- Email: [email protected]
Colinear synthesis of an antigen-specific B-cell epitope with a ‘promiscuous’ tetanus toxin T-cell epitope: a synthetic peptide immunocontraceptive
Vaccine, Vol. 16, No. 15, pp. 1761-1766, 1997 0 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0264-410X/97 $17+0.00
PII: SO264-410X(97)00105-9? ELSEVIER
Colinear synthesis of an antigenspecific B-cell epitope with a 6promiscuous’ tetanus toxin T-cell epitope: a synthetic peptide immunocontraceptive Patricia A. O’Hern*, Zhi-Guo Liang”, Charanjit Goldberg*$
S. Barnbra? and Erwin
Carrier conjugation is commonly used to provide T-cell help for small, linear peptides containing antigen-specific B-cell epitopes. However; carrier conjugation is expensive, variable and often results in adverse side effects if the conjugate is administered repeatedly. To eliminate the need for carrier conjugation, we examined two synthetic peptides for their ability to elicit sustained antibody titres in female rabbits and baboons. One peptide (hCl-20) was based on the sequence of the sperm-specific isozyme of human lactate dehydrogenase (LDH-CJ. This peptide stimulates helper T-cell responses. The other peptide (bC519:TT) was a chimera between an LDH-C, B-cell epitope and a ‘promiscuous’ T-cell epitope from tetanus toxin which has been shown to bind to and stimulate many different major histocompatibility complex alleles. Both peptides were immunogenic in rabbits and baboons. The chimera elicited consistently high antibody titres and was immunogenic in 19119 wild-caught female baboons. When 14 bC.5-19:TT immunized baboons were mated, their fertility was reduced by 62% compared with controls (P~0.02). This cam’er-free construct can be incorporated into biodegradable microspheres which may provide long-tetm protection from pregnancy with a single dose. 0 1997 Elsevier Science Ltd. Keywords: immunocontraceptive,
T-cell
epitope,
lactate
dehydrogenase
A long-term goal of our research is the development of a safe, effective contraceptive vaccine for use by women. We have tested the sperm-specific isozyme of lactate dehydrogenase (LDH-C4) as our model antigen. In previous studies’, purified mouse LDH-C, was shown to suppress fertility in female mice, rabbits and baboons. The crystal structure of the protein was determined to 2.8 A resolution, and the B-cell epitopes of the antigen were mapped to four discrete regions on the surface of the LDH tetrame?. Computer-assisted sequence analysis was used to predict the immunodominant B-cell epitope of human LDH-C44. This peptide (hC9-20) was synthesized, conjugated to
*Center for Recombinant Gamete Vaccinogens and Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208, USA. tlnstitute of Primate Research, National Museums of Kenya, Karen, Naiobi, Kenya. $To whom correspondence should be addressed. el.: (847) 491-5416; fax: (847) 467-1380; e-mail: [email protected]. (Received 9 January 1997; revised version received 1 April 1997; accepted 2 April 1997)
diphtheria toxoid (DT) as a carrier molecule, and used as a contraceptive vaccine in female baboons. The fertility of the immunized animals was reduced by 70% compared with controls, and the contraceptive effect was reversed within a year of the last immunization. These results demonstrated the feasibility of using an LDH-&based vaccine for immunocontraception. However, carrier conjugation is neither desirable nor practical for general use. While providing a source of T-cell help for weakly immunogenic synthetic peptides, antigen specificity is sometimes altered by this procedure, and immune suppression or hypersensitivity due to repeated injection of carrier protein can occur’.‘. The conjugation chemistry is not trivialx, and the ratio of peptide to carrier protein varies from one batch to the next. Finally, most peptideicarrier conjugates are not stable to the organic solvents used in sustained release vaccine formulations”.“‘, or to extended storage at ambient temperatures. The goal of the present study was to avoid carrier conjugation by identifying one or more T-cell epitopes which could be incorporated into a completely synthetic peptide vaccine. This construct should be
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Carrier-free contraceptive vaccine in non-human primates: P.A. O’Hern et al. effective in a broad range of major histocompatibility complex (MHC) haplotypes and should significantly suppress fertility in non-human primates. We studied both an endogenous LDH-C, T-cell epitope (hCl-20), and a 39 amino acid chimeric peptide containing the B-cell epitope of baboon LDH-C4 (bC5-19) synthesized in tandem with a ‘promiscuous’ T cell epitope from tetanus toxin (TT580-599)“. The immunogenicity of these constructs in rabbits and baboons, and the suppression of fertility in female baboons immunized with the chimeric peptide construct are reported here.
MATERIALS
AND METHODS
Antigen preparation
Peptides hCl-20, hC9-20 and bC5-19:TT were synthesized at the Salk Institute (under Contract NOl-HD-0-2906 with the NIH) and made available by the Contraceptive Development Branch, Center for NICHD. The lyophilized Population Research, peptides were SO-90% pure as determined by high pressure liquid chromatography. The design of the chimeric peptide incorporating a four amino acid linker sequence between the B cell and T cell epitopes is essentially as described by Kaumaya et al.“.
Immunizations
and antibody titre determinations
Female rabbits (two per group) were immunized intradermally with 200 pg of peptide hCl-20, hC9-20 or bC5-19:TT emulsified in Freund’s Complete Adjuvant (FCA). Booster injections were administered intravenously 6 weeks later and consisted of 200 pg peptide in PBS. For immunogenicity testing, female baboons (five per group) were immunized i.m. with 400 pg peptide hCl-20 or bC5-19:TT or a combination of both peptides (200 /lg each) emulsified with Squalene:Arlace1 A (4:l) containing 200 L’g of the synthetic muramyl dipeptide CGP11637 (a gift of Ciba-Geigy Pharmaceuticals, Base], Switzerland) as adjuvant in a total volume of 0.50 ml. Booster immunizations administered i.m. on days 10 and 40 consisted of 200 Llg antigen and 100 pg adjuvant in 0.25 ml Squalene:Arlacel A. Antibody titres were determined by ELISA on microtitre plates coated with lo-’ M purified, recombinant human LDH-Cq13. Titre is defined as the reciprocal of the serum dilution that gives 0.5 absorbance units over background. Average tertiary titre is defined as the average ELISA titre for five individual bleeds taken during weeks 9-23 of the fertility trial (i.e. after the third injection of antigen).
horseradish peroxidase conjugated secondary antiserum (goat anti-human or goat anti-rabbit; Zymed, San Francisco, CA) diluted 1:50000 in blocking buffer, and developed with the enhanced chemiluminescent system following the manufacturer’s instructions (Amersham Life Sciences, Arlington Heights, IL).
Baboon fertility trial
Wild-caught female baboons (Papio cynocephalus, 15 per group) were housed in individual cages at the Institute of Primate Research, Nairobi, Kenya. The animals were allowed to acclimatize and resume cycling before the experiments began. Experimental animals were immunized i.m. with 400 pg peptide bC5-19:TT, 200 /lg adjuvant (CGPI 1637) emulsified with Squalene:Arlacel A as described above; control animals received vehicle and adjuvant only. Booster injections (200 /lg peptide, 100 pg adjuvant) were administered on days 10 and 70. Serum was collected every 2 weeks and tested for antibody titre by ELISA assay against purified recombinant human LDH-C,. Matings were begun during the first cycle following the third injection of antigen. Cohabitation with males was permitted for 5 days at the time of maximum sex skin turgescence when ovulation was predicted. This regime was followed for four cycles or until pregnancy was achieved. Statistical analyses were performed based on a one-way Gaussian analysis of variance (ANOVA). Two animals from the control group and one from the immunized group were eliminated from the trials because they stopped cycling after they had been acclimatized and immunized but before the first mating had occurred.
RESULTS Immunogenicity
testing
The amino acid sequences of the synthetic peptide vaccinogens are presented in Figure 1. Previously4 we predicted that hCl-20 (Figure Z(A)) contained T-cell helper activity. Detailed analyses of the human T-cell response to tetanus toxin”.‘4 indicated that TT contains
A. 1 STVK
B.
10
20
EOLIEKLIEDDENSQC
5
19
NH2-EQLIEKLIEDDKNSQ G Western blot analyses
Approximately 50 ,ug total protein from human sperm or liver, or 1 /lg purified, recombinant human LDH-C4 were resolved by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membranes, air dried and blocked in 0.1 M PBS containing 2% normal goat serum, 1% bovine serum albumin (BSA) and 0.2% Tween-20. The filters were incubated with appro-
priately diluted primary antiserum (baboon or rabbit),
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P
LS COOH-VSPFYSYYKKSNNLAADV Figure 1 Amino acid sequences of peptide immunogens. (A) Residues l-20 of human LDH-C, (hCl-20). The known B-cell epitope is underlined. (B) Chimeric peptide bC5-19:TT. Residues 5-19 of baboon LDH-C, were synthesized colinearly with a non-natural P-bend (GPSL) and a T-cell epitope from tetanus toxin (residues 580499). The known B-cell epitope is underlined; the T-cell epitope is shown in boldface type. Human and baboon LDH-C, differ at residue 16 (glutamic acid-lysine)
Carrier-free contraceptive vaccine in non-human primates: P.A. O’Hern et al. a booster injection was detected. Peptides hCl-20 and bC5-19:TT elicited strong and sustained antibody titres in both rabbits, with clear mnemonistic responses to a booster injection 40 days later. In addition, the tetanus toxin construct elicited antibody titres approximately one order of magnitude higher than did peptide hCl-20. We concluded that peptides hCl-20 and bC5-19:TT contain one or more T-cell epitopes which are recognized by rabbits, but peptide hC9-20 does not. We next tested the immunogenicity of peptides hCl-20 and bC5-19:TT in female baboons that were to be used in a fertility study. We immunized groups of five wild-caught female baboons with peptide hCl-20 or with peptide bC5-19:TT. To test whether these two antigenic peptides could act synergistically, we immunized a third group with a combination of both hCl-20 and bC5-19:TT. The results are presented in 7irhle I. Peptide hCl-20 was weakly immunogenic in female baboons. Peptide bC5-19:TT was strongly immunogenic in all five animals, particularly after the third injection, eliciting peak titres at least an order a magnitude higher than did peptide hCl-20. The combination of both peptides was not significantly better than the tetanus toxin construct alone, and certainly was not synergistic. Since peptide bC5-19:TT elicited moderate to good antibody titres and was effective in a broad range of genetic backgrounds, it was selected as the immunogen to use in fertility trials in female baboons.
several T-cell epitopes, termed ‘promiscuous’ epitopes, which are able to stimulate MHC class II responses in a wide variety of human haplotypes. We chose to test one such broadly-recognized T-cell epitope (IT residues 580-599, Figure Z(B)) for its ability to substitute for diphtheria toxoid as a component of our contraceptive vaccine. The B-cell epitope in this case was based on the baboon homologue of LDH-C,, which is identical to the human sequence except for a single amino acid substitution at position 16 (human Glu to baboon Lys)‘. Female rabbits (two per group) were immunized with either the B-cell epitope alone (hC9-20), the B-cell epitope synthesized colinearly with the putative LDH T-cell epitope (hCl-20) or the chimeric peptide containing the baboon B-cell epitope synthesized in tandem with the T-cell epitope from tetanus toxin (bC5-19:TT). The immune response of the rabbits is presented in Figure 2. All three peptides were able to elicit circulating antibodies which recognized purified, recombinant LDH-CJ by ELISA assay. Peptide hC9-20 elicited a strong response in one rabbit, and a weak response in the other rabbit. No secondary response to
Weeks
Post
Baboon fertility trial In the fertility trial with hC9-20:DT’, both control and treated animals were housed as a troop with males so that mating would occur naturally, presumably whenever a female reached mid-cycle. The present trial was modified by housing animals in individual cages during the trial. Female baboons cohabited with a male during the period of maximal sex skin turgescence at least 3 days prior to and 2 days after expected ovulation. Due to a limited number of cages, the fertility trial was performed in two stages. Group 1 consisted of eight immunized and seven control females. When the
Primory
Figure 2 Rabbit immune response to synthetic peptides. Two female rabbits were immunized with peptide hC9-20 (circles), peptide hCl-20 (triangles) or peptide bC5-19:TT (squares). Booster injections were administered in week 5 (arrows). Serum antibody titres were determined by ELISA on microtitre plates coated with purified, recombinant LDH-C,
Table 1
Baboon immune response to peptide vaccines Weeks following
the first injection
Animal 0
no. Vaccination 1766 1784 1706 1665 1663
with hC7-20
Vaccination
with X5-79:77
1435 1585 1657 1660 1659 Vaccination 1661 1662 1668 1616 1547
4
2
6
14
9
225 225 30 45 170
505 225 50 70 630
725 200 170 65 250
575 145 370 70 120
1960 1115 10 965 1544 2175
1960 485 8965 944 1775
760 140 2465 164 125
250 800 11 750 29 930 550
180 5400 2550 1530 640
100 1000 950 1530 210
0 0 0 0 0
75 35 20 15 0
0 65 0 655 30
0 575 -0 30
-
0 0 0 0 0
160 10 0 34 0
60 23 65 134 22
260 13 235 194 135
-
0 40 125 134 195
0 0 0 0 0
30 0 0 40 0
80 2800 310 490 470
100 1300 1350 1180 80
-
90 550 0 850 0
-
12
16
8 (third injection
with hCl-20+bC5-19:77
Vaccine 1997 Volume 15 Number 16
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Carrier-free contraceptive vaccine in non-human primates: P.A. O’Hern et al. breeding protocol was completed (approximately 6 months later), the second half of the study (Group 2: eight control and seven experimental animals) commenced. The kinetics of the immune response for three representative animals is shown in Figure 3. The magnitude and duration of the immune response differed significantly between individuals. Two of the animals (Nos. 1254 and 1705) produced a strong and rapid response to the vaccine, while animal 1.557 produced a slower and more modest response. As observed during the preliminary immunogenic& study, the serum titres declined rapidly after the tertiary injection, then stabilized at low but detectable levels for several months. As observed previously with hC9-ZO:DT, there was no correlation between serum antibody titre and infertility. One of the high responder animals (No. 1254) did not become pregnant, while another high responder animal (No. 1705) did. Furthermore, No. 1557, one of the lowest responder animals, was also protected from pregnancy.
‘OS5 10’ L
9)
E
4 wl i W
103
lo2
10’
10
i123450709
Months
Post
11
12
Primary
Figure 3 Immune response profile of three female baboons immunized with peptide bC5-19:lT. Three wild-caught female baboons were immunized with 400 pg of peptide C5-19:TT and 200 pg of CGPll637 on day 0. Booster injections were administered on days 10 and 70 (arrows). Cohabitation with males (male symbol) commenced on the first ovulatory cycle following the third injection and continued for four cycles or until pregnancy was established (P). (.), baboon 1254; l, baboon 1705; A, baboon 1557
Anrmal
While individual titres varied considerably from animal to animal, and from week to week, the average titre during the first 12 weeks following the tertiary injection was remarkably consistent. Figure 4 shows the average tertiary ELISA titre for all 14 experimental animals (one of the 15 original animals was removed from the study because she stopped cycling before the matings began). All 14 animals produced moderate to good antibody responses to the vaccine, and no significant titre differences between Groups 1 and 2 were apparent. No anti-LDH-C, titres were detected among the control animals (data not shown). For purposes of comparison, the average tertiary titre for the female baboons immunized in our previous study with hC9-20:DT is shown in Figure 5. With this construct, the average titre during the post-tertiary period was highly variable, ranging from less than 20 to over 3000, and four animals produced antibody titres which were barely above background. Matings were initiated during the first ovulatory cycle following the third injection of antigen. As shown in Table 2, lo/13 (77%) of the control animals and 4/14 (29%) of the experimental animals became pregnant, a 62% reduction in fertility. A one-way analysis of variance (ANOVA) showed this to be a statistically significant difference (PcO.02). Of the four pregnant animals in the experimental group, three miscarried during the first trimester (Figure 4). (The miscarriages are counted as pregnancies in the statistical analyses.) None of the control animals miscarried. The pregnancies and miscarriages were evenly distributed between Group 1 and Group 2 animals, and do not appear to be related to serum antibody titre. To determine whether the antibodies elicited by bC5-19:TT were specific for the Cq isozyme of lactate dehydrogenase, Western blots of human tissue extracts were probed with sera from immunized baboons. No reactivity was detected with baboon preimmune serum (F&ire 6). Serum from a pregnant/miscarried female detected a single 34 kDa band in human sperm and testes extracts, consistent with the size of LDH-C monomers, but did not react with human liver extracts (a rich source of the LDH-A, isozyme). As a control, rabbit anti-LDH-Ad did detect the A, isozyme in
Number
Figure 4 Immune response and fertility outcome of all female baboons immunized with peptide bC5-19:TT. Fifteen female baboons were randomly divided into two groups (Group 1, cross-hatched bars; Group 2, horizontally hatched bars) and injected three times with bC5-19:TT. The average ELISA titre during the first 12 weeks following the tertiary immunization was calculated for each individual and presented in rank order as lowest responder (left) to highest responder (right). Animals which conceived during the fertility trial are indicated by P (pregnant) or M (pregnant but miscarried)
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Carrier-free contraceptive vaccine in non-human primates: P.A. O’Hern et al.
7
Animal
Number
Figure 5 Immune response and fertility outcome of female baboons immunized with an hC9-20:DT conjugate. Thirteen female baboons were injected three times with peptide hC9-20 conjugated to diphtheria toxoid (hC9-20:DT). The average post-tertiary ELBA titre was calculated for each individual and presented as described in the legend to Figure 4. P, pregnant
liver. Therefore, lack of specificity of antibodies to the vaccine is not likely to be the cause of the observed miscarriages. human
DISCUSSION In the present study, we describe a completely synthetic immunocontraceptive vaccine (bC5-19:TT) which suppressed the fertility of wild-caught female baboons by 62% compared with controls (P10.02). The vaccine was immunogenic in 19 of 19 immunized animals, regardless of their genetic background. Peptide hCl-20 is also immunogenic in rabbits and baboons. FCA was used for the primary immunizations in rabbits, while the baboons received Squalene:Arlacel A containing a synthetic muramyl dipeptide as Table 2 Treatment
Anti-fertility group
Control Experimental
effect of bC519:TT No. of animals
No. pregnant
% pregnant
13 14
10 4a
77 29*
aP 2 0.02 b62% reduction
sp
c4
Li
sp
c4 Li
sp
c4 Li
kD - 84 -
5a
- 18 - 36 - 26 antiserum. Figure 6 Tissue specificity of baboon anti-bC5-19:lT Human sperm (Sp), human liver (Li) and purified, recombinant human LDH-C, (C4) were electrophoresed by SDS-PAGE, transferred to nitrocellulose filters and probed with baboon preimmune serum (left panel), baboon anti-bC5-19:m (centre panel), or rabbit anti-human LDH-A, (right panel). The position of the molecular weight standards is shown on the right
adjuvant. When rabbits are immunized with synthetic peptide antigens emulsified in Squalene:Arlacel A containing CGPll637, the resulting serum antibody titres are still of the same order of magnitude as those observed here with FCA as adjuvant (O’Hern and Goldberg, unpublished data). The immune response to the chimeric peptide was remarkably consistent among all the animals studied, particularly after the third injection. Since one of the major challenges in developing synthetic peptide-based vaccines is to elicit an effective immune response regardless of the MHC haplotype of the individual, this result is very encouraging. Certainly, the immune response to bC5-19:TT is more robust and consistent than was the response to the hC9-20:DT construct (cf. Figure 4 and Figure 5). Although the tetanus toxin epitope used is known to be effective in a broad range of haplotypes, it is not universally antigenic “,‘5. Since peptide hCl-20 does contain at least one T-cell epitope which is recognized by rabbits and baboons (Figure 2, Table I), it is possible that bC519:TT contains an antigen-specific T-cell epitope in addition to the promiscuous T-cell epitope. This combination may result in a more effective vaccine than would the TT epitope alone. It is important to note that three of the four pregnant experimental animals miscarried during the first trimester; two from Group 1 and one from Group 2 (Figure 4). None of the ten pregnant control animals miscarried. This is the first time the authors have observed miscarriages among baboons immunized with LDH-C, or its peptides. However, in the previous trial with hC9-20:DT, the animals were housed as a troop in a single large cage, and spontaneous abortions may not have been detected. Western blots using serum from one of these animals did not reveal any non-specific or autoantigenic immune reactivity (Figure 6), nor did the serum recognize the tetanus toxin T-cell epitope itself by ELBA assay (data not shown). The authors now have a single, synthetic peptide which is highly immunogenic without carrier conjugation, regardless of the MHC haplotype of the recipient. Immunization with this antigen significantly reduces fertility (62% reduction; P < 0.02) in non-human
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Carrier-free contraceptive vaccine in non-human primates: P.A. OHem et al. primates. Experiments are in progress to incorporate bC5-19:TT into biodegradable polylactide/polyglycolide microspheres’0.‘h.‘7. A single dose of microspheres containing the carboxy-terminal peptide of human chorionic gonadotrophin conju ated to diphtheria toxoid” or tetanus toxoid alone’ 5 has been shown to elicit sustained T-cell and antibody responses equal to or greater than those obtained with three standard injections. A similar strategy could greatly improve the efficacy and acceptability of a contraceptive vaccine.
6
7
8
9
ACKNOWLEDGEMENTS This work was supported by NIH Sub-5-U54-HD29099, by P30HD28048, and by the Contraceptive Research and Development Program (CSA-92-099) under a Cooperative Agreement with the US Agency for International Development (U.S.A.I.D.) (DPE-3044-A00-6063-00) which in turn receives funds for AIDS research from an interagency agreement with the National Institute of Child Health and Human Development. The views expressed by the authors do not necessarily reflect the views of U.S.A.I.D. and CONRAD.
REFERENCES Goldberg, E. Lactate dehydrogenase C, as an immunocontraceptive model. In Gamete Interaction: Prospects for lmmunocontracepfion (Eds Alexander, N. J., Griffin, D., Spieler, J. M. and Waites, G. M. H.). Wiley-Liss, New York, 1990, pp. 63-73 Hogrefe, H. H., Griffith, J. P., Rossmann, M. G. and Goldberg, E. Characterization of antigenic sites on the refined 3-A resolution structure of mouse testicular lactate dehydrogenase-Cd. Journai of Biological Chemistry 1987, 262, 13155-13162. Hogrefe, H. H., Kaumaya, P. T. P. and Goldberg, E. Immunogenicity of synthetic peptides corresponding to flexible and antibody-accessible segments of mouse lactate dehydrogenase (LDH-C,). Journaf of Biofogical Chemistry 1989, 264, 10513-10519. O’Hern. P. A. and Goldberg, E. The use of molecular modelling to delineate B-cell and T-cell epitopes of human spermspecific LDH-C,. In Techniques in Protein Chemistry IV (Ed Angelette, R. H.). Academic Press, New York, 1993, pp. 481-490 O’Hern, P. A., Bambra, C. S., Isahakia, M. and Goldberg, E. Reversible contraception in female baboons immunized with a synthetic epitope of sperm-specific lactate dehydrogenase. Biology of Reproduction 1995, 52, 331-339.
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14
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Schutze, M. P., Leclerc, C., Jolivet, M., Audibert, F. and Chedid, L. Carrier-induced epitopic suppression, a major issue for future synthetic vaccines. Journal of immunology 1985,135,2319-2322. Kaliyaperumal, A., Chauhan, V. S., Talwar, G. P. and Raghupathy, R. Carrier-induced epitope-specific regulation and its bypass in a protein-protein conjugate. European Journal of Immunology 1995, 25,3375-3380. Lee, E. C., Powell, J. E., Tregear, G. W., Niall, H. D. and Stevens, V. C. A method for preparing beta-hCG COOH peptide-carrier conjugates of predictable composition. Molecular fmmunology 1980, 17, 749-756. Eldridge, J. H., Staas, J. K., Meulbroek, J. A., McGhee, J. R., Tice, T. R. and Gilley, R. M. Biodegradable microspheres as a vaccine delivery system. Molecular immunology 1991, 28, 287-294. O’Hagan, D. T., Jeffery, H., Roberts, M. J., McGee, J. P. and Davis, S. S. Controlled release microparticles for vaccine development. Vaccine 1991, 9, 768-771. Ho, P. C., Mutch, D. A., Winkel, K. D., Saul, A. J., Jones, G. L., Doran, T. J. and Rzepczyk, C. M. Identification of two promiscuous T cell epitopes from tetanus toxin. European Journal of Immunology 1990,20,477-483. Kaumaya, P. T., Kobs-Conrad, S., Seo, Y. H., Lee, H., VanBuskirk, A. M., Feng, N., Sheridan, J. F. and Stevens, V. Peptide vaccines incorporating a ‘promiscuous’ T-cell epitope bypass certain haplotype restricted immune responses and provide broad spectrum immunogenicity. Journal of Molecular kecognition 1993, 6, 81-94. LeVan. K. M. and Goldbera. E. Properties of human testisspecific lactate dehydrogelase expressed from Escherichia co/i. Biochemistry Journal 1991, 273, 587-592. Karr, R. W., Panina-Bordignon, P., Yu, W. Y. and Lanzavecchia, A. Antigen-specific T cells with monogamous or promiscuous restriction patterns are sensitive to different HLA-CR beta chain substitutions. Journal of immunology 1991, 146, 4242-4247. Panina-Bordignon, P., Tan, A., Termijtelen, A., Demotz, S., Corradin, G. and Lanzavecchia, A. Universally immunogenic T cell epitopes: promiscuous binding to human MHC class II and promiscuous recognition by T cells. European Journal of Immunology 1989,19,2237-2242. Goldberg, E. and O’Hern, P. A. Identification of cDNA clones reactive with sera from infertile patients. In Reproductive Immunology-Serono Symposia Publication from Raven Press (Eds Dondero, F. and Johnson, P. M.). Raven Press, New York, 1993, pp. 35-39 Stevens, V. C., Powell, J. E., Rickey, M., Lee, A. C. and Lewis, D. H. Studies of various delivery systems for a human chorionic gonadotropin vaccine. In Gamete Interaction: Prospects for lmmunocontracepfion (Eds Alexander, N. J., Griffin, D., Spieler, J. M. and Waites. G. M.H.). Wiley-Liss, New York, 1990, pp. 549-563 Men, Y., Thomasin, C., Merkle, H. P., Gander, B. and Corradin, G. A single administration of tetanus toxoid in biodegradable microspheres elicits T cell and antibody responses similar or superior to those obtained with aluminum hydroxide. Vaccine 1995, 13, 683-689.
PII: SO264-410X(97)00105-9? ELSEVIER
Colinear synthesis of an antigenspecific B-cell epitope with a 6promiscuous’ tetanus toxin T-cell epitope: a synthetic peptide immunocontraceptive Patricia A. O’Hern*, Zhi-Guo Liang”, Charanjit Goldberg*$
S. Barnbra? and Erwin
Carrier conjugation is commonly used to provide T-cell help for small, linear peptides containing antigen-specific B-cell epitopes. However; carrier conjugation is expensive, variable and often results in adverse side effects if the conjugate is administered repeatedly. To eliminate the need for carrier conjugation, we examined two synthetic peptides for their ability to elicit sustained antibody titres in female rabbits and baboons. One peptide (hCl-20) was based on the sequence of the sperm-specific isozyme of human lactate dehydrogenase (LDH-CJ. This peptide stimulates helper T-cell responses. The other peptide (bC519:TT) was a chimera between an LDH-C, B-cell epitope and a ‘promiscuous’ T-cell epitope from tetanus toxin which has been shown to bind to and stimulate many different major histocompatibility complex alleles. Both peptides were immunogenic in rabbits and baboons. The chimera elicited consistently high antibody titres and was immunogenic in 19119 wild-caught female baboons. When 14 bC.5-19:TT immunized baboons were mated, their fertility was reduced by 62% compared with controls (P~0.02). This cam’er-free construct can be incorporated into biodegradable microspheres which may provide long-tetm protection from pregnancy with a single dose. 0 1997 Elsevier Science Ltd. Keywords: immunocontraceptive,
T-cell
epitope,
lactate
dehydrogenase
A long-term goal of our research is the development of a safe, effective contraceptive vaccine for use by women. We have tested the sperm-specific isozyme of lactate dehydrogenase (LDH-C4) as our model antigen. In previous studies’, purified mouse LDH-C, was shown to suppress fertility in female mice, rabbits and baboons. The crystal structure of the protein was determined to 2.8 A resolution, and the B-cell epitopes of the antigen were mapped to four discrete regions on the surface of the LDH tetrame?. Computer-assisted sequence analysis was used to predict the immunodominant B-cell epitope of human LDH-C44. This peptide (hC9-20) was synthesized, conjugated to
*Center for Recombinant Gamete Vaccinogens and Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208, USA. tlnstitute of Primate Research, National Museums of Kenya, Karen, Naiobi, Kenya. $To whom correspondence should be addressed. el.: (847) 491-5416; fax: (847) 467-1380; e-mail: [email protected]. (Received 9 January 1997; revised version received 1 April 1997; accepted 2 April 1997)
diphtheria toxoid (DT) as a carrier molecule, and used as a contraceptive vaccine in female baboons. The fertility of the immunized animals was reduced by 70% compared with controls, and the contraceptive effect was reversed within a year of the last immunization. These results demonstrated the feasibility of using an LDH-&based vaccine for immunocontraception. However, carrier conjugation is neither desirable nor practical for general use. While providing a source of T-cell help for weakly immunogenic synthetic peptides, antigen specificity is sometimes altered by this procedure, and immune suppression or hypersensitivity due to repeated injection of carrier protein can occur’.‘. The conjugation chemistry is not trivialx, and the ratio of peptide to carrier protein varies from one batch to the next. Finally, most peptideicarrier conjugates are not stable to the organic solvents used in sustained release vaccine formulations”.“‘, or to extended storage at ambient temperatures. The goal of the present study was to avoid carrier conjugation by identifying one or more T-cell epitopes which could be incorporated into a completely synthetic peptide vaccine. This construct should be
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1997 Volume
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Carrier-free contraceptive vaccine in non-human primates: P.A. O’Hern et al. effective in a broad range of major histocompatibility complex (MHC) haplotypes and should significantly suppress fertility in non-human primates. We studied both an endogenous LDH-C, T-cell epitope (hCl-20), and a 39 amino acid chimeric peptide containing the B-cell epitope of baboon LDH-C4 (bC5-19) synthesized in tandem with a ‘promiscuous’ T cell epitope from tetanus toxin (TT580-599)“. The immunogenicity of these constructs in rabbits and baboons, and the suppression of fertility in female baboons immunized with the chimeric peptide construct are reported here.
MATERIALS
AND METHODS
Antigen preparation
Peptides hCl-20, hC9-20 and bC5-19:TT were synthesized at the Salk Institute (under Contract NOl-HD-0-2906 with the NIH) and made available by the Contraceptive Development Branch, Center for NICHD. The lyophilized Population Research, peptides were SO-90% pure as determined by high pressure liquid chromatography. The design of the chimeric peptide incorporating a four amino acid linker sequence between the B cell and T cell epitopes is essentially as described by Kaumaya et al.“.
Immunizations
and antibody titre determinations
Female rabbits (two per group) were immunized intradermally with 200 pg of peptide hCl-20, hC9-20 or bC5-19:TT emulsified in Freund’s Complete Adjuvant (FCA). Booster injections were administered intravenously 6 weeks later and consisted of 200 pg peptide in PBS. For immunogenicity testing, female baboons (five per group) were immunized i.m. with 400 pg peptide hCl-20 or bC5-19:TT or a combination of both peptides (200 /lg each) emulsified with Squalene:Arlace1 A (4:l) containing 200 L’g of the synthetic muramyl dipeptide CGP11637 (a gift of Ciba-Geigy Pharmaceuticals, Base], Switzerland) as adjuvant in a total volume of 0.50 ml. Booster immunizations administered i.m. on days 10 and 40 consisted of 200 Llg antigen and 100 pg adjuvant in 0.25 ml Squalene:Arlacel A. Antibody titres were determined by ELISA on microtitre plates coated with lo-’ M purified, recombinant human LDH-Cq13. Titre is defined as the reciprocal of the serum dilution that gives 0.5 absorbance units over background. Average tertiary titre is defined as the average ELISA titre for five individual bleeds taken during weeks 9-23 of the fertility trial (i.e. after the third injection of antigen).
horseradish peroxidase conjugated secondary antiserum (goat anti-human or goat anti-rabbit; Zymed, San Francisco, CA) diluted 1:50000 in blocking buffer, and developed with the enhanced chemiluminescent system following the manufacturer’s instructions (Amersham Life Sciences, Arlington Heights, IL).
Baboon fertility trial
Wild-caught female baboons (Papio cynocephalus, 15 per group) were housed in individual cages at the Institute of Primate Research, Nairobi, Kenya. The animals were allowed to acclimatize and resume cycling before the experiments began. Experimental animals were immunized i.m. with 400 pg peptide bC5-19:TT, 200 /lg adjuvant (CGPI 1637) emulsified with Squalene:Arlacel A as described above; control animals received vehicle and adjuvant only. Booster injections (200 /lg peptide, 100 pg adjuvant) were administered on days 10 and 70. Serum was collected every 2 weeks and tested for antibody titre by ELISA assay against purified recombinant human LDH-C,. Matings were begun during the first cycle following the third injection of antigen. Cohabitation with males was permitted for 5 days at the time of maximum sex skin turgescence when ovulation was predicted. This regime was followed for four cycles or until pregnancy was achieved. Statistical analyses were performed based on a one-way Gaussian analysis of variance (ANOVA). Two animals from the control group and one from the immunized group were eliminated from the trials because they stopped cycling after they had been acclimatized and immunized but before the first mating had occurred.
RESULTS Immunogenicity
testing
The amino acid sequences of the synthetic peptide vaccinogens are presented in Figure 1. Previously4 we predicted that hCl-20 (Figure Z(A)) contained T-cell helper activity. Detailed analyses of the human T-cell response to tetanus toxin”.‘4 indicated that TT contains
A. 1 STVK
B.
10
20
EOLIEKLIEDDENSQC
5
19
NH2-EQLIEKLIEDDKNSQ G Western blot analyses
Approximately 50 ,ug total protein from human sperm or liver, or 1 /lg purified, recombinant human LDH-C4 were resolved by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membranes, air dried and blocked in 0.1 M PBS containing 2% normal goat serum, 1% bovine serum albumin (BSA) and 0.2% Tween-20. The filters were incubated with appro-
priately diluted primary antiserum (baboon or rabbit),
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P
LS COOH-VSPFYSYYKKSNNLAADV Figure 1 Amino acid sequences of peptide immunogens. (A) Residues l-20 of human LDH-C, (hCl-20). The known B-cell epitope is underlined. (B) Chimeric peptide bC5-19:TT. Residues 5-19 of baboon LDH-C, were synthesized colinearly with a non-natural P-bend (GPSL) and a T-cell epitope from tetanus toxin (residues 580499). The known B-cell epitope is underlined; the T-cell epitope is shown in boldface type. Human and baboon LDH-C, differ at residue 16 (glutamic acid-lysine)
Carrier-free contraceptive vaccine in non-human primates: P.A. O’Hern et al. a booster injection was detected. Peptides hCl-20 and bC5-19:TT elicited strong and sustained antibody titres in both rabbits, with clear mnemonistic responses to a booster injection 40 days later. In addition, the tetanus toxin construct elicited antibody titres approximately one order of magnitude higher than did peptide hCl-20. We concluded that peptides hCl-20 and bC5-19:TT contain one or more T-cell epitopes which are recognized by rabbits, but peptide hC9-20 does not. We next tested the immunogenicity of peptides hCl-20 and bC5-19:TT in female baboons that were to be used in a fertility study. We immunized groups of five wild-caught female baboons with peptide hCl-20 or with peptide bC5-19:TT. To test whether these two antigenic peptides could act synergistically, we immunized a third group with a combination of both hCl-20 and bC5-19:TT. The results are presented in 7irhle I. Peptide hCl-20 was weakly immunogenic in female baboons. Peptide bC5-19:TT was strongly immunogenic in all five animals, particularly after the third injection, eliciting peak titres at least an order a magnitude higher than did peptide hCl-20. The combination of both peptides was not significantly better than the tetanus toxin construct alone, and certainly was not synergistic. Since peptide bC5-19:TT elicited moderate to good antibody titres and was effective in a broad range of genetic backgrounds, it was selected as the immunogen to use in fertility trials in female baboons.
several T-cell epitopes, termed ‘promiscuous’ epitopes, which are able to stimulate MHC class II responses in a wide variety of human haplotypes. We chose to test one such broadly-recognized T-cell epitope (IT residues 580-599, Figure Z(B)) for its ability to substitute for diphtheria toxoid as a component of our contraceptive vaccine. The B-cell epitope in this case was based on the baboon homologue of LDH-C,, which is identical to the human sequence except for a single amino acid substitution at position 16 (human Glu to baboon Lys)‘. Female rabbits (two per group) were immunized with either the B-cell epitope alone (hC9-20), the B-cell epitope synthesized colinearly with the putative LDH T-cell epitope (hCl-20) or the chimeric peptide containing the baboon B-cell epitope synthesized in tandem with the T-cell epitope from tetanus toxin (bC5-19:TT). The immune response of the rabbits is presented in Figure 2. All three peptides were able to elicit circulating antibodies which recognized purified, recombinant LDH-CJ by ELISA assay. Peptide hC9-20 elicited a strong response in one rabbit, and a weak response in the other rabbit. No secondary response to
Weeks
Post
Baboon fertility trial In the fertility trial with hC9-20:DT’, both control and treated animals were housed as a troop with males so that mating would occur naturally, presumably whenever a female reached mid-cycle. The present trial was modified by housing animals in individual cages during the trial. Female baboons cohabited with a male during the period of maximal sex skin turgescence at least 3 days prior to and 2 days after expected ovulation. Due to a limited number of cages, the fertility trial was performed in two stages. Group 1 consisted of eight immunized and seven control females. When the
Primory
Figure 2 Rabbit immune response to synthetic peptides. Two female rabbits were immunized with peptide hC9-20 (circles), peptide hCl-20 (triangles) or peptide bC5-19:TT (squares). Booster injections were administered in week 5 (arrows). Serum antibody titres were determined by ELISA on microtitre plates coated with purified, recombinant LDH-C,
Table 1
Baboon immune response to peptide vaccines Weeks following
the first injection
Animal 0
no. Vaccination 1766 1784 1706 1665 1663
with hC7-20
Vaccination
with X5-79:77
1435 1585 1657 1660 1659 Vaccination 1661 1662 1668 1616 1547
4
2
6
14
9
225 225 30 45 170
505 225 50 70 630
725 200 170 65 250
575 145 370 70 120
1960 1115 10 965 1544 2175
1960 485 8965 944 1775
760 140 2465 164 125
250 800 11 750 29 930 550
180 5400 2550 1530 640
100 1000 950 1530 210
0 0 0 0 0
75 35 20 15 0
0 65 0 655 30
0 575 -0 30
-
0 0 0 0 0
160 10 0 34 0
60 23 65 134 22
260 13 235 194 135
-
0 40 125 134 195
0 0 0 0 0
30 0 0 40 0
80 2800 310 490 470
100 1300 1350 1180 80
-
90 550 0 850 0
-
12
16
8 (third injection
with hCl-20+bC5-19:77
Vaccine 1997 Volume 15 Number 16
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Carrier-free contraceptive vaccine in non-human primates: P.A. O’Hern et al. breeding protocol was completed (approximately 6 months later), the second half of the study (Group 2: eight control and seven experimental animals) commenced. The kinetics of the immune response for three representative animals is shown in Figure 3. The magnitude and duration of the immune response differed significantly between individuals. Two of the animals (Nos. 1254 and 1705) produced a strong and rapid response to the vaccine, while animal 1.557 produced a slower and more modest response. As observed during the preliminary immunogenic& study, the serum titres declined rapidly after the tertiary injection, then stabilized at low but detectable levels for several months. As observed previously with hC9-ZO:DT, there was no correlation between serum antibody titre and infertility. One of the high responder animals (No. 1254) did not become pregnant, while another high responder animal (No. 1705) did. Furthermore, No. 1557, one of the lowest responder animals, was also protected from pregnancy.
‘OS5 10’ L
9)
E
4 wl i W
103
lo2
10’
10
i123450709
Months
Post
11
12
Primary
Figure 3 Immune response profile of three female baboons immunized with peptide bC5-19:lT. Three wild-caught female baboons were immunized with 400 pg of peptide C5-19:TT and 200 pg of CGPll637 on day 0. Booster injections were administered on days 10 and 70 (arrows). Cohabitation with males (male symbol) commenced on the first ovulatory cycle following the third injection and continued for four cycles or until pregnancy was established (P). (.), baboon 1254; l, baboon 1705; A, baboon 1557
Anrmal
While individual titres varied considerably from animal to animal, and from week to week, the average titre during the first 12 weeks following the tertiary injection was remarkably consistent. Figure 4 shows the average tertiary ELISA titre for all 14 experimental animals (one of the 15 original animals was removed from the study because she stopped cycling before the matings began). All 14 animals produced moderate to good antibody responses to the vaccine, and no significant titre differences between Groups 1 and 2 were apparent. No anti-LDH-C, titres were detected among the control animals (data not shown). For purposes of comparison, the average tertiary titre for the female baboons immunized in our previous study with hC9-20:DT is shown in Figure 5. With this construct, the average titre during the post-tertiary period was highly variable, ranging from less than 20 to over 3000, and four animals produced antibody titres which were barely above background. Matings were initiated during the first ovulatory cycle following the third injection of antigen. As shown in Table 2, lo/13 (77%) of the control animals and 4/14 (29%) of the experimental animals became pregnant, a 62% reduction in fertility. A one-way analysis of variance (ANOVA) showed this to be a statistically significant difference (PcO.02). Of the four pregnant animals in the experimental group, three miscarried during the first trimester (Figure 4). (The miscarriages are counted as pregnancies in the statistical analyses.) None of the control animals miscarried. The pregnancies and miscarriages were evenly distributed between Group 1 and Group 2 animals, and do not appear to be related to serum antibody titre. To determine whether the antibodies elicited by bC5-19:TT were specific for the Cq isozyme of lactate dehydrogenase, Western blots of human tissue extracts were probed with sera from immunized baboons. No reactivity was detected with baboon preimmune serum (F&ire 6). Serum from a pregnant/miscarried female detected a single 34 kDa band in human sperm and testes extracts, consistent with the size of LDH-C monomers, but did not react with human liver extracts (a rich source of the LDH-A, isozyme). As a control, rabbit anti-LDH-Ad did detect the A, isozyme in
Number
Figure 4 Immune response and fertility outcome of all female baboons immunized with peptide bC5-19:TT. Fifteen female baboons were randomly divided into two groups (Group 1, cross-hatched bars; Group 2, horizontally hatched bars) and injected three times with bC5-19:TT. The average ELISA titre during the first 12 weeks following the tertiary immunization was calculated for each individual and presented in rank order as lowest responder (left) to highest responder (right). Animals which conceived during the fertility trial are indicated by P (pregnant) or M (pregnant but miscarried)
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Vaccine 1997 Volume 15 Number 16
Carrier-free contraceptive vaccine in non-human primates: P.A. O’Hern et al.
7
Animal
Number
Figure 5 Immune response and fertility outcome of female baboons immunized with an hC9-20:DT conjugate. Thirteen female baboons were injected three times with peptide hC9-20 conjugated to diphtheria toxoid (hC9-20:DT). The average post-tertiary ELBA titre was calculated for each individual and presented as described in the legend to Figure 4. P, pregnant
liver. Therefore, lack of specificity of antibodies to the vaccine is not likely to be the cause of the observed miscarriages. human
DISCUSSION In the present study, we describe a completely synthetic immunocontraceptive vaccine (bC5-19:TT) which suppressed the fertility of wild-caught female baboons by 62% compared with controls (P10.02). The vaccine was immunogenic in 19 of 19 immunized animals, regardless of their genetic background. Peptide hCl-20 is also immunogenic in rabbits and baboons. FCA was used for the primary immunizations in rabbits, while the baboons received Squalene:Arlacel A containing a synthetic muramyl dipeptide as Table 2 Treatment
Anti-fertility group
Control Experimental
effect of bC519:TT No. of animals
No. pregnant
% pregnant
13 14
10 4a
77 29*
aP 2 0.02 b62% reduction
sp
c4
Li
sp
c4 Li
sp
c4 Li
kD - 84 -
5a
- 18 - 36 - 26 antiserum. Figure 6 Tissue specificity of baboon anti-bC5-19:lT Human sperm (Sp), human liver (Li) and purified, recombinant human LDH-C, (C4) were electrophoresed by SDS-PAGE, transferred to nitrocellulose filters and probed with baboon preimmune serum (left panel), baboon anti-bC5-19:m (centre panel), or rabbit anti-human LDH-A, (right panel). The position of the molecular weight standards is shown on the right
adjuvant. When rabbits are immunized with synthetic peptide antigens emulsified in Squalene:Arlacel A containing CGPll637, the resulting serum antibody titres are still of the same order of magnitude as those observed here with FCA as adjuvant (O’Hern and Goldberg, unpublished data). The immune response to the chimeric peptide was remarkably consistent among all the animals studied, particularly after the third injection. Since one of the major challenges in developing synthetic peptide-based vaccines is to elicit an effective immune response regardless of the MHC haplotype of the individual, this result is very encouraging. Certainly, the immune response to bC5-19:TT is more robust and consistent than was the response to the hC9-20:DT construct (cf. Figure 4 and Figure 5). Although the tetanus toxin epitope used is known to be effective in a broad range of haplotypes, it is not universally antigenic “,‘5. Since peptide hCl-20 does contain at least one T-cell epitope which is recognized by rabbits and baboons (Figure 2, Table I), it is possible that bC519:TT contains an antigen-specific T-cell epitope in addition to the promiscuous T-cell epitope. This combination may result in a more effective vaccine than would the TT epitope alone. It is important to note that three of the four pregnant experimental animals miscarried during the first trimester; two from Group 1 and one from Group 2 (Figure 4). None of the ten pregnant control animals miscarried. This is the first time the authors have observed miscarriages among baboons immunized with LDH-C, or its peptides. However, in the previous trial with hC9-20:DT, the animals were housed as a troop in a single large cage, and spontaneous abortions may not have been detected. Western blots using serum from one of these animals did not reveal any non-specific or autoantigenic immune reactivity (Figure 6), nor did the serum recognize the tetanus toxin T-cell epitope itself by ELBA assay (data not shown). The authors now have a single, synthetic peptide which is highly immunogenic without carrier conjugation, regardless of the MHC haplotype of the recipient. Immunization with this antigen significantly reduces fertility (62% reduction; P < 0.02) in non-human
Vaccine 1997 Volume 15 Number 16
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Carrier-free contraceptive vaccine in non-human primates: P.A. OHem et al. primates. Experiments are in progress to incorporate bC5-19:TT into biodegradable polylactide/polyglycolide microspheres’0.‘h.‘7. A single dose of microspheres containing the carboxy-terminal peptide of human chorionic gonadotrophin conju ated to diphtheria toxoid” or tetanus toxoid alone’ 5 has been shown to elicit sustained T-cell and antibody responses equal to or greater than those obtained with three standard injections. A similar strategy could greatly improve the efficacy and acceptability of a contraceptive vaccine.
6
7
8
9
ACKNOWLEDGEMENTS This work was supported by NIH Sub-5-U54-HD29099, by P30HD28048, and by the Contraceptive Research and Development Program (CSA-92-099) under a Cooperative Agreement with the US Agency for International Development (U.S.A.I.D.) (DPE-3044-A00-6063-00) which in turn receives funds for AIDS research from an interagency agreement with the National Institute of Child Health and Human Development. The views expressed by the authors do not necessarily reflect the views of U.S.A.I.D. and CONRAD.
REFERENCES Goldberg, E. Lactate dehydrogenase C, as an immunocontraceptive model. In Gamete Interaction: Prospects for lmmunocontracepfion (Eds Alexander, N. J., Griffin, D., Spieler, J. M. and Waites, G. M. H.). Wiley-Liss, New York, 1990, pp. 63-73 Hogrefe, H. H., Griffith, J. P., Rossmann, M. G. and Goldberg, E. Characterization of antigenic sites on the refined 3-A resolution structure of mouse testicular lactate dehydrogenase-Cd. Journai of Biological Chemistry 1987, 262, 13155-13162. Hogrefe, H. H., Kaumaya, P. T. P. and Goldberg, E. Immunogenicity of synthetic peptides corresponding to flexible and antibody-accessible segments of mouse lactate dehydrogenase (LDH-C,). Journaf of Biofogical Chemistry 1989, 264, 10513-10519. O’Hern. P. A. and Goldberg, E. The use of molecular modelling to delineate B-cell and T-cell epitopes of human spermspecific LDH-C,. In Techniques in Protein Chemistry IV (Ed Angelette, R. H.). Academic Press, New York, 1993, pp. 481-490 O’Hern, P. A., Bambra, C. S., Isahakia, M. and Goldberg, E. Reversible contraception in female baboons immunized with a synthetic epitope of sperm-specific lactate dehydrogenase. Biology of Reproduction 1995, 52, 331-339.
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Schutze, M. P., Leclerc, C., Jolivet, M., Audibert, F. and Chedid, L. Carrier-induced epitopic suppression, a major issue for future synthetic vaccines. Journal of immunology 1985,135,2319-2322. Kaliyaperumal, A., Chauhan, V. S., Talwar, G. P. and Raghupathy, R. Carrier-induced epitope-specific regulation and its bypass in a protein-protein conjugate. European Journal of Immunology 1995, 25,3375-3380. Lee, E. C., Powell, J. E., Tregear, G. W., Niall, H. D. and Stevens, V. C. A method for preparing beta-hCG COOH peptide-carrier conjugates of predictable composition. Molecular fmmunology 1980, 17, 749-756. Eldridge, J. H., Staas, J. K., Meulbroek, J. A., McGhee, J. R., Tice, T. R. and Gilley, R. M. Biodegradable microspheres as a vaccine delivery system. Molecular immunology 1991, 28, 287-294. O’Hagan, D. T., Jeffery, H., Roberts, M. J., McGee, J. P. and Davis, S. S. Controlled release microparticles for vaccine development. Vaccine 1991, 9, 768-771. Ho, P. C., Mutch, D. A., Winkel, K. D., Saul, A. J., Jones, G. L., Doran, T. J. and Rzepczyk, C. M. Identification of two promiscuous T cell epitopes from tetanus toxin. European Journal of Immunology 1990,20,477-483. Kaumaya, P. T., Kobs-Conrad, S., Seo, Y. H., Lee, H., VanBuskirk, A. M., Feng, N., Sheridan, J. F. and Stevens, V. Peptide vaccines incorporating a ‘promiscuous’ T-cell epitope bypass certain haplotype restricted immune responses and provide broad spectrum immunogenicity. Journal of Molecular kecognition 1993, 6, 81-94. LeVan. K. M. and Goldbera. E. Properties of human testisspecific lactate dehydrogelase expressed from Escherichia co/i. Biochemistry Journal 1991, 273, 587-592. Karr, R. W., Panina-Bordignon, P., Yu, W. Y. and Lanzavecchia, A. Antigen-specific T cells with monogamous or promiscuous restriction patterns are sensitive to different HLA-CR beta chain substitutions. Journal of immunology 1991, 146, 4242-4247. Panina-Bordignon, P., Tan, A., Termijtelen, A., Demotz, S., Corradin, G. and Lanzavecchia, A. Universally immunogenic T cell epitopes: promiscuous binding to human MHC class II and promiscuous recognition by T cells. European Journal of Immunology 1989,19,2237-2242. Goldberg, E. and O’Hern, P. A. Identification of cDNA clones reactive with sera from infertile patients. In Reproductive Immunology-Serono Symposia Publication from Raven Press (Eds Dondero, F. and Johnson, P. M.). Raven Press, New York, 1993, pp. 35-39 Stevens, V. C., Powell, J. E., Rickey, M., Lee, A. C. and Lewis, D. H. Studies of various delivery systems for a human chorionic gonadotropin vaccine. In Gamete Interaction: Prospects for lmmunocontracepfion (Eds Alexander, N. J., Griffin, D., Spieler, J. M. and Waites. G. M.H.). Wiley-Liss, New York, 1990, pp. 549-563 Men, Y., Thomasin, C., Merkle, H. P., Gander, B. and Corradin, G. A single administration of tetanus toxoid in biodegradable microspheres elicits T cell and antibody responses similar or superior to those obtained with aluminum hydroxide. Vaccine 1995, 13, 683-689.