Enhanced immunogenicity of a contraceptive vaccine using diverse synthetic carriers with permissible adjuvant

Enhanced immunogenicity of a contraceptive vaccine using diverse synthetic carriers with permissible adjuvant
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  Vaccine 19 (2001) 3384–3389 Enhanced immunogenicity of a contraceptive vaccine using diversesynthetic carriers with permissible adjuvant Anushree Gupta  1 , Rahul Pal, Sarita Ahlawat, Prakash Bhatia, Om Singh * Immunoendocrinology Laboratory ,  National Institute of Immunology ,  New Delhi   110067  ,  India Received 27 July 2000; received in revised form 19 February 2001; accepted 27 February 2001 Abstract A vaccine directed against human chorionic gonadotropin (hCG) has previously undergone clinical testing demonstrating thefeasibility of the approach in preventing pregnancy in women. Some individuals, however, did not respond adequately despiteemploying highly immunogenic bacterial toxoids as carriers. We investigated the potential of three promiscuous pathogen-derivedTh peptides as carriers, employing alum as the adjuvant. While conjugation with each peptide improved the antibody responseagainst hCG in mice of different haplotypes, immunisation with a combination of these peptide-conjugates generated anti-hCGresponses higher than those achieved with the individual peptides or tetanus toxoid (TT). Antibodies were of high affinity andcapable of neutralising the bioactivity of hCG but were devoid of anti-peptide reactivity. These results have implication for thedesign of hCG vaccine with improved immunogenicity for diverse population. © 2001 Published by Elsevier Science Ltd. Keywords :   Human chorionic gonadotropin; Immunocontraception; Th / locate / vaccine 1. Introduction Human chorionic gonadotropin (hCG) is a hormonethat is produced by the peri-implantation embryo andplays a critical role in the establishment and mainte-nance of early pregnancy. Chemical linkage to a proteincarrier has been used as a strategy to overcome im-munological tolerance against hCG. The   -subunit of hCG (  hCG) conjugated to tetanus toxoid (TT) in-duced anti-hCG antibody response not only in animalsbut also in humans [1,2]. Carrier recruits Th cells thatprovide help to antibody producing hCG specific Bcells. Clinical studies with a   hCG based contraceptivevaccine demonstrated the lack of notable adverse ef-fects [3], the generation of neutralising antibodies [2,4]and the feasibility of the approach in preventing preg-nancy in fertile women [5]. Protective levels of antibod-ies were however not attained in some individuals.While one reason for diminished response may berelated to carrier-mediated epitopic suppression [6–12],it has also been shown that the T cell response to acomplex protein antigen is usually focused on a few,and frequently a single immunodominant epitope[13,14]. Being given that carrier linkage is not only acritical strategy for contraceptive vaccines but is alsoessential for evoking antibody responses to subunitpeptide vaccines, it becomes imperative to devise analternative approach that can ensure generation of anadequate anti-ligand antibody response in most if notall recipients. In the present study, we investigated thefeasibility of using promiscuous Th epitopes as carriers.pMVF corresponds to residues 288–302 of the measlesvirus fusion protein [15], pIVH corresponds to residues307–319 of the influenza virus hemagglutinin [16], andpHIVT represents residues 38–52 of the HIV-1 reversetranscriptase [17]. These peptides were synthesised withan additional cysteine at the N-terminal and individu-ally linked to   hCG. Employing a permissible adjuvant,the conjugates were investigated individually as well asin combination for their immunogenic potential in in-bred mice of different haplotypes. * Corresponding author. Tel.:  + 91-11-6188305; fax:  + 91-11-6162125. E  - mail address : (O. Singh). 1 Present address: Department of Pathology, Wayne State Univer-sity School of Medicine, 540 E Canfield Avenue, Detroit, MI 48201,USA.0264-410X / 01 / $ - see front matter © 2001 Published by Elsevier Science Ltd.PII: S0264-410X(01)00079-2  A .  Gupta et al  .  /   Vaccine  19 (2001) 3384  –  3389   3385 2. Materials and methods 2  . 1 .  Design of immunogens Amino acid sequences of the peptides used in thestudy are: pMVF (CLSEIKGVIVHRLEGV), pIVH(CPKYVKQNTLKLAT) and pHIVT (CTEMEKE-GKISKIGP). The peptides were synthesised by QualityControlled Biochemicals (Hopkinton, USA). The purityof the peptides (  95%) was determined by mass spec-troscopy.   hCG was coupled to individual peptidesusing  N  -succinimidyl-3-(2-pyridyl dithio) propionate(SPDP, Pierce) [18]. Brie fl y,   hCG (5 mg dissolved in100 mM PBS, pH 7.2) was treated at 25 ° C for 1 h with10 M excess of SPDP. Unreacted SPDP was removedby gel  fi ltration on a Sephadex G-25 column. Theaverage number of lysine residues activated per   hCGmolecule were estimated to be 3. Activated   hCG andpeptides were mixed in a molar ratio of 1:6 and incu-bated for 2 h at 25 ° C and overnight at 4 ° C. By measur-ing the release of pyridine-2-thione at 343 nm, thenumber of peptide molecules conjugated to each   hCGmolecule were computed to be   3. TT-  hCG wasprepared as described previously [19]. 2  . 2  .  Immunisation Female 8  –  10-week-old BALB / c (H-2 d ), C57Bl / 6 (H-2 b ), FVB (H-2 q ) and SJL (H-2 s ) mice were used for thestudy. Groups of eight to ten mice each were immu-nised with antigens adsorbed on alum, Al(OH) 3  (Super-fos, Denmark). A total of three injections, at 4-weekintervals, were given intramuscularly at two contra-lat-eral sites in a total volume of 100   l. The mice werebled from the retro-orbital plexus. Sera were dilutedappropriately and stored at  − 70 ° C until assayed. 2  . 3  .  Characterisation of antibody response Anti-hCG antibody titres expressed as hCG bindingcapacity were determined by a direct binding RIA.hCG was iodinated (sp.act. 40  –  60   Ci /  g) by the Iodo-gen method [20]. The assay protocol consisted of 100   lof diluted antiserum; 100   l of   125 I-hCG (15000 dpm),100   l of 20% horse serum and 200   l of assay buffer(50 mM PBS with 0.1% BSA and 0.1% sodium azide,pH 7.2). After incubation at 4 ° C for 48 h, the antibodybound fraction was precipitated by adding 500   l PEG8000 (12.5%  fi nal concentration) and centrifugation at1500 ×  g   for 20 min. The pellet (radioactivity) wascounted in a multi-  -counter (LKB 1260). After cor-recting for non-speci fi c binding, hCG binding capacitywas calculated at a point(s) where proportionality wasobtained between antiserum dilutions and iodinatedhCG binding. Association constant ( K  a ) of the Ab-hCGinteraction was determined by cold displacement analy-sis essentially as described [2]. Brie fl y, the serum dilu-tions (binding 20  –  30% in absence of cold hormone)were incubated for 48 h at 4 ° C with  125 I-hCG (15000dpm) and increasing concentrations (0.1, 0.3, 1, 3, 10,30, 100 ng) of unlabeled hCG in a total volume 500   l.The antibody bound fraction was separated as aboveand counted for radioactivity. Association constant( K  a ) of hCG-antibody interaction was computed usinga programme, Ligand [21].hCG neutralisation capacity of antibodies was esti-mated as a function of the ability of sera to inhibit thebinding of   125 I-hCG to testicular receptors as described[5]. Brie fl y, rat testicular homogenate was incubatedwith different dilutions of antiserum and  125 I-hCG(20000 dpm) for 2 h at 37 ° C. The reaction was termi-nated by addition of cold Tris buffer. Tubes werecentrifuged at 2000 ×  g   and the pellet counted for ra-dioactivity. Neutralisation capacity was calculated byregression analysis at the 50% inhibition point andexpressed as ng / ml of serum.Anti-TT or anti-peptide antibody titres were deter-mined by standard ELISA systems. A 96-well plate wascoated with TT or individual peptides (1   g / well) inPBS (pH 7.4) and non-speci fi c sites saturated with 1%of low fat milk powder. After incubation with variousserum dilutions, goat anti-mouse-HRPO conjugate wasadded as the revealing antibody. Orthophenylene di-amine was employed as substrate. 2  . 4  .  Statistical analysis Statistical analysis of data was carried out on logtransformed values of antibody titres. The signi fi cancelevel was calculated by the Student ’ s unpaired  t -test,and considered signi fi cant if   P  0.05. 3. Results 3  . 1 .  Immunogenicity of conjugates with Th -  peptides The effect of conjugation of peptides on the immuno-genicity of    hCG was evaluated in mice of four differ-ent H-2 haplotypes (b, d, q and s). Each peptide wasindividually linked to   hCG. Groups of eight to tenmice each were injected with   hCG alone or peptide-  hCG conjugates. One group in each strain received  hCG linked to TT (TT-  hCG). A total of three injec-tions of alum adsorbed antigen were given at an inter-val of 4 weeks. Mice were bled at intervals andanti-hCG antibody titres (hCG binding capacity) deter-mined in individual sera. Results summarised in Fig. 1indicate that conjugation of    hCG to peptides en-hanced anti-hCG antibody responses in all the fourmouse strains tested. All mice receiving   hCG-peptideconjugates responded with formation of anti-hCG anti-  A .  Gupta et al  .  /   Vaccine  19 (2001) 3384  –  3389  3386 bodies. Geometric mean titres induced by each of thepeptides in BALB / c and FVB mice were signi fi cantlyhigher ( P  0.01) compared to mean responses in   hCGimmunised groups. The increase in the immune re-sponse was comparable to that achieved with TT. Sim-ilar results were obtained in C57Bl / 6 mice, althoughlevels of antibody responses were lower. In SJL mice,all the peptide-  hCG conjugates elevated ( P  0.01) themean antibody titres over those attainable with uncon- jugated   hCG with pMVF giving the maximum en-hancement; the difference was however not statisticallysigni fi cant from that obtained with TT. 3  . 2  .  Efficacy of a combination of peptides conjugates We next tested whether a combination of the threepeptide-conjugates would lead to an additive effect inenhancing the antibody responses. An equimolar physi-cal mixture of peptide-conjugates adsorbed on alumwas administered to groups of mice as a compositepreparation. A group in each mouse strain was immu-nised with TT-  hCG conjugate on alum. Results shownin Fig. 2 reveal that each of the four different mousestrains responded with an elevated anti-hCG antibodyresponse, clearly establishing the advantage of the mix-ture over individual peptide conjugates. While antibodytitres generated by the peptide mixture were signi fi -cantly higher ( P  0.05) than those elicited by the TT-conjugated antigen in BALB / c, FVB, and SJL mice, theresponse in C57Bl / 6 mice was similar to that observedin group immunised with TT-  hCG. When the peakantibody response in all the four strains were pooled(akin to analysing the immune response in heteroge-nous population), there was a signi fi cant difference Fig. 1. Carrier effect of Th peptides on immunogenicity of    hCG in different mouse strains. Groups of eight to ten mice in each strain wereimmunised with pIVH-  hCG, pHIVT-  hCG and pMVF-  hCG, TT-  hCG conjugates or   hCG alone. Three injections of alum adsorbed antigens(5   g gonadotropin equivalent) were given intramuscularly at 0, 4 and 8 weeks. Antibody titres at 9 and 12 weeks in individual animals are shownas points and the bar indicates the geometric mean of responses. For all groups, peptide conjugates: vs.   hCG ( P  0.01), vs. TT (not signi fi cant).  A .  Gupta et al  .  /   Vaccine  19 (2001) 3384  –  3389   3387Fig. 2. Enhanced immunogenicity of a cocktail mixture of peptide conjugates. Mice were immunised with a physical mixture of pIVH-  hCG,pHIVT-  hCG and pMVF-  hCG conjugates adsorbed on alum. Three injections each containing an equimolar mixture of conjugates (5   g each)were given intramuscularly at an interval of 4 weeks. Groups of mice were also immunised with TT-  hCG. Antibody titres at 9 and 12 weeks areshown in individual sera as points with bar as geometric mean of the group. Mean peak titres with peptide mixture is signi fi cantly higher( P  0.05) than corresponding TT-  hCG groups of all strains except C57Bl / 6. ( P  0.01) between mean responses generated by pep-tide mixture and TT (geometric mean hCG bindingcapacity 2510 vs. 1400 ng / ml). Antibodies were foundcompetent to inhibit the bioactivity of hCG in vitro.We have earlier demonstrated a good correlation be-tween the in vitro neutralisation capacity of antibodiesand contraceptive ef  fi cacy of the vaccine [5]. Althoughthe bioneutralistion capacity per ml serum was consis-tently higher for all peptide-groups when compared tothe capacity of corresponding TT groups (Fig. 3), thedifference for C57Bl / 6 mice was however not signi fi -cant. Antibodies generated by the cocktail   hCG-pep-tide conjugates in all strains had high af  fi nity for hCG;association constants ( K  a ), computed by Scatchardanalysis, were of the order of 10 10 M − 1 . While animalsimmunised with the TT-  hCG conjugate responded bygenerating an anti-TT antibody response, antibodieswere not detectable (  1 U) to any of the three pep-tides in animals immunised with the peptide cocktail(data not shown). 4. Discussion In this study, we demonstrate the potential of threepathogen derived non-B promiscuous Th peptides toprovide T cell help for in vivo antibody productionagainst a covalently linked antigen (  hCG) without theuse of CFA.   hCG being  ‘ foreign ’ , mice do respond byproducing anti-hCG antibodies, albeit of low order,when it is injected after adsorption on alum. If, how-ever, the molecule is linked to pMVF, pIVH or pHIVT,anti-hCG response increases several-fold. Our data in-dicate that the nature of the conjugated B cell antigen isprobably not an important factor in determining thehaplotype-dependent hierarchy of antibody responsesand that adequate processing and presentation of Thpeptides occur even when the peptide is linked to  hCG, a 23 kDa protein.Although the three epitopes we employed are promis-cuous i.e. they are recognised in context of a variety of MHC / HLA alleles, they were not found equally reac-tive with different alleles. A given peptide may not beable to bind to all MHC alleles in the same orientationor with the same sequence [22,23], resulting in variable Fig. 3. Neutralising ef  fi cacy of the antibodies elicited by peptidemixture. Capacity to neutralise hCG bioactivity was measured by areceptor binding inhibition assay.  A .  Gupta et al  .  /   Vaccine  19 (2001) 3384  –  3389  3388 af  fi nity to MHC and presentation to T cells. Interactionbetween MHC-peptide-TCR may signi fi cantly in fl uencethe induction of humoral responses. We reasoned thatthe use of a combination of Th peptides instead of alarge carrier might help overcome this limitation of genetic restriction for production of anti-hCG antibod-ies. Our data show that co-injecting an equimolar mix-ture of three peptide-conjugates led to a signi fi cantenhancement of the immune responses in four differentstrains of mice. Antibodies were bioneutralising in na-ture, indicating that peptide linkage did not affectcrucial B cell epitopes. Earlier studies demonstratingimproved immunogenicity with Th epitope conjugationemployed CFA [11,15,22  –  30]. Here we employed onlyalum, an adjuvant approved for human use. It is possi-ble that immunisation with the cocktail leads to therecruitment of a larger repertoire of T cells. It has beenshown that the simultaneous presence of immunodomi-nant T cell epitopes does not result in competitionwhen the peptides are injected together [31]. Such co-dominance is anticipated among peptides that bindwith relatively high af  fi nity to the presenting moleculesand thus have a chance to occupy a signi fi cant numberof binding sites for T cell activation. An additive syner-gism, where the presence of a peptide enhances theresponse to another, is reported [31].Epitopic suppression, a phenomenon observed in avariety of hapten-carrier systems [6  –  12], has been at-tributed to the presence of anti-carrier antibodies [10],to induction and activation of suppressor T cells [7  –  10]or to antigenic competition between carrier-speci fi c andligand-speci fi c B cells [9]. The peptides investigated herecontain information for helper function and do notinduce antibodies against themselves. Responses elicitedby the peptide-conjugates should not be susceptible tocarrier speci fi c B and Ts cell mediated suppression. Infact, for peptide-conjugate vaccines, prior natural expo-sure to the infectious agents from which the peptidesare derived may prove to be bene fi cial rather thandetrimental. TT primed mice, when immunised with theB cell epitope (NANP) 4  linked to a Th peptide fromTT, generated an antibody response to (NANP) 4 . Theresponse was greater than when they received the B cellepitope linked to TT [11].In conclusion, our data show that an appropriatecombination of promiscuous Th cell peptides can en-hance the responsiveness to a conjugate vaccine usingonly permissible adjuvant. Absence of B and Ts epi-topes in these sequences would eliminate the possibilityof carrier-induced epitopic suppression. These  fi ndingshave implications for the development of contraceptivevaccines in particular and subunit vaccines in general,which aim at inducing protective levels of antibodytitres in a majority of individuals of a diversepopulation. Acknowledgements We thank Dayanand, Ashok, Ramesh and Deepti forexpert technical assistance. This work was supported bygrants from the Department of Biotechnology, Govern-ment of India, and the International Development Re-search Centre (IDRC) of Canada. References [1] Talwar GP, Sharma NC, Dubey SK, et al. Isoimmunizationagainst human chorionic gonadotropin with conjugates of pro-cessed   -subunit of the hormone and tetanus toxoid. Proc NatlAcad Sci USA 1976;73:218  –  22.[2] Singh O, Rao LV, Gaur A, Sharma NC, Alam A, Talwar GP.Antibody response and characteristics of antibodies in womenimmunized with three contraceptive vaccines inducing antibodiesagainst human chorionic gonadotropin. Fertil Steril 1989;52:739  –  44.[3] Talwar GP, Hingorani V, Kumar S, et al. Phase I clinical trialswith three formulations of anti-human chorionic gonadotropinvaccine. Contraception 1990;41:301  –  16.[4] Pal R, Singh O, Rao LV, Talwar GP. Bioneutralization capacityof the antibodies generated in women by the beta subunit of human chorionic gonadotropin (  hCG) and   hCG associatedwith alpha subunit of ovine luteinizing hormone linked to carri-ers. Am J Reprod Immunol 1990;22:124  –  6.[5] Talwar GP, Singh O, Pal R, et al. A vaccine that pre-vents pregnancy in women. Proc Natl Acad Sci USA 1994;91:8532  –  6.[6] Herzenberg LA, Tokuhisa T, Herzenberg LA. Carrier-primingleads to hapten-speci fi c suppression. Nature 1980;285:664  –  7.[7] Herzenberg LA, Tokuhisa T. Epitope-speci fi c regulation: I. Car-rier-speci fi c induction of suppression for IgG anti-hapten anti-body responses. J Exp Med 1982;155:1730  –  40.[8] Schutze MP, Leclerc C, Vogel FR, Chedid L. Epitopic suppres-sion in synthetic vaccine models: analysis of effector mecha-nisms. Cell Immunol 1987;104:79  –  90.[9] Schutze MP, Deriaud E, Przewlocki G, Lecrec C. Carrier-in-duced epitopic suppression is initiated through clonal domi-nance. J Immunol 1989;142:2635  –  40.[10] Di John D, Wasserman SS, Torres JR, et al. Effect of primingwith carrier on response to conjugate vaccine. Lancet1989;2:1415  –  8.[11] Etlinger HM, Gillessen D, Lahm HW, Matile H, Schonfeld HJ,Trzeciak A. Use of prior vaccinations for the development of new vaccines. Science 1990;249:423  –  5.[12] Renjifo X, Wolf S, Pastoret PP, et al. Carrier-induced, hapten-speci fi c suppression: a problem of antigen presentation? J Im-munol 1998;161:702  –  6.[13] Katz ME, Maizels RM, Wicker L, Miller A, Sercarz EE. Im-munological focusing by the mouse major histocompatibilitycomplex: mouse strains confronted with distantly relatedlysozymes con fi ne their attention to very few epitopes. Eur JImmunol 1982;12:535  –  40.[14] Berkower I, Matis LA, Buckenmeyer GK, Gurd FR, Longo DL,Berzofsky JA. Identi fi cation of distinct predominant epitopesrecognized by myoglobin-speci fi c T cells under the control of different Ir genes and characterization of representative T cellclones. J Immunol 1984;132:1370  –  8.[15] Partidos CD, Stanley CM, Steward MW. Immune responses inmice following immunization with chimeric synthetic peptides
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