Antigenic and immunogenic properties of totally synthetic peptide-based anti-fertility vaccines
Souravi Ghosh and
David C. Jackson
Co-Operative Research Centre for Vaccine Technology, Department of Microbiology and Immunology, University of Melbourne, Parkville 3052, Victoria, Australia
Correspondence to:
D. C. Jackson
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Abstract
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In this study we describe the results of experiments in which a variety of totally synthetic luteinizing hormone releasing hormone (LHRH) vaccines were assembled and examined for their abilities to elicit antibody responses and induce sterility in mice. It is shown that totally synthetic vaccines consisting of a 15 residue-defined T cell epitope and the 10 residue LHRH epitope not only induced high titers of antibody but also induced sterility. This effect did not appear to correlate with antibody titer, antibody isotype or comparative antibody affinity, but may be related to the length of time for which antibodies are present to exert their influence.
Keywords: immunocastration, luteinizing hormone releasing hormone, synthetic vaccine
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Introduction
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Luteinizing hormone releasing hormone (LHRH) is a peptide hormone 10 amino acids in length which is secreted by the hypothalamus. It initiates a cascade of events leading to the control of reproduction in males and females of most mammals in which the sequence is conserved. LHRH is not immunogenic on its own, but when animals are immunized with LHRH conjugated to a carrier protein they produce anti-LHRH antibodies and animals producing high titers of antibodies become sterile (1,2). Because LHRH holds enormous potential in the design of immunocastration vaccines, it is one of the most extensively studied molecules as an anti-fertility agent (
37).
The choice of appropriate carrier proteins is very important in designing peptide vaccines, and their selection is limited by factors such as toxicity and feasibility of their large-scale production for in vivo use. There are other limitations to this approach including the peptide load that can be coupled and the dose of carrier that can be safely administered (8). Although carrier molecules allow the induction of a strong immune response they are also associated with undesirable effects such as suppression of the anti-peptide antibody response (911). To avoid the problems associated with the use of carrier proteins, the concept of using defined Th epitopes in vaccines directed against other targets has been investigated (12,13). Among Th epitopes that have been identified in other systems are those that have been reported not to be restricted to a single MHC haplotype (1417). Theoretically, such a promiscuous helper epitope could serve as a source of T cell help in an outbred population.
The use of defined Th epitopes which do not themselves induce significant antibody responses has the attraction that carrier-induced epitope suppression of antibody responses may be avoided. In this study we therefore assembled a number of totally synthetic vaccines based on LHRH and containing defined Th epitopes, some of which have been reported to be promiscuous for a variety of species and strains. Another epitope used was chosen because it is a very potent Th epitope and it is restricted by MHC class II to a particular strain of mouse. Previous work with LHRH has indicated that the orientation of LHRH in carrier proteinLHRH conjugates is important for eliciting antibodies to LHRH (18,19), and our earlier studies have demonstrated that the geometry and valency of attachment of defined T cell and B cell epitopes has an effect on the magnitude of the immune response (20). The immunogens were therefore assembled in a variety of different configurations in order to study the effect that the geometry of assembly and the orientation of the Th epitope and LHRH have on the immunogenicity and antigenicity of totally synthetic peptide-based immunocontraceptive vaccine candidates.
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Methods
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Synthetic peptide immunogens
Peptides and peptide-based immunogens were assembled in the solid phase using Fmoc chemistry either manually or in a Milligen 9050 Plus automatic peptide synthesizer as described by Fitzmaurice et al. (20). PAL PEG PS resin (PerSeptive Biosystems, Hamburg, Germany; substitution 0.15 mmol/g) or Novasyn KR 100 resin (Calbiochem-Novabiochem P/L, Alexandria, Australia) was used for the syntheses to yield the carboxamide form of the peptide. Side chain protecting groups were: Arg, PMC; Ser, tBu; Gln, Trt; Asn, Trt; Lys, Boc or Dde; Glu, Otbu; Tyr, tbu; H, Trt; Trp, Boc. All amino acids were incorporated as the free acid in the presence of equimolar amounts of O-benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBt) and two equivalents of diisopropylethylamine (DIPEA). Acylation was carried out for 30 min and each cycle monitored at 365 nm in the 9050 Plus synthesizer or by the trinitrobenezene sulfonic acid test (21) if syntheses were done manually.
Branched synthetic immunogens were assembled according to the method reported earlier (20). Briefly, one or two residues of the orthogonally protected amino acid Fmoc-lysine-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene ethyl) [Fmoc lys(Dde)] was incorporated at the C-terminus. The Fmoc group was then removed and the sequence of T4, ALNNRFQIKGVELKS, assembled followed by acetylation of the N-terminal alanine using N-acetylimidazole. The Dde groups present on the
amino group(s) of the lysine residues were then removed with 2.5% anhydrous hydrazine in dimethylformamide, thereby exposing the amino groups and allowing the LHRH peptide sequence to be assembled at this point.
Peptides were cleaved from the resin and side chain protecting groups simultaneously removed by treatment with 88% TFA containing 5% phenol, 2% tri-isopropylsilane and 5% water. Crude peptides were precipitated and washed in cold diethyl ether, and purified by reverse phase chromatography using a C4 PepRPC column (1.6x10 cm) installed in an FPLC system (Pharmacia, Uppsala, Sweden). Chromatograms were developed in a gradient of 0.1% TFA in water with 0.1% TFA in acetonitrile as the limit solvent. Appropriate fractions were collected, and the purity and authenticity of peptides assessed by analytical reverse-phase chromatography using a C4 Vydac column (0.46x25 cm) installed in a Waters HPLC system. Peptides were also examined by mass spectrometry.
The immunogens assembled using these methods are represented schematically in Fig. 1
: T1 is a reportedly promiscuous Th epitope (22) from tetanus toxin and has the sequence FNNFTVSFWLRVPKVSASHLE; T2 and T3 are Th epitopes from a membrane protein of Escherichia coli which have been reported to stimulate T cells obtained from different species of animals, their sequences are STETGNQHHYQTRVVSNANK and GLQGKIADAVKAKG respectively (23); T4 is a Th epitope from the light chain (HA2) of influenza virus hemagglutinin (24) and has the sequence ALNNRFQIKGVELKS. LHRH has the sequence EHWSYGLRPG.

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Fig. 1. Sequences of Th epitopes, T1, T2, T3 and T4, and schematic representation of vaccine candidates used. Th epitopes, T4 and T2, were assembled in various configurations with LHRH (EHWSYGLRPG); immunogen 1 is a linear tandem construct with T4 at the N-terminus and LHRH at the C-terminus; immunogen 2 is a linear tandem construct of the same epitopes but with T4 at the C-terminus; immunogen 3 contains one copy of each of T4 and LHRH in a branched orientation; and immunogen 4 contains a single copy of T4 and two copies of LHRH in a branched orientation. In the case of immunogens 3 and 4, the C-terminus of LHRH is occupied in covalent linkage to the branching lysine residue. Immunogen 5 is a linear construct of T2 and LHRH. In these cases where the C-terminus of LHRH is a free carboxamide group this is represented as LHRH-CONH2.
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Immunization of mice
Female BALB/c (H-2d) mice were immunized with each of the peptide immunogens at a dose equivalent to 20 nmol of LHRH for antibody studies. Peptide-based, immunogens were emulsified in complete Freund's adjuvant (CFA) and injected s.c. at the back of the hind leg for the primary immunization. Secondary inoculations were carried out after 4 weeks using immunogens emulsified in incomplete Freund's adjuvant and injected by the same route. For T cell studies, mice were inoculated s.c. in the hind footpads with a total of 20 nmol of T cell determinant emulsified in CFA.
T cell clone 12V1
The derivation, properties and maintenance of 12V1 which proliferates in response to peptide T4 have been described elsewhere (24).
T cell proliferation assays
T cells obtained from popliteal and inguinal lymph nodes were enriched using nylon wool (25). An enriched population of T cells, 3x105 or 104 cells of T cell clone 12V1, was incubated in the presence of syngeneic
-irradiated (2200 rad, 60Co source) spleen cells (3x105) as a source of antigen-presenting cells together with antigen in 96-well microtiter plates. The total volume of culture medium was 250 µl/well. The culture medium consisted of RPMI supplemented with 10% heat-inactivated FCS, 2 mM glutamine, 2 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, 30 µg/ml gentamycin, 100 IU/ml penicillin and 100 µg/ml streptomycin. Cells were cultured for 4 days at 37°C in an atmosphere which was 5% in CO2. [3H]Thymidine, 1 µCi/well, was added during the last 18 h of the culture period, and cells were then harvested onto glass fiber filters and the level of cellular thymidine incorporation measured using a Hewlett Packard Matrix 9600 direct ß-counter.
ELISA
These assays were carried out essentially as described by Brown et al. (26). Briefly, polyvinyl flat-bottom microtiter plates (Dynex Technologies Inc., VA) were coated with 50 µl/well of a solution of antigen (5 µg/ml) in PBS, pH 7.3, for 1820 h at room temperature in a humidified atmosphere. Antigen was then removed from the wells and 100 µl of a solution (10 mg/ml) of BSA in PBS added for 1 h. Plates were washed with PBS/Tween-20 (0.05%) and then 50 µl of serial dilutions of individual sera obtained from immunized mice added to the wells. Following overnight incubation at room temperature in a humidified atmosphere the sera were removed and the plates washed with PBS/Tween-20 and 50 µl of a 1/400 dilution of horseradish peroxidase-conjugated rabbit antibody directed against mouse IgG (Dako, Glostrup, Denmark), in BSA (5 mg/ml), PBS/Tween-20 (0.05%) added. After 1 h at room temperature the plates were washed and 100 µl of enzyme substrate (0.2 mM 2,2'-azino-bis 3-ethylbenzthiazoline-sulfonic acid in 50 mM citric acid pH 4.0 containing 0.004% v/v hydrogen peroxide) added. The absorbance of the solutions was then determined at a wavelength of 405 nm using a Labsystems Multiscan Multisoft microplate reader (Pathtech Diagnostics, Melbourne, Australia). Antibody titers were expressed as the reciprocal of that serum dilution which gave an absorbance 3 times that obtained in wells lacking antisera but containing all other components.
Determination of relative avidity of antibodies by ELISA
This procedure was similar to that described by Macdonald et al. (27). Following incubation of sera with antigen, plates were washed and 50 µl aliquots of serial dilutions of ammonium thiocyanate in 0.1 M phosphate buffer, pH 7.0, added to cover the range 09 M. The plates were allowed to stand for 15 min at room temperature before washing 3 times with PBS/Tween-20. The assay was then developed as described for the ELISA.
Estimation of antibody isotype
This was done essentially as described for the ELISA except that following incubation of antigen with antisera and washing, rabbit antisera directed either to mouse IgM, IgG1, IgG2a, IgG2b, IgG3 or IgA (ICN Pharmaceuticals, Costa Mesa, CA) were added and plates incubated for 2 h. After washing, 50 µl (1/400 dilution) horseradish peroxidase-conjugated swine antibody directed to rabbit Ig was added. The assay was then completed as described for the ELISA.
Fertility studies in mice
Female mice immunized with peptide immunogens were tested for their ability to reproduce by mating with syngeneic male mice. A single male mouse was introduced to a cage containing two or three females. Male mice were rotated between the females such that each group of females was exposed to each male mouse. Males were kept with females for a total of 3 weeks at the end of which time males were removed and females kept under observation.
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Results
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Evaluation of Th epitopes in a murine model
We examined the various Th epitopes for their ability to induce T cell proliferation as a prelude to their selection for incorporation into LHRH vaccines. The reportedly promiscuous Th epitopes T1 from tetanus toxin (22), T2 and T3 from outer membrane protein of E. coli (23), and also a potent murine H-2d restricted Th epitope T4 (24) were synthesized and tested for their T cell stimulatory activities in BALB/c mice. The T cell stimulatory activities of the Th epitopes measured as proliferation of T cells is shown in Fig. 2
. It can be seen that the H-2d-restricted Th epitope, T4, showed the highest activity followed by T1 and T2 respectively. T3 showed little or no detectable stimulatory effect on H-2d murine T cells.
Antibody responses to immunogens containing LHRH and the T4 epitope
The Th epitope T4 exhibited the highest T cell stimulatory activity and was therefore incorporated with LHRH into peptide vaccine candidates. The immunogens (14, Fig. 1
) were assembled in various configurations each containing at least one LHRH and one Th cell epitope, and their abilities to elicit antibodies to LHRH examined.
Groups of BALB/c mice which had received the various peptide immunogens were bled periodically and sera assayed individually for the presence of anti-LHRH antibodies. Only those mice immunized with immunogen 1 produced high titers of anti-LHRH antibodies in the primary response; sera obtained from the same animals 2 weeks after the secondary immunization showed a marked increase in anti-LHRH antibody titers (Fig. 3
). The group of mice which received immunogen 4 produced significant but much lower titers of anti-LHRH antibodies and only following a second dose of immunogen. Immunogens 2 and 3 did not generate any significant amounts of anti-LHRH antibodies.

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Fig. 3. Elicitation of antibodies by peptide immunogens based on T4 and LHRH. Groups of five mice received peptide immunogens equivalent to 20 nmol of LHRH, and were bled 4 weeks following the first inoculation and again 2 weeks following the second immunization. The sera were tested for the presence of antibodies to LHRH and also to T4 by ELISA. The antibody titers are expressed as the difference of the reciprocal of the dilution of antisera raised against the immunogen and that raised against the formulation in the absence of immunogen. The results are shown as the mean of antibody titers directed to LHRH or T4 obtained from the first (shaded bars) and second (solid bars) bleeds respectively. A paired two tailed t-test was also carried out and the results shown as P values.
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Immunogens 1, 2 and 4 also elicited anti-T4 antibodies during the primary response. Although this titer increased during the secondary response in those groups which received immunogens 2 or 4, no increase in anti-Th epitope antibody was apparent in mice inoculated with immunogen 1 during the secondary response.
The anti-LHRH antibody titers of sera obtained from each animal were examined again at 12 and 24 weeks following the secondary immunization (Table 1
). Although there was a drop in anti-LHRH titers in all groups 12 weeks following the second inoculation, there was no further decrease in antibody titers up to and including week 24.
T cell proliferative responses in mice immunized with peptide immunogens
The above results clearly indicate that totally synthetic peptide immunogens can elicit and maintain a lasting antibody response to the epitope of choice in mice but in the case of combinations of T4 and LHRH which were incorporated in different formats, the anti-LHRH antibody responses they elicited varied significantly. The best of these was the linear construct, immunogen 1. In order to determine if the differences in immunogenicity could be attributed to the activity of the individual immunogens' abilities to recruit T cells these immunogens were examined in T cell proliferation assays.
The results of these experiments (Fig. 4
) show that of all those tested, immunogens 1 and 3 were better T cell stimulators than the original T4 epitope itself. Immunogen 2 was just as efficient as the helper epitope alone but the ability of immunogen 4 to stimulate T cells was the least of all.
A T cell clone, 12V1, specific for T4 was also used to test the antigenicity of peptide immunogens 14. The clone proliferated equally well when presented with T4 alone or any of the chimeric immunogens tested (data not shown).
Antibody responses to LHRH immunogens incorporating promiscuous Th epitopes
We have demonstrated that it is possible to elicit high titers of anti-LHRH antibodies with immunogens where carrier proteins are replaced with Th epitopes. The advantages of using a promiscuous T cell epitope therefore prompted us to examine the immunogenic activity of T2 incorporated into a linear tandem format (immunogen 5). Our decision to pursue T2 and not the apparently stronger T1 helper epitope was based on the fact that T2 has been reported to be promiscuous for a larger number of different animal species than is T1. Because one long-term goal for an immunocontraceptive vaccine would be to cover the animal universe, we felt that an examination of a more promiscuous epitope was warranted. As shown in Fig. 5
, no significant antibody directed to LHRH was induced although antibody directed to the Th epitope was elicited and in fact the anti-LHRH response was apparently suppressed in the secondary response. These results indicate that this promiscuous T cell epitope can have a deleterious effect on the required immune response when associated with the B cell epitope of interest.

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Fig. 5. Antibody responses of mice to LHRH and T2. Mice immunized with 20 nmol of immunogen 5 were bled 4 and 2 weeks after the first and second inoculation respectively. Sera were tested for the presence of anti-LHRH and anti-T2 antibodies by ELISA. The results are presented as the mean of five individual sera. Antibody titers to LHRH and T2 after the first (shaded bars) and second bleeds (solid bars) are expressed as the difference of the reciprocal of the dilution of antisera raised against the immunogen and that raised against the formulation without the immunogen. A two-tailed paired t-test was used to determine if the differences between the two bleeds were significant.
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Fertility status of mice immunized with immunogens containing LHRH and T4
Mice immunized with each of the four peptide immunogens containing T4 and LHRH in various orientations were tested for their ability to produce litters at different intervals following either one or two inoculations of immunogen and also for the anti-LHRH titers of antibody present in sera. Mice were mated 2, 12 and 24 weeks after the second immunization, and the results obtained are shown in Table 1
. Where litters were produced, they were apparently normal with the litter size varying from three to eight.
Inoculation with immunogen 1 prevented pregnancies in all mice receiving two doses of the vaccine. Even 24 weeks after receiving the second dose of immunogen, no litters were dropped. Immunogens 2 and 4 also prevented pregnancies 2 weeks following the second dose of vaccine but by 12 weeks most animals in these groups had dropped litters indicating a wane in efficacy with time. Immunogen 3 appeared to have little or no immunocontraceptive effect.
To validate these observations, another trial with a larger number of mice was undertaken. Two groups of female mice with 10 animals in each group were either immunized with immunogen 1 or with saline. First and second inocula were administered 4 weeks apart and 2 weeks later the mice were mated. No litters were produced by mice which received the immunogen, confirming that immunogen 1 induces high levels of anti-LHRH antibodies and renders the animals infertile.
Because the antibody titers to LHRH decreased over a 24 week period to levels comparable with those obtained after primary immunization, we carried out a study to determine if a single dose of immunogen 1 would have an immunocontraceptive effect.
Four weeks after receiving a single inoculation of immunogen 1, sterility was not achieved in all the animals despite the fact that the mean anti-LHRH antibody titer of 4.5 was comparable with those titers associated with complete sterility prevailing during the secondary responses (Table 1
). Because the apparent lack of correlation between titer of anti-LHRH antibodies and status of fertility observed during the primary and secondary antibody responses could be due to qualitative differences in the antibodies produced, we compared the titers of individual antibody isotypes directed towards LHRH and also compared the avidities of anti-LHRH antibodies. The results of the antibody isotype studies (Fig. 6
) indicate that the most significant differences between isotype titers present in the primary and secondary bleeds occurs with IgG1 and IgG2b.

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Fig. 6. Isotypes of anti-LHRH antibodies present during primary and secondary antibody responses. Mice were immunized with 20 nmol of immunogen 1 on two occasions, and sera obtained from mice 4 (open circles) and 12 (solid circles) weeks after the first and second inoculations respectively. The isotypes of anti-LHRH antibodies generated were then determined by ELISA. Each panel shows the isotype titer of sera obtained from individual mice. A two-tailed paired t-test was used to determine P values signifying any differences between the primary and secondary bleeds of each isotype.
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The relative avidities of anti-LHRH antibodies in mice inoculated with immunogen 1 were also examined to investigate if higher affinity antibodies were generated following the second dose of vaccine. There was no significant difference in the avidities of antibodies directed to LHRH obtained after one or two doses of immunogen 1 (Fig. 7
).

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Fig. 7. Affinity of anti-LHRH antibodies. Sera from 10 mice immunized with immunogen 1 obtained 4 weeks after the first immunization and 12 weeks after the second immunization were examined for their binding avidity to LHRH in a modified ELISA. The results are expressed as that concentration of ammonium thiocyanate required to disrupt antibody binding to LHRH. Any significance in differences between affinities obtained from the two bleeds was determined by a paired two-tailed t-test.
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Discussion
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We have synthesized a series of simple peptide immunogens based on LHRH and defined T
h epitopes. Of the Th epitopes examined, a 15 residue sequence (T4) restricted by I-Ed gave the best T cell proliferative response in BALB/c mice and the reportedly promiscuous epitopes elicited poor proliferation of T cells. Subsequent examination of the immunogenic and antigenic properties of vaccine candidates incorporating T4 and LHRH assembled in various geometries demonstrated that even though each of the immunogens contained a copy of the same Th epitope, some exhibited better T cell stimulatory activities than others. Immunogens 1 and 3, with T4 at the N-terminus of the construct and a single copy of LHRH, elicited greater T cell proliferation than the T4 alone. Immunogens 1 and 2 differ only in the relative orientation of the T and B cell epitope, and have quite different immunogenic properties indicating that the polarity of the Th epitope with respect to LHRH has an important effect on immunogenicity, a fact which has been reported for other immunogen systems (2831).
The anti-LHRH antibody responses elicited by the different immunogens appeared to be at least partly dependent on whether the C-terminus of LHRH was free or involved in covalent linkage rather than on the polarity of LHRH with respect to the Th epitope. This is clearly demonstrated by immunogens 1 and 3, both of which have LHRH C-terminal to T4 and both of which exhibited high T cell stimulatory activities, but only immunogen 1 which contains LHRH with a free C-terminal carboxyamide group induced high titers of anti-LHRH antibodies. These results support those reported by Ladd et al. (19) where immunogens incorporating LHRH with a free C-terminus were shown to be more efficient in generating high titers of neutralizing anti-LHRH antibodies. In the present study, however, we have shown that this non-responsive status is overcome to some extent by incorporating two copies of LHRH in the `wrong' orientation. Although mice in this group did not demonstrate detectable anti-LHRH antibodies during the primary response significant titers of antibodies were elicited after the secondary immunization.
Immunogen 2 proved to be the least efficient vaccine demonstrating low T cell responses and minimal levels of anti-LHRH antibodies; this immunogen not only contains LHRH in which the C-terminus is involved in covalent linkage but the Th epitope is C-terminal to LHRH. Although orientation and polarity affect immunogenicity, generic rules may not be applicable to immunogen design, e.g. the finding that branched immunogens are better than linear immunogens (20) does not appear to be the case here.
One of the major problems associated with coupling peptides to proteins is the phenomenon of carrier-induced epitope suppression (9,10). If such suppression is due to antibody being directed to the carrier in preference to the B cell epitope of interest, it is possible that use of T cell epitopes which do not elicit antibody would be an advantage. Although the best vaccine candidate, immunogen 1 described here, generated significant amounts of anti-T4 antibody in the primary response, titers were lower than those directed to LHRH. Furthermore following the second immunization, anti-T4 antibody titer did not increase despite a 10-fold increase in anti-LHRH antibody titer. This finding is in contrast to observations with LHRHprotein conjugates (32) and also the results we observed with immunogens 5 and 2 in which anti-T2 and anti-T4 antibody titers respectively were higher than those generated against LHRH. The ability of a Th epitope to provide help without diverting antibody responses towards itself should be regarded as one of the biggest advantages of using defined Th epitopes in vaccine candidates. Moreover, such an immunogen design approach allows us to manipulate the properties of synthetic immunogens so as to direct antibody response towards the B cell epitope of interest in preference to the T cell epitope.
Although a comparison of the antibody induced during primary and secondary responses indicated that there may be a correlation between anti-LHRH antibody titers and infertility, litters were nevertheless produced by animals inoculated with a single dose of immunogen 1 which elicited antibody titers as high as those observed in animals following a secondary antibody response and which were infertile. We are unable to say whether the differences that we observed in IgG1 (P = 0.01) and IgG2b (P = 0.02) titers between the primary and the secondary antibody responses are significant in terms of the biological effect of the immunogen, but Meloen et al. (33) were unable to correlate infertility in piglets with antibody titer following inoculation with LHRHprotein vaccine candidates. Our inability to correlate efficacy with antibody avidity also indicates that the efficacy of this anti-fertility vaccine is related to other effects rather than simply antibody quality; such factors may include, for example, the length of time for which the endocrine system is bathed by antibody.
Sad et al. (34) and Ferro and Stimson (35) have reported the induction of anti-LHRH antibodies using a totally synthetic peptide immunogen consisting of Th epitope and LHRH; furthermore, Ferro and Stimson (35) present evidence that a synthetic antigen causes a decrease in testicle size. This current report demonstrates for the first time that a completely synthetic vaccine not only elicits high antibody titers to LHRH but also sterilizes animals for at least 6 months. It seems likely that such totally synthetic vaccines may have advantages in quality control and in avoiding carrier-induced epitope suppression.
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Abbreviations
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CFA | complete Freund's adjuvant |
LHRH | luteinizing hormone releasing hormone |
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Notes
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Transmitting editor: A. Kelso
Received 22 February 1999,
accepted 25 March 1999.
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