti-peptide antibodies were preferentially directed at the antigenically common B-cell epitope LNPTIAG within the VDIV sequence, recognized this epitope in its native configuration, and were functional antibodies capable of neutralizing the infectivity of those C. trachomatis serovars that are epidemiologically important as causative agents of chlamydial STDs.
Synthetic peptide and recombinant subunit immunogens tend to be poorly immunogenic, antibodies produced against them may not recognize the targeted B-ceJl epitope within the peptide's primary sequence, and the anti-peptide antibodies may fail to react with the targeted B-cell epitope in its native configuration on the pathogens' surface.
In addition, the number of T-helper cell epitopes that can be incorporated into synthetic peptide immunogens is restricted. This too can colllplolllise the immunogenicity of the peptide within the general population since HLA class 11 diversity is known to influence the recognition of T-cell antigens (Schwartz, R.H. Curr. Top. Microbiol. ImmunoL 130:79, 1986).
The results of these studies in mice in~ ated that six of eight H-2 congenic mouse strains that were immunized with peptide A8-VDIV produced IgG antibodies reactive with the B-cell portion of the peptide and these findings are consistent with the hypothesis that the TH-cell epitope(s) contained in the A8 sequence is recognized by multiple MHC class Il haplotypes. Two of the responding strains of mice, C57BL/10 and BlO.M also produced antibodies following immunization with peptide VDIV alone; in~lic~ing that in addition to B-cell epitopes, the VDIV sequence also contained a TH-cell epitope whose recognition is restricted by H-2b and H-2f haplotypes. Thus, peptide A8-VDIV contains at least two distinct epitopes that elicit functional TH activity. The combination of these two sites enhance the possibility of the peptide being generally recognized in the heterogenous human population. Consistent with this hypothesis is the finding that all three cynomolgus monkeys immunized with peptide A8-VDIV produced IgG antibodies against the targeted LNPTlAG
B-cell epitope. We have recently immunized four other cynomolgus monkeys with peptide A8-VDIV as part of a separate study to evaluate the protective efficacy of the peptide immunogen. All four of the vaccinated monkeys produced lgG antibodies against the LNPTIAG neutralizing epitope further supporting the potential of the peptide immunogen to be generally recognized.
- Surprisingly, our findings suggest that peptide A8-VDIV does not share the unfavorable immunological characteristics that have been commonly associated with synthetic peptide immunogens. It is not understood why immunization with peptide A8-VDJV was so effective in evoking antihody responsiveness to the LNPTlAG epitope within W O 94/06827 -2 I ~ 4`8 8 2 PC~r/US93/08739 the VDIV sequence. It is possible, because of the size of the VDIV sequence (1/' amino acids) incorporated in the A8-VDIV peptide, that the peptide maintained structural elements important in determining its immunogenicity. Our findings convincingly demonstrate that the A8-VDIV peptide is a very effective immunogen that is capable of S preferentially evoking antibody responsiveness to the antigenically common LNPTlAG
neutralizing epitope contained within the VDIV sequence.
An unexpected finding of this work was that both the mouse and monkey anti-A8-VDIV antisera were reactive against serovar K (Fig. 3 and Table 1). These data are not c~n~ictent with the immunoreactivity of mAb DIII-A3 which does not react with the MOMP
of serovar K by western blotting (see Zhang et al., Infect. Immur~., ( 1989) supra). The lack of immunoreactivity of mAb DIII-A3 with serovar K can be explained by the threonine for alanine substitution at position 303 of the LNPTlA(T)G sequence. Unlike mAb DIII-A3 which reacts with a single epitope, polyclonal anti-A8-VDl V antibodies reacted with multiple B-cell epitopes contained in the VDIV sequence. This is evident from the pepscan analysis of the polyclonal anti-A8-VDIV response (Figs. 2 and S) which demonstrated in addition to octapeptides containing the LNPTIAG epitope, both mice and monkeys produced antibodies reactive with other octapeptides corresponding to VDIV sequences. Although it is difficult to determine which epitope(s) is important for the reactivity of the anti-peptide antibodies with serovar K, an epitope(s) contained within the 296TTLNPTI302 sequence is a likely possibility. This sequence is present in VDIV of serovar K and the anti-peptide antibodies were immunoreactive with octapeptides containing the TTLNPTI sequence by pepscan analysis. The TTLNPTI sequence is also common to the C-complex serovars H, I and J, however, like the LNPTIAG epitope, it is apparently not accessible to antibody on the native EB surfaces of these serovars. Nonetheless, the f`act that immunization with 2'. peptide produced antibodies reactive with serovar K is clearly advantageous because it increases the number of C. trachomatis serovars to which peptide A8-VDIV can evoke neutralizing antibodies.
A more precise evaluation of the peptides vaccine potential will require phase Ivaccine studies in humans to define its immunogenicity and toxicity. ln this context, we observed that none of the primates immunized with the oligopeptide developed clinical sigm.
of allergenic or toxigenic reactivities suggesting that the peptide could be safely administered to humans.
Particular embodiments of the invention will be discussed in detail and reference will be made to possible variations within the scope of the invention. There are a variety ol ~_ W094/06827 2 1 448 ~ 2 PCI'/US93/08739 alternative techniques and procedures available to those of skill in the art which would similarly permit one to succrssfully perform the intended invention.
- FY~ , IC 1 Construction of a Synthetic Vaccine Containin~ a Conserved B cell Neutralizing Epitopes - S and a conserved T cell Epitope Peptides VDIV, and A8-VDIV were synthesized using an automated peptide synthesizer (Model 431A Synthesizer, Applied Biosystems, Inc., Foster City, CA) as described previously by Su et al., supra. Peptides were purified by reverse phase HPLC on a C18 column (Ber~m~n Ins~ ents, Inc., Fullerton, CA). The accuracy of the synthesis reaction was defined by amino acid sequ~nrin~ Peptide VDIV corresponding to serovar B MOMP residues 293-309 (FDV'ITLNPTIAGAGDVK) and containing the sequence invariant LNPTIAG septmeric epitope that is recognized by the neutrali~ing mAb DIIl-A3.
Peptide A8 corresponds to serovar A MOMP residues 106-130 (ALNIWDRFDVFCTLGAl-rGYLKGNS) which contains a MOMP TH-cell epitope that effectively primes mice to produce an anamnestic IgG response specific to MOMP following secondary immunization with the native protein. Peptide A8-VDIV consists of peptide A8 and VDIV colinearly synthesized with the A8 sequence at its N-terminus and the VDIV
sequence at its carboxyl terminal end.
FY~ le 2 Vaccination of Mice with Synthetic Peptide C57BL/lOSnJ (H-2b), B10.A/SgSnJ (H-2a), B10.D2/oSnJ (H-2d), B10.M/Sn (H-2f), B10.WB/Sn (H-2ia), B 10.Br/SgSnJ (H-2k), B 10.PL(73NS)/Sn (H-2U ) and B 10.SM (70NS)/Sn (H-2V) mice were purchased from Jackson Laboratory (Bar Harbor, ME). Both sexes of mice at 8-12 weeks of age were used for experimentation. Groups of five mice were immunized by intraperitoneal injection of 50 llg of peptide A8-VDIV or peptide VDIV
alone emulsified in complete Freund's adjuvant (CFA) and boosted once three weeks later with the same dose of peptide in incomplete Freund's adjuvant (IFA). .~Iice were bled two weeks after the secondary immunization.
Example 3 Vaccination of Primates with Synthetic Peptide Cynomolgus (Macaca fasicular7s) monkeys of Mauritius Island origin were used in these studies. Monkeys were part of a ful~y condition colony that had been stable tor over two years. All examinations of experimental monkeys were conducted using ketamine W0 94/06827 2 1 4 4~ ~ 2 PCI/US93/08739 hydrochloride sedation. The work was conducted in full compliance with the "Guide for Case and Use of Laboratory Animals of the Institute of Laboratory Animal Resources Commission on Life Sciences National Research Council," as well as all applicable federal laws and regulations. The facilities are fully accredited by the American Association for S Accreditation of Laboratory Animal Care (AAALAC). Three cynomolgus monkeys (705, 752, and 907) were immunized intramuscularly with 1 mg of peptide A8-VDIV emulsified with Ribi adjuvant (MPL+TDM+CWS emulsion, Ribi lmmunochem Research. Inc., Hamilton, MT). Two control monkeys (842 and 880) were immunized with adjuvant alone.
Immunization was repeated twice with 500 ~ g of peptide and adjuvant at four week intervals. Monkeys were bled two weeks after the third immunization.
FY~ , le 4 Seroloeic Evaluation of Antibody Response C trachom~tis serovars D (UW-3/Cx), E (Bour), F (IC-Cal-13), G (UW-57/Cx), H
(UW-4/Cx), I (UW-12/Ur), J (WU-36/Cx), and K (UW-31/Cx) were grown in Hel~ 22~
cells and chlamydial elementary bodies (EBs) were purified from infected cells by d~nsity gradient centrifugation as previously described by Caldwell et al., supra.
Serum antibody responses were assayed by enzyme linked immunoabsorbent assay (ELISA) followingpreviously described methods (Su et al., (1990)supra). Briefly, microtiter plates (Immunolon 2; Dynatech Laboratories, lnc. Alexandria, VA) were coated overnight at 40C with 100 Ill of synthetic peptide (5 llg/ml), purified MOMP (0.5 ~g/ml), or formalin killed C. trachornatis EBs (10 ~g/ml) in 0.05 M Tris buffer (pH 7.5) containing 0.15 M NaCl.
Serial twofold diluted mouse or monkey sera were tested in duplicate. Mouse and monkey IgG were detected using anti-mouse and anti-human IgG alkaline phosphatase conjugate respectively (y chain specific, Zymed Laboratories, Inc., San Francisco, CA) followed by substrate (5 mg p-nitrophenyl phosphate in 10 ml. of 0.1 M 2,2 amino-2-methyl-1,3-propandiol, pH 10.3). Absorbance at 405 nm was measured with an ELISA reader (Bio-Rad Laboratories, Richmond, CA). Pooled pre-immune sera was used as the negative controls and ELISA titers were expressed as the highest serum dilution giving an absorbance of 0.3 OD units.
Example 5 Defining Linear Epitopes Associated with the Immune Response Linear epitopes from MOMP that bound serum antibody from vaccinated mice and monkeys were identified by pepscan assay as described by Geysen, et al. (J. Ir7lmu~01.

- ~ W 0 94/06827 21g;~8g2 PC~r/US93/08739 Methods 102:259, 1989). Sequential and overlapping octapeptides corresponding to serovar B MOMP VDIV (residues 288-314) were synthesized on prederivatized pins using a commercially available kit (Epitope Sç~nning Kit, Cambridge Research Biochemicals, Inc., Wilmin~ton, DE) following the instructions of the manufacturer. Reactivity of mouse and S monkey IgG antibodies to the solid phase octapeptides were determined by ELISA using the same anti-mouse or human IgG alkaline phosphatase conjugate as described above for the ELISA assay.
Example 6 In vttro neutralization of C. trachomatts infectivitv.
In vitro neutralization of C. trachomatis infectivity by mouse or monkey anti-peptide antibodies was assayed on Syrian hamster kidney (HaK) cells grown in 96-well microtiter plates (Linbro, 96 flat-bottomed wells, Flow Laboratories, Inc., McLean, VA) as described previously (Su, H. and H.D. Caldwell., J. ~xp. Me~ 175:227 (1992). Briefly, lOs HaK cells were seeded in 96-well plates 24 hours prior to the neutralization assay. C. trachomatts serovars D, G, and H were diluted in 250 mM sucrose, lO mM sodium phosphate, S mM
L-~lut~mic acid, pH 7.2 (SPG) to give a final concentration of 3 x lOs - 3 x lo6 inclusion-forming units (IFUs)/ml. Two fold dilutions of pooled mouse sera or individual monkey sera were mixed with an equal volume of chlamydiae and incubated at 370C for 30 minutes.
The mixtures (50111/well) were inoculated in triplicate onto HaK cell monolayers. After 2 hours incubation at 370C, the inocula were removed, the monolayers washed and refed with media. The monolayers were fixed with methanol after incubation at 370C for 48 hours and IFUs were identified by indirect fluorescent antibody staining. Serum neutralizing titers were expressed as percent reduction in chlamydial IFUs and were calculated as follows:
[(IFUs control sera - IFUs experimental sera)/IFUs control sera] X 100.
While particular embodiments of the invention have been described in detail, it will be apparent to those skilled in the art that these embodiments are exemplary rather than limitinP, and the true scope of the invention is that defined in the following claims.

W094/~27 ~ 1 4 ~ ~ 8 PCT/US93/0~39 SEQUENCE LISTING

1) GENERAL lN~O~IATION:
(i) APPLICANT: The Government of the United States of America as represented ~y the Secretary of the Department of Health and Human Services (ii) TITLE OF lNv~NlloN: S~.L~11C PEPTIDE VACCINE FOR CHLAMYDIA
TRACHOMATIS
(iii) NuM~K OF S:Q~CES: 3 (v) COII~ul ~:K R~n~T~ FORM:
(A) MEDIUM TYPE: Floppy disk (B) COII~ul~:K: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUM~K: 07/947, 671 US
(B) FILING DATE: 18 SEP 93 2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) H~Ol~llCAL: NO
(iv) ANTI-SENSE: NO
(v) FRA~GMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Ala Leu Asn Ile Trp Asp Arg Phe Asp Val Phe Cys Thr Leu Gly Ala Thr Thr Gly Tyr Leu Lys Gly Asn Ser 2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids ~ wo g4/0~27 2 1 g ~ 8 8 2 PCT/US93/08739 (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Phe Asp Val Thr Thr Leu Asn Pro Thr Ile Ala Gly Ala Gly Asp Val Lys 2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide tiii) HYPO~ CAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Ala Leu Asn Ile Trp Asp Arg Phe Asp Val Phe Cys Thr Leu Gly Ala Thr Thr Gly Tyr Leu Lys Gly Asn Ser Phe Asp Val Thr Thr Leu Asn Pro Thr Ile Ala Gly Ala Gly Asp Val Lys

Claims (16)

WHAT IS CLAIMED IS:

1. A synthetic peptide capable of producing an immunological response to C.
trachomatis in a vertebrate, comprising:
a conserved T-helper cell stimulating epitope from the major outer membrane protein of C. trachomatis; and a serovar conserved B-cell neutralizing antibody stimulating epitope from the major outer membrane protein of C. trachomatis.

2. The synthetic peptide of Claim 1, wherein said T-helper cell stimulating epitope and said B-cell neutralizing antibody stimulating epitope are colinear.

3. The synthetic peptide of Claim 1, wherein said synthetic peptide contains the sequence identified as SEQ ID NO:3.

4. The synthetic peptide of Claim 1, wherein said T-helper cell stimulating epitope has a sequence located within SEQ ID NO: 1.

5. The synthetic peptide of Claim 1, wherein said B-cell neutralizing antibody stimulating epitope has a sequence located within SEQ ID NO: 2.

6. The synthetic peptide of either Claim 4 or 5, wherein said T-helper stimulating epitope has a sequence located within SEQ ID NO:1 and said B-cell neutralizing antibody stimulating epitope has a sequence located within SEQ ID NO:2.

7. The synthetic peptide of Claim 6, wherein said T-helper stimulating epitope is on the N-terminus side of said B-cell neutralizing antibody stimulating epitope.

8. The synthetic peptide of Claim 6, additionally comprising a linker peptide sequence separating the sequence of SEQ ID NO:1 from the sequence of SEQ ID NO:2.

9. The synthetic peptide of Claim 6, wherein said peptide additionally comprises at least one species specific B-cell neutralizing antibody stimulating epitope.

10. The synthetic peptide of Claim 7. wherein said synthetic peptide additionally comprises a known T-helper stimulating epitope from a protein other than the major outer membrane protein of C. trachomatis.

11. Use of a synthetic peptide according to Claim 1 in the preparation of a vaccine for C. trachomatis.

12. The use of Claim 11, wherein said conserved T-helper cell stimulating epitope is an epitope having a sequence located within SEQ ID NO:1 and said serovar conserved B-cell neutralizing antibody stimulating epitope is an epitope having a sequence located within SEQ ID NO:2.

13. An immunoassay for detecting the presence of antibody to C. trachomatis in a sample, comprising a synthetic peptide according to Claim 1 and means for detecting antibody bound to said peptide.

14. A method for inducing a protective immune response to C trachomatis in a vertebrate comprising introducing a synthetic peptide according to Claim 1 into said vertebrate, said peptide being introduced along with a pharmaceutically acceptable buffer.

15. The method of Claim 14, additionally comprising, after the introduction step, the step of determining the presence of neutralizing antibody to C. trachomatis in said vertebrate

16. The method of Claim 14, wherein the introducing step comprises injecting said synthetic peptide into said vertebrate intramuscularly.