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Incorporation of T and B epitopes of the circumsporozoite protein in a chemically defined synthetic vaccine against malaria

Incorporation of T and B epitopes of the circumsporozoite protein in a chemically defined... INCORPORATION OF T AND B EPITOPES OF THE CIRCUMSPOROZOITE PROTEIN IN A CHEMICALLY DEFINED SYNTHETIC VACCINE AGAINST MALARIA BY JAMES P TAM," PEDRO CLAVIJOJ YI-AN LU,` VICTOR NUSSENZWEIG,S RUTH NUSSENZWEIG,I AND FIDEL ZAVALAI From `The Rockefeller University, New York, New York 10021; and New York University School of Medicine, (Department of Medical and Molecular Parasitology and DDpparmment of Pathology, New York, New York 10010 Multiple antigen peptide for peptide-based were systems (MAPs) 1 vaccines de- signed one of us overcome the ambiguity requirement by to and of conjugation to protein carrier (1, 2) . The MAP consists a by synthetic peptides system of an oligo- meric branching core, usually of lysines, eight lysine comprised seven and dendritic arms of peptides containing antigenic epitopes . Since each arm of peptide may con- sist of 10 to more than 20 amino acids, the overall appearance of the MAP system is of a macromolecule with a high density of surface peptide antigen and a molec- ular weight exceeding 10,000, surrounding a lysine scaffolding core of molecular weight of <800 . The immunogenicity of monoepitope MAPs has been tested in animals (1, 2) . All model MAPs elicited high-titered antisera, most of which recognized the cog- nate protein from which the epitopes were derived . However, some issues concerning the possible use of the MAP system for production of vaccines were not resolved . One important remaining question is whether the MAPs can be engineered to con- tain not only B cell epitopes but also functional T cell epitopes, selected from protein molecules that are candidates for development of subunit vaccines . In addition, the effect of the stoichiometry, orientation, and arrangement of the B and T epitopes on the immunogenicity of the MAPs has not been determined . To MAP T B study these problems we prepared 10 models containing and epi- topes of the circurnsporozoite (CS) protein of the rodent malaria parasite, Plasmo- dium berghei. The immunodominant B cell epitope of the P. berghei CS protein is con- tained within its repeat domain and can be represented by a 16-residue peptide (PPPNPND)z (3, 4) . Monoclonal and polyclonal antibodies against the repeat do- mains of the CS protein neutralize in vitro and in vivo the infectivity of malaria This work was supported by the Agency for International Development (DPE 0453-A-00-5012-00), Na- tional Institutes of Health grant AI-28701, the MacArthur Foundation, and Bachem, USA. F. Zavala is a recipient of an Irma T. Hirschl Career Development Award. Address correspondence to Dr. James P. Tam, The Rockefeller University, 1230 York Avenue, Box 294, NY New York, 10021 . in : 1 Abbreviations used this paper CS, circumsporozoite; IFA, immunofluorescence assay ; IRMA, im- munoradiometric assay ; MAPs, multiple antigen . peptide systems J . Exp. MED. 0 The Rockefeller University Press - 0022-1007/90/01/0299/08 .00 $2 Volume 171 January 1990 299-306 MULTIPLE ANTIGEN PEPTIDE SYSTEM IN PEPTIDE-BASED VACCINES sporozoites (reviewed in reference 5) . Recently, several T helper epitopes ofthe P . berghei protein CS have been identified, thus providing the opportunity not only to develop di-epitope but as a MAPs, also to test their efficacy vaccines in well-characterized rodent malaria T 265 model. One of these helper epitopes, between residues and (KQIRDSITEEWS), was selected for inclusion in the di-epitope MAPs because it was recognized by several strains inbred of mice and particularly because it dis- played helper activity in vivo (6) . Materials and Methods Synthesis and Characterization ofMAPs . The monomeric BT peptide was synthesized as de- scribed (6) by a stepwise solid-phase peptide synthesis method of Merrifield (7) MAPS were synthesized as described, on a tertbutoxycarbonyl (Boc)-Ala-Pam resin with a low loading (0 .1 mmol/g of resin) (1) . In the MAP models containing four copies of the peptide antigens [T-(4), B-(4), BT(4), and TB-(4)], the core was synthesized with only three lysines prepared from branching with two levels ofBoc-Lys(Boc), while in the MAP models containing eight copies of the antigens [T(8), B-(8), BT-(8), and TB-(8)], the core was synthesized with seven lysines prepared from branching with three levels of Boc-Lys(Boc) . In the MAP models con- taining eight copies of or T T or B antigen T(8)-B], B antigens and one copy of [B-(8)-T, the B or T antigen was first synthesized linearly on the Boc-Ala-Pam resin and then branched lysine to give eight copies T or B antigens All peptides with three levels of the subsequent of . and were cleaved from the resin by the low/high HF procedure (8) to mini- MAPS supports the peptides were extracted from the resin with, and extensively mize side reactions, and 8 All products were character- dialyzed in, 8 M urea in 0 .1 M Tris/HCI buffer, pH .0 . MAP ized gel chromatography . Amino acid analysis gave satisfactory results by high-performance that agreed with the expected composition . H-2a Immune ofMAPs. Groups of five mice of the haplotype (A/J strain) were Responses with 50 i .p. a given MAP, or a mixture of MAPs [T(8) + B-(8)], emulsified injected of Kg in CFA on day 0, and boosted with of the same antigen in IFA on day 21 . Sera were 50,ug later. Sera were pooled and antibody titers were determined by an immunoradio- collected 21 d assay using recombinant protein as antigen or by indirect immunofluorescence metric a CS (IFA) using glutaraldehyde-fixed sporozoites as antigen. The titers were expressed as the reciprocal of the highest positive serum dilution. Results and Discussion B T epitopes, The 10 MAP models (Fig . 1) included tandemly connecting and BT(8)], and similar with four or eight peptide antigen arms [models BT(4), and was reversed [models TB- models in which the orientation of the B and T epitopes stoichiometry ofthe B and T cell epitopes, (4), TB-(8)] . To test the importance ofthe of B epitope and only one copy of the T model B-(8)-T contained eight copies the copies of the T epitope and only one epitope, while model T(8)-B contained eight prepared four different models of MAPs con- copy of the B epitope. As controls, we alone . Two of the control models [T(4), B-(4)] taining either B or T cell epitopes con- B or T epitopes, and the other two [T(8), B-(8)] contained four copies of either [BT] either epitope. For comparison, a monomeric peptide tained eight copies of one copy each of the B and T epitopes was prepared . containing only the 3D11 mono- presence of the B epitope in the MAPs, we used To assay for the which reacts with the raised against P . berghei sporozoites and clonal antibody (9) epitopes were strong in- B epitope (10) . All MAPs containing the B 16 amino acid sporozoites immobilized in wells of hibitors of the binding of radiolabeled 3D11 to binding was observed at MAPs concentrations microtiter plates . 50% inhibition of TAM ET AL . 30 1 represen- FIGURE 1. Schematic tation of the structure of the CS protein ofP . berghei of the mono- meric form of a peptide con- taining tandem B and T cell epi- topes and of 10 MAP models (see text for explanation), -t3 degree required of 10 -t° to 10 M, while the same of inhibition -10-s M of the BT shown) . The containing only T epitope monomeric peptide (data not MAN the noninhibitory. These results indicated that the B epitopes in the MAPs func- were tioned as antigens . We had earlier shown that immunization with theP . berghei B epitope alone, either as a monomer or as an octameric MAP, did not elicit antibody responses in A/J and several other inbred strains of mice (6) . To test for the capacity of the newly designedMAP antigens to elicit the production of antibodies to this B epitope, groups of five A/J mice were immunized with each of the 10 models of MAPs . Additional control groups were immunized with a BT monomer, or with a mixture of equal amounts of B-(8) and TI(8) . The pooled sera from each group of animals were as- sayed for their reactivities with the recombinant CS protein by immunoradiometric MULTIPLE ANTIGEN PEPTIDE SYSTEM IN PEPTIDE-BASED VACCINES assay (IRMA) and with glutaraldehyde-fixed sporozoites by indirect immunofluores- cence assay (IFA) . The primary antibody responses, measured 3 wk after the first antigen dose, showed that the MAPS that contained equimolar T and B epitopes, linked in tandem, were highly immunogenic. The best immunogen, BT(4), produced serum antibody levels that were detectable at dilutions greater than 10 5 . The secondary antibody responses observed 21 d (Fig . 2) and 34 d after administration of a second dose of the MAPS were significantly higher, but the ranking order of immunogenicity was similar to that observed in the primary responses . The serum titers of mice injected with BT (4), greater x were than 4 105 , while the other three di-epitope MAPs containing equimolar T and B peptides, x elicited titers between 10 5 and 4 10 5 . The animals immunized with the mono-epitope B-(4) and B-(8) MAPS did not respond . There was a poor antibody response to T(8)-B, B-(8)-T, T(4), and T(8) . TB monomers or a mixture of equimolar amounts of B-(8) and T-(8) failed to elicit an antibody response Thus, a . covalent high molecular weight structure containing multiple copies of tandemly arranged B and T for epitopes was required good immunogenicity . The IFA titers of the sera 34 after d the booster injection are shown in Table I . The BT(4)-injected mice had IFA titers 1.28 x 10 5 against of sporozoites. These titers are at least 10 times higher than those usually found in the serum of mice hyperim- munized with irradiated sporozoites . Immunization of A/J mice with similar doses of recombinant P . berghei CS protein incorporated in CFA, encompassing amino acids 81-277, resulted in much lower IFA titers x 103 ) (6) . It should be pointed out (2 that although the present results were obtained with the P. berghei MAPS incorpo- rated in CFA, high antibody titers were elicited with other MAPs when the adjuvant was alum, or even in the absence of adjuvant (in preparation) . A plausible explana- tion for the greater immunogenicity of the MAPs may be that they contain a high 8- 6- FIGURE 2. Secondary antibody responses of groups of mice immunized with different MAP models . 2- 8 32 64 256 1024 4096 Reciprocal Serum Dilution X10-2 of TAM ET AL . TABLE I Protective Efficacy of Different MAP Models in Mice Challenged with 2, 000 P. berghei Sporozoites Antisporozoite Number: Immunogen (IFA titer x 10-3 )' protected/challenged Protection BT-(4) 128 4/5 80 TB-(4) 32 3/5 60 TB-(8) 32 3/5 60 BT-(8) 8 2/4 50 T-(4) <0 .2 0/4 0 B-(4) <0 .2 0/5 0 B-(8) <0 .2 0/5 0 BT monomer <0 .2 0/5 0 No immunogen - 0/5 0 Titer determined by IFA using glutaraldehyde-fixed sporozoites, 34 d after the booster injection of antigen . : Mice were challenged by intravenous inoculations of 2,000 sporozoites 35 d after the booster injection . Peripheral blood smears were examined daily for parasitized erythrocytes . Protection is defined as absence of parasites from day 3 to 12 after challenge . density multiple copies of peptide antigens . In the recombinant CS protein, and there are single copies of the T epitope and multiple B epitopes . Such an arrange- simulated in the design of the B-(8)-T model that was a poor immunogen . ment was In addition, as reported for other antigens, the CS protein may contain epitopes suppress antibody responses (11) . that Because the tandemly connected B and T cell epitopes in the MAP are relatively long, it is conceivable that the steric interactions between branches, and the epitope orientations of MAPS, may influence their immunogenicity . For example, the less bulky model BT (4) produced N50-fold higher antibody titers than BT(8) . How- ever, there was little difference in immune response between the reversely oriented models TB-(4) and TB-(8) . Although the present results indicate that there is no advantage to increasing the number ofMAP branches from 4 to 8, the effects on the immune response of the number of the B and T epitopes in the MAP models may be sequence dependent, and may require optimization in a given experimental . noteworthy that the B cell epitope tested in this study is exceptionally system It is proline (50%), while theT cell epitope has a strong propensity for amphipathic rich in formation . helix the MAPS as vaccines was evaluated by intravenous challenge of The efficacy of immunized mice with 2,000P . berghei sporozoites 35 d after the booster injection . the 80% of the mice immunized with BT(4) were protected, and in the groups of mice immunized with the other three di-epitopes MAPS, there was 50-60% protection (Table I) . The levels of antibodies to sporozoites (IFA) in the vaccinated mice cor- related well with the degree of protection . No protection was observed in mice im- munized with the mono-epitope MAPS or the monomer BT Although the IFA titers ofmice hyperimmunized with irradiated P . berghei sporozoites are much lower than those after immunization with the di-epitope MAPs, the mice MULTIPLE ANTIGEN PEPTIDE SYSTEM IN PEPTIDE-BASED VACCINES resist challenge with much higher doses ofsporozoites, underscoring the importance of T cell effector (12) . mechanisms in protection Early experiments had shown that Ft-suppressed mice, make which do not antibodies, can be effectively vaccinated with irradiated sporozoites In (13) . this model of immunization, CD8' cytotoxic T cells are required for protection and (14, 15), their target is most likely the liver stage of the parasite (16, 17) . In fact, passive transfer to naive mice of CD8' cloned T cells recognizing protein . an epitope of the CS of P berghei can confer a high degree of protection against challenge with sporozoites (18) . While considerable progress has been made in understanding the mechanisms of protection in murine malaria models, the relative importance of serum antibodies and effector T cells in mediating protection against human malaria sporozoites has not (5, Two . been established 19) . Pfalciparum malaria vaccines have undergone human trials . was a One recombinant fusion protein containing multiple copies of NANP (20), the B P. falciparum epitope of the CS protein (21), and the other was a synthetic vaccine . consisting of (NANP)3 coupled to a tetanus toxoid carrier (22) In both trials, one out of six and one out of three volunteers were protected, although the serum levels of antisporozoite antibodies were low . The frequency and magnitude of the in volunteers receiving antibody response the the synthetic vaccine increased with the dose of the conjugate . Unfortunately, however, the peptide vaccine dose could not be raised the toxicity carrier due to ofthe tetanus toxoid . It is also conceiv- able that to was the immune response the peptide suppressed because the volunteers had been previously vaccinated with tetanus toxoid (23) . These two problems, car- rier toxicity and epitopic suppression, may severely limit the effectiveness of this synthetic malaria vaccine and of other similarly designed vaccines . Both obstacles In can be overcome by di-epitope MAPS . addition, as shown here, these chemically unambiguous structures can be highly immunogenic and engineered to contain at two . least and perhaps several different functional B andT epitopes MAPs may there- vaccines fore serve as a basis for developing subunit for diseases in which circulating antibodies play role in protection . Summary We show here an effective and novel approach to engineer peptide-based vaccines using a chemically defined system, known as multiple peptide antigen systems (MAPs), to protect an inbred mouse strain from infection against rodent malaria . 10 mono- and di-epitope MAP models containing different arrangements and stoichiometry of functional B and/or T helper cell epitopes from the circumsporozoite protein of Plasmodium berghei were used to immunize A/J mice. While these mice did not re- spond to the mono-epitope MAP bearing only the B or T epitope, very high titers of antibody and protective immunity against sporozoite challenge were elicited by di-epitope MAPs, particularly those with the B andT epitopes in tandem and present in equimolar amounts . These results, obtained in a well-defined rodent malaria model, indicate that MAPs may overcome some of the difficulties in the development of synthetic vaccines, not only for malaria but also for other infectious diseases . for We thank thejoint UNDP/World Bank/WHO Special Programme Research and Training TAM ET AL . in Tropical Diseases and Rockefeller Foundation for their continued support, and Christina Sang for the preparation of the manuscript. Received for publication 16 August 1989. References 1 . P. Synthetic peptide vaccine design : synthesis and properties of a high- Tam, J . 1988. Proc. Nail. Acad. Sci. USA . 85:5409 . density multiple antigen system . N ., H . McGrath, and J . P Tam . 1988 . A novel method for producing anti- 2 . Posnett, D. antibodies using a peptide derived from the T cell antigen receptor /3-chain con- peptide stant region . Biol. Chem . 263 :1719 . J . 3 . Eichinger, D. J ., D. E . Arnot, J . P Tam, V. Nussenzweig, and V. Enea. 1986 . Circum- sporozoite protein of Plasmodium berghei : gene cloning and identification of the im- munodominant epitopes . Mol. Cell. Biol. 6:3965 . 4 . Weber, J . L ., J . E . Egan, J . A . Lyon, R . A . Wirtz, Y. Charoenvit, W. L . Maloy, and W. T. Hockmeyer. 1987 . Plasmodium berghei : cloning ofthe circumsporozoite protein gene . Exp. Parasitol. 63 :295 . 5 . Nussenzweig, V., and R . S . Nussenzweig . 1989 . Rational e for the development of an engineered sporozoites malaria vaccine . Adv. Immunol. 45:283 . 6 . Romero, P J ., J . P. Tam, D. Schlesinger, P. Clavijo, P. J . Barr, R . S. Nussenzweig, V. Nussenzweig, and F. Zavala. 1988 . Multiple T-helper cell epitopes ofthe circumsporozoite protein of Plasmodium berghei . Eur. Immunol . 18 :1951 . 7 . Merrifield, R . B . 1986 . Solid phase synthesis . Science (Wash . DC). 232 :341 . 8 . Tam, J . P., W. F. Heath, and R . B. Merrifield . 1983 . SN 2 deprotection of synthetic pep- tides with a low concentration ofHF in dimethylsulfide : evidence and application in pep- tide analysis . Am. Chem. Soc. 105:6442 . J. 9 . Yoshida, N ., R . S . Nussenzweig, P. Potocnjak, V. Nussenzweig, and M . Aikawa . 1980 . Hybridoma produces protective antibodies directed against the sporozoite stage ofmalaria parasite . Science (Wash. DC) . 207 :71 . 10 . Zavala, E, J . P Tam, P. J . Barr, P. J . Romero, V. Ley, R . S . Nussenzweig, and V. Nus- senzweig . 1987 . Synthetic peptide vaccine confers protection against murine malaria. J. Exp. Med. 166 :1591. 11 . Adorini, L ., M . A . Harvey, A . Miller, and E . E . Sercarz . 1979 . Fin e specificity of regula- tory T cells . II . Suppressor and helper T cells are induced by different regions of hen egg white lysozome in a genetically non-responder mouse strain . . Exp. Med . 150 :293 . 12 . Spitalny, G. L., and R . S. Nussenzweig . 1973 . Plasmodium berghei: relationship between protective immunity and anti-sporozoite (CSP) antibody in mice . Exp. Parasitol. 33 :168. 13 . Chen, D. H ., R. E . Tigelaar, and F. I . Weinbaum . 1977 . Immunit y to sporozoite-induced malaria infection in mice . I . The effect ofimmunization of T and B cell-deficient mice. f. Immunol. 118 :1322 . 14 . Schofield, L., J . Villaquiran, A . Ferreira, H . Schellekens, R. Nussenzweig, and V. Nus- senzweig . 1987 . Gamma interferon, CD8' T cells and antibodies required for immu- nity to malaria sporozoites . Nature (Loud.) . 330 :664 15 . Weiss, W. R ., M . Sedegah, R . L . Beaudoin, L . H . Miller, andM . F. Good. 1988 . CD8' T cells (cytotoxic/suppressors) are required for protection in mice immunized with malaria sporozoites . . . . Proc. Nail Acad. Sci. USA 85 :573 16 . Ferreira, Enea, van E . A ., L . Schofield, V. H . Schellekens, P der Meide, W. Collins, R. S . Nussenzweig, and V. Nussenzweig . 1986 . Inhibition ofdevelopment ofexoerythro- cytic forms of malaria parasites gamma-interferon Science (Wash. by . DC). 232 :881 . MULTIPLE ANTIGEN PEPTIDE SYSTEM IN PEPTIDE-BASED VACCINES . W P Puri, B . N . Dhawan, and R. M . 17 . Maheshwari, R K ., C . Czarniecki, G . Dutta, S . K . interferon malaria. Infect. Friedman . 1986 . Recombinan t human gamma inhibits simian Immun. 53:628 . Nussenzweig, V. Nussenzweig, and 18 . Romero, P, J . L . Maryanski, G . Corradin, R . S . Tcells recognize an epitope in the circumsporozoite F Zavala. 1989 . Cloned cytotoxic against malaria. Nature (Loud.). In press . (CS) protein and protect ., . W. Hollingdale, W R . Majarian, D. M . 19 . Egan, J . E J . L Weber, R . Ballou, M . R . . L . A. Wirtz, I . Schneider, G . R. Woolett, J . F Gordon, W. L . Maloy, S. Hoffman, R . W. . 1987 . Efficac ofmurine malaria sporozoite vaccines : Im- Young, and T. Hockmeyer y for vaccine development . Science (Wash . DC). 236 :453 . plications human Hollingdale, A . H . Cochrane, I . Quakyi, R . S. Nussenz- 20 . Zavala, F., J . P Tam, M . R . weig, V. Nussenzweig . 1985. Rationale for the development of a synthetic vaccine and DC). :1436. against P . falciparum malaria. Science (Wash . 228 A . M . R . Hollingdale, F. A . Neva, W. T. 21 . Ballou, W. R ., S. L . Hoffman, J . Sherwood, G . F. Wasserman, Hockmeyer, D. M . Gordon, I . Schneider, R . A . Wirtz, J . F Young, recombinant DNA P Reeve, C . L . Diggs, andJ . D. Chulay. 1987 . Safety and efficacy ofa Plasmodium falciparum sporozoite vaccine. Lancet. i :1277. R. Murphy, J . Davis, S . . Herrington, D. A ., D. F Clyde, G . Losonsky, M . Cortesia, J . E. S. Nussenzweig, V. Nus- Baqar, A . M . Felix, E. P Heimer, D. Gillessen, Nardin, R . . Safet and immunogenicity in senzweig, M. R . Hollingdale, and M . M . Levine . 1987 y sporozoites . man of a synthetic peptide malaria vaccine against Plasmodium falciparum Nature (Loud.). 328 :257 . Carrier-specific . Herzenberg, L. A ., and T Tokuhisa . 1983 . Epitope-specific regulation . I . Exp. Med. 155 :1730 . induction ofsuppression for IgG anti-hapten antibody responses . J. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Experimental Medicine Pubmed Central

Incorporation of T and B epitopes of the circumsporozoite protein in a chemically defined synthetic vaccine against malaria

The Journal of Experimental Medicine , Volume 171 (1) – Jan 1, 1990

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Abstract

INCORPORATION OF T AND B EPITOPES OF THE CIRCUMSPOROZOITE PROTEIN IN A CHEMICALLY DEFINED SYNTHETIC VACCINE AGAINST MALARIA BY JAMES P TAM," PEDRO CLAVIJOJ YI-AN LU,` VICTOR NUSSENZWEIG,S RUTH NUSSENZWEIG,I AND FIDEL ZAVALAI From `The Rockefeller University, New York, New York 10021; and New York University School of Medicine, (Department of Medical and Molecular Parasitology and DDpparmment of Pathology, New York, New York 10010 Multiple antigen peptide for peptide-based were systems (MAPs) 1 vaccines de- signed one of us overcome the ambiguity requirement by to and of conjugation to protein carrier (1, 2) . The MAP consists a by synthetic peptides system of an oligo- meric branching core, usually of lysines, eight lysine comprised seven and dendritic arms of peptides containing antigenic epitopes . Since each arm of peptide may con- sist of 10 to more than 20 amino acids, the overall appearance of the MAP system is of a macromolecule with a high density of surface peptide antigen and a molec- ular weight exceeding 10,000, surrounding a lysine scaffolding core of molecular weight of <800 . The immunogenicity of monoepitope MAPs has been tested in animals (1, 2) . All model MAPs elicited high-titered antisera, most of which recognized the cog- nate protein from which the epitopes were derived . However, some issues concerning the possible use of the MAP system for production of vaccines were not resolved . One important remaining question is whether the MAPs can be engineered to con- tain not only B cell epitopes but also functional T cell epitopes, selected from protein molecules that are candidates for development of subunit vaccines . In addition, the effect of the stoichiometry, orientation, and arrangement of the B and T epitopes on the immunogenicity of the MAPs has not been determined . To MAP T B study these problems we prepared 10 models containing and epi- topes of the circurnsporozoite (CS) protein of the rodent malaria parasite, Plasmo- dium berghei. The immunodominant B cell epitope of the P. berghei CS protein is con- tained within its repeat domain and can be represented by a 16-residue peptide (PPPNPND)z (3, 4) . Monoclonal and polyclonal antibodies against the repeat do- mains of the CS protein neutralize in vitro and in vivo the infectivity of malaria This work was supported by the Agency for International Development (DPE 0453-A-00-5012-00), Na- tional Institutes of Health grant AI-28701, the MacArthur Foundation, and Bachem, USA. F. Zavala is a recipient of an Irma T. Hirschl Career Development Award. Address correspondence to Dr. James P. Tam, The Rockefeller University, 1230 York Avenue, Box 294, NY New York, 10021 . in : 1 Abbreviations used this paper CS, circumsporozoite; IFA, immunofluorescence assay ; IRMA, im- munoradiometric assay ; MAPs, multiple antigen . peptide systems J . Exp. MED. 0 The Rockefeller University Press - 0022-1007/90/01/0299/08 .00 $2 Volume 171 January 1990 299-306 MULTIPLE ANTIGEN PEPTIDE SYSTEM IN PEPTIDE-BASED VACCINES sporozoites (reviewed in reference 5) . Recently, several T helper epitopes ofthe P . berghei protein CS have been identified, thus providing the opportunity not only to develop di-epitope but as a MAPs, also to test their efficacy vaccines in well-characterized rodent malaria T 265 model. One of these helper epitopes, between residues and (KQIRDSITEEWS), was selected for inclusion in the di-epitope MAPs because it was recognized by several strains inbred of mice and particularly because it dis- played helper activity in vivo (6) . Materials and Methods Synthesis and Characterization ofMAPs . The monomeric BT peptide was synthesized as de- scribed (6) by a stepwise solid-phase peptide synthesis method of Merrifield (7) MAPS were synthesized as described, on a tertbutoxycarbonyl (Boc)-Ala-Pam resin with a low loading (0 .1 mmol/g of resin) (1) . In the MAP models containing four copies of the peptide antigens [T-(4), B-(4), BT(4), and TB-(4)], the core was synthesized with only three lysines prepared from branching with two levels ofBoc-Lys(Boc), while in the MAP models containing eight copies of the antigens [T(8), B-(8), BT-(8), and TB-(8)], the core was synthesized with seven lysines prepared from branching with three levels of Boc-Lys(Boc) . In the MAP models con- taining eight copies of or T T or B antigen T(8)-B], B antigens and one copy of [B-(8)-T, the B or T antigen was first synthesized linearly on the Boc-Ala-Pam resin and then branched lysine to give eight copies T or B antigens All peptides with three levels of the subsequent of . and were cleaved from the resin by the low/high HF procedure (8) to mini- MAPS supports the peptides were extracted from the resin with, and extensively mize side reactions, and 8 All products were character- dialyzed in, 8 M urea in 0 .1 M Tris/HCI buffer, pH .0 . MAP ized gel chromatography . Amino acid analysis gave satisfactory results by high-performance that agreed with the expected composition . H-2a Immune ofMAPs. Groups of five mice of the haplotype (A/J strain) were Responses with 50 i .p. a given MAP, or a mixture of MAPs [T(8) + B-(8)], emulsified injected of Kg in CFA on day 0, and boosted with of the same antigen in IFA on day 21 . Sera were 50,ug later. Sera were pooled and antibody titers were determined by an immunoradio- collected 21 d assay using recombinant protein as antigen or by indirect immunofluorescence metric a CS (IFA) using glutaraldehyde-fixed sporozoites as antigen. The titers were expressed as the reciprocal of the highest positive serum dilution. Results and Discussion B T epitopes, The 10 MAP models (Fig . 1) included tandemly connecting and BT(8)], and similar with four or eight peptide antigen arms [models BT(4), and was reversed [models TB- models in which the orientation of the B and T epitopes stoichiometry ofthe B and T cell epitopes, (4), TB-(8)] . To test the importance ofthe of B epitope and only one copy of the T model B-(8)-T contained eight copies the copies of the T epitope and only one epitope, while model T(8)-B contained eight prepared four different models of MAPs con- copy of the B epitope. As controls, we alone . Two of the control models [T(4), B-(4)] taining either B or T cell epitopes con- B or T epitopes, and the other two [T(8), B-(8)] contained four copies of either [BT] either epitope. For comparison, a monomeric peptide tained eight copies of one copy each of the B and T epitopes was prepared . containing only the 3D11 mono- presence of the B epitope in the MAPs, we used To assay for the which reacts with the raised against P . berghei sporozoites and clonal antibody (9) epitopes were strong in- B epitope (10) . All MAPs containing the B 16 amino acid sporozoites immobilized in wells of hibitors of the binding of radiolabeled 3D11 to binding was observed at MAPs concentrations microtiter plates . 50% inhibition of TAM ET AL . 30 1 represen- FIGURE 1. Schematic tation of the structure of the CS protein ofP . berghei of the mono- meric form of a peptide con- taining tandem B and T cell epi- topes and of 10 MAP models (see text for explanation), -t3 degree required of 10 -t° to 10 M, while the same of inhibition -10-s M of the BT shown) . The containing only T epitope monomeric peptide (data not MAN the noninhibitory. These results indicated that the B epitopes in the MAPs func- were tioned as antigens . We had earlier shown that immunization with theP . berghei B epitope alone, either as a monomer or as an octameric MAP, did not elicit antibody responses in A/J and several other inbred strains of mice (6) . To test for the capacity of the newly designedMAP antigens to elicit the production of antibodies to this B epitope, groups of five A/J mice were immunized with each of the 10 models of MAPs . Additional control groups were immunized with a BT monomer, or with a mixture of equal amounts of B-(8) and TI(8) . The pooled sera from each group of animals were as- sayed for their reactivities with the recombinant CS protein by immunoradiometric MULTIPLE ANTIGEN PEPTIDE SYSTEM IN PEPTIDE-BASED VACCINES assay (IRMA) and with glutaraldehyde-fixed sporozoites by indirect immunofluores- cence assay (IFA) . The primary antibody responses, measured 3 wk after the first antigen dose, showed that the MAPS that contained equimolar T and B epitopes, linked in tandem, were highly immunogenic. The best immunogen, BT(4), produced serum antibody levels that were detectable at dilutions greater than 10 5 . The secondary antibody responses observed 21 d (Fig . 2) and 34 d after administration of a second dose of the MAPS were significantly higher, but the ranking order of immunogenicity was similar to that observed in the primary responses . The serum titers of mice injected with BT (4), greater x were than 4 105 , while the other three di-epitope MAPs containing equimolar T and B peptides, x elicited titers between 10 5 and 4 10 5 . The animals immunized with the mono-epitope B-(4) and B-(8) MAPS did not respond . There was a poor antibody response to T(8)-B, B-(8)-T, T(4), and T(8) . TB monomers or a mixture of equimolar amounts of B-(8) and T-(8) failed to elicit an antibody response Thus, a . covalent high molecular weight structure containing multiple copies of tandemly arranged B and T for epitopes was required good immunogenicity . The IFA titers of the sera 34 after d the booster injection are shown in Table I . The BT(4)-injected mice had IFA titers 1.28 x 10 5 against of sporozoites. These titers are at least 10 times higher than those usually found in the serum of mice hyperim- munized with irradiated sporozoites . Immunization of A/J mice with similar doses of recombinant P . berghei CS protein incorporated in CFA, encompassing amino acids 81-277, resulted in much lower IFA titers x 103 ) (6) . It should be pointed out (2 that although the present results were obtained with the P. berghei MAPS incorpo- rated in CFA, high antibody titers were elicited with other MAPs when the adjuvant was alum, or even in the absence of adjuvant (in preparation) . A plausible explana- tion for the greater immunogenicity of the MAPs may be that they contain a high 8- 6- FIGURE 2. Secondary antibody responses of groups of mice immunized with different MAP models . 2- 8 32 64 256 1024 4096 Reciprocal Serum Dilution X10-2 of TAM ET AL . TABLE I Protective Efficacy of Different MAP Models in Mice Challenged with 2, 000 P. berghei Sporozoites Antisporozoite Number: Immunogen (IFA titer x 10-3 )' protected/challenged Protection BT-(4) 128 4/5 80 TB-(4) 32 3/5 60 TB-(8) 32 3/5 60 BT-(8) 8 2/4 50 T-(4) <0 .2 0/4 0 B-(4) <0 .2 0/5 0 B-(8) <0 .2 0/5 0 BT monomer <0 .2 0/5 0 No immunogen - 0/5 0 Titer determined by IFA using glutaraldehyde-fixed sporozoites, 34 d after the booster injection of antigen . : Mice were challenged by intravenous inoculations of 2,000 sporozoites 35 d after the booster injection . Peripheral blood smears were examined daily for parasitized erythrocytes . Protection is defined as absence of parasites from day 3 to 12 after challenge . density multiple copies of peptide antigens . In the recombinant CS protein, and there are single copies of the T epitope and multiple B epitopes . Such an arrange- simulated in the design of the B-(8)-T model that was a poor immunogen . ment was In addition, as reported for other antigens, the CS protein may contain epitopes suppress antibody responses (11) . that Because the tandemly connected B and T cell epitopes in the MAP are relatively long, it is conceivable that the steric interactions between branches, and the epitope orientations of MAPS, may influence their immunogenicity . For example, the less bulky model BT (4) produced N50-fold higher antibody titers than BT(8) . How- ever, there was little difference in immune response between the reversely oriented models TB-(4) and TB-(8) . Although the present results indicate that there is no advantage to increasing the number ofMAP branches from 4 to 8, the effects on the immune response of the number of the B and T epitopes in the MAP models may be sequence dependent, and may require optimization in a given experimental . noteworthy that the B cell epitope tested in this study is exceptionally system It is proline (50%), while theT cell epitope has a strong propensity for amphipathic rich in formation . helix the MAPS as vaccines was evaluated by intravenous challenge of The efficacy of immunized mice with 2,000P . berghei sporozoites 35 d after the booster injection . the 80% of the mice immunized with BT(4) were protected, and in the groups of mice immunized with the other three di-epitopes MAPS, there was 50-60% protection (Table I) . The levels of antibodies to sporozoites (IFA) in the vaccinated mice cor- related well with the degree of protection . No protection was observed in mice im- munized with the mono-epitope MAPS or the monomer BT Although the IFA titers ofmice hyperimmunized with irradiated P . berghei sporozoites are much lower than those after immunization with the di-epitope MAPs, the mice MULTIPLE ANTIGEN PEPTIDE SYSTEM IN PEPTIDE-BASED VACCINES resist challenge with much higher doses ofsporozoites, underscoring the importance of T cell effector (12) . mechanisms in protection Early experiments had shown that Ft-suppressed mice, make which do not antibodies, can be effectively vaccinated with irradiated sporozoites In (13) . this model of immunization, CD8' cytotoxic T cells are required for protection and (14, 15), their target is most likely the liver stage of the parasite (16, 17) . In fact, passive transfer to naive mice of CD8' cloned T cells recognizing protein . an epitope of the CS of P berghei can confer a high degree of protection against challenge with sporozoites (18) . While considerable progress has been made in understanding the mechanisms of protection in murine malaria models, the relative importance of serum antibodies and effector T cells in mediating protection against human malaria sporozoites has not (5, Two . been established 19) . Pfalciparum malaria vaccines have undergone human trials . was a One recombinant fusion protein containing multiple copies of NANP (20), the B P. falciparum epitope of the CS protein (21), and the other was a synthetic vaccine . consisting of (NANP)3 coupled to a tetanus toxoid carrier (22) In both trials, one out of six and one out of three volunteers were protected, although the serum levels of antisporozoite antibodies were low . The frequency and magnitude of the in volunteers receiving antibody response the the synthetic vaccine increased with the dose of the conjugate . Unfortunately, however, the peptide vaccine dose could not be raised the toxicity carrier due to ofthe tetanus toxoid . It is also conceiv- able that to was the immune response the peptide suppressed because the volunteers had been previously vaccinated with tetanus toxoid (23) . These two problems, car- rier toxicity and epitopic suppression, may severely limit the effectiveness of this synthetic malaria vaccine and of other similarly designed vaccines . Both obstacles In can be overcome by di-epitope MAPS . addition, as shown here, these chemically unambiguous structures can be highly immunogenic and engineered to contain at two . least and perhaps several different functional B andT epitopes MAPs may there- vaccines fore serve as a basis for developing subunit for diseases in which circulating antibodies play role in protection . Summary We show here an effective and novel approach to engineer peptide-based vaccines using a chemically defined system, known as multiple peptide antigen systems (MAPs), to protect an inbred mouse strain from infection against rodent malaria . 10 mono- and di-epitope MAP models containing different arrangements and stoichiometry of functional B and/or T helper cell epitopes from the circumsporozoite protein of Plasmodium berghei were used to immunize A/J mice. While these mice did not re- spond to the mono-epitope MAP bearing only the B or T epitope, very high titers of antibody and protective immunity against sporozoite challenge were elicited by di-epitope MAPs, particularly those with the B andT epitopes in tandem and present in equimolar amounts . These results, obtained in a well-defined rodent malaria model, indicate that MAPs may overcome some of the difficulties in the development of synthetic vaccines, not only for malaria but also for other infectious diseases . for We thank thejoint UNDP/World Bank/WHO Special Programme Research and Training TAM ET AL . in Tropical Diseases and Rockefeller Foundation for their continued support, and Christina Sang for the preparation of the manuscript. Received for publication 16 August 1989. References 1 . P. Synthetic peptide vaccine design : synthesis and properties of a high- Tam, J . 1988. Proc. Nail. Acad. Sci. USA . 85:5409 . density multiple antigen system . N ., H . McGrath, and J . P Tam . 1988 . A novel method for producing anti- 2 . Posnett, D. antibodies using a peptide derived from the T cell antigen receptor /3-chain con- peptide stant region . Biol. Chem . 263 :1719 . J . 3 . Eichinger, D. J ., D. E . Arnot, J . P Tam, V. Nussenzweig, and V. Enea. 1986 . Circum- sporozoite protein of Plasmodium berghei : gene cloning and identification of the im- munodominant epitopes . Mol. Cell. Biol. 6:3965 . 4 . Weber, J . L ., J . E . Egan, J . A . Lyon, R . A . Wirtz, Y. Charoenvit, W. L . Maloy, and W. T. Hockmeyer. 1987 . Plasmodium berghei : cloning ofthe circumsporozoite protein gene . Exp. Parasitol. 63 :295 . 5 . Nussenzweig, V., and R . S . Nussenzweig . 1989 . Rational e for the development of an engineered sporozoites malaria vaccine . Adv. Immunol. 45:283 . 6 . Romero, P J ., J . P. Tam, D. Schlesinger, P. Clavijo, P. J . Barr, R . S. Nussenzweig, V. Nussenzweig, and F. Zavala. 1988 . Multiple T-helper cell epitopes ofthe circumsporozoite protein of Plasmodium berghei . Eur. Immunol . 18 :1951 . 7 . Merrifield, R . B . 1986 . Solid phase synthesis . Science (Wash . DC). 232 :341 . 8 . Tam, J . P., W. F. Heath, and R . B. Merrifield . 1983 . SN 2 deprotection of synthetic pep- tides with a low concentration ofHF in dimethylsulfide : evidence and application in pep- tide analysis . Am. Chem. Soc. 105:6442 . J. 9 . Yoshida, N ., R . S . Nussenzweig, P. Potocnjak, V. Nussenzweig, and M . Aikawa . 1980 . Hybridoma produces protective antibodies directed against the sporozoite stage ofmalaria parasite . Science (Wash. DC) . 207 :71 . 10 . Zavala, E, J . P Tam, P. J . Barr, P. J . Romero, V. Ley, R . S . Nussenzweig, and V. Nus- senzweig . 1987 . Synthetic peptide vaccine confers protection against murine malaria. J. Exp. Med. 166 :1591. 11 . Adorini, L ., M . A . Harvey, A . Miller, and E . E . Sercarz . 1979 . Fin e specificity of regula- tory T cells . II . Suppressor and helper T cells are induced by different regions of hen egg white lysozome in a genetically non-responder mouse strain . . Exp. Med . 150 :293 . 12 . Spitalny, G. L., and R . S. Nussenzweig . 1973 . Plasmodium berghei: relationship between protective immunity and anti-sporozoite (CSP) antibody in mice . Exp. Parasitol. 33 :168. 13 . Chen, D. H ., R. E . Tigelaar, and F. I . Weinbaum . 1977 . Immunit y to sporozoite-induced malaria infection in mice . I . The effect ofimmunization of T and B cell-deficient mice. f. Immunol. 118 :1322 . 14 . Schofield, L., J . Villaquiran, A . Ferreira, H . Schellekens, R. Nussenzweig, and V. Nus- senzweig . 1987 . Gamma interferon, CD8' T cells and antibodies required for immu- nity to malaria sporozoites . Nature (Loud.) . 330 :664 15 . Weiss, W. R ., M . Sedegah, R . L . Beaudoin, L . H . Miller, andM . F. Good. 1988 . CD8' T cells (cytotoxic/suppressors) are required for protection in mice immunized with malaria sporozoites . . . . Proc. Nail Acad. Sci. USA 85 :573 16 . Ferreira, Enea, van E . A ., L . Schofield, V. H . Schellekens, P der Meide, W. Collins, R. S . Nussenzweig, and V. Nussenzweig . 1986 . 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Cochrane, I . Quakyi, R . S. Nussenz- 20 . Zavala, F., J . P Tam, M . R . weig, V. Nussenzweig . 1985. Rationale for the development of a synthetic vaccine and DC). :1436. against P . falciparum malaria. Science (Wash . 228 A . M . R . Hollingdale, F. A . Neva, W. T. 21 . Ballou, W. R ., S. L . Hoffman, J . Sherwood, G . F. Wasserman, Hockmeyer, D. M . Gordon, I . Schneider, R . A . Wirtz, J . F Young, recombinant DNA P Reeve, C . L . Diggs, andJ . D. Chulay. 1987 . Safety and efficacy ofa Plasmodium falciparum sporozoite vaccine. Lancet. i :1277. R. Murphy, J . Davis, S . . Herrington, D. A ., D. F Clyde, G . Losonsky, M . Cortesia, J . E. S. Nussenzweig, V. Nus- Baqar, A . M . Felix, E. P Heimer, D. Gillessen, Nardin, R . . Safet and immunogenicity in senzweig, M. R . Hollingdale, and M . M . Levine . 1987 y sporozoites . man of a synthetic peptide malaria vaccine against Plasmodium falciparum Nature (Loud.). 328 :257 . Carrier-specific . Herzenberg, L. A ., and T Tokuhisa . 1983 . Epitope-specific regulation . I . Exp. Med. 155 :1730 . induction ofsuppression for IgG anti-hapten antibody responses . J.

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The Journal of Experimental MedicinePubmed Central

Published: Jan 1, 1990

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