Note: Descriptions are shown in the official language in which they were submitted.
21.q4882
~ W O 94/06827 ~.~ P(~r/US93/08739
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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
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2144882 -2-
(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.
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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-
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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
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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 _
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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~
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TABLE 1
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
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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.
TABLE 2
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
TABLE 3
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
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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
-15-
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
-16-
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
1 5 10 15
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
--19--
(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
1 5 10 15
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
1 5 10 15
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
35 40
