Peptide-based subunit vaccines against pre-erythrocytic stages of malaria parasites

Molecular Immunology 38 (2001) 433–442

Review

Peptide-based subunit vaccines against pre-erythrocytic stages of malaria parasites Moriya Tsuji, Fidel Zavala∗ Department of Medical and Molecular Parasitology, New York University School of Medicine, 341 East 25th Street, New York, NY 10010, USA

Abstract Malaria currently ranks among the most prevalent infections in tropical and sub-tropical areas throughout the world with relatively high morbidity and mortality particularly in young children. The widespread occurrence and the increased incidence of malaria in many countries, caused by drug-resistant parasites (Plasmodium falciparum and P. vivax) and insecticide-resistant vectors (Anopheles mosquitoes), indicate the need to develop new methods of controlling this disease. Experimental vaccination with radiation-attenuated sporozoites can protect animals and humans against the disease, demonstrating the feasibility of developing an effective malaria vaccine. However, vaccines based on radiation-attenuated sporozoites are not feasible for large scale application due to lack of in vitro culture system. Therefore, the development of peptide-based subunit vaccines has been undertaken as an alternative approach. Synthetic peptides containing defined Band T-cell epitopes of different antigens expressed in sporozoites and/or liver stages have been used as subunit vaccines in experimental animal models. They have been shown to be highly immunogenic and capable of inducing protective immunity mediated by antibodies, as well as CD4+ and CD8+ T-cells. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Synthetic peptides; Malaria; Sporozoite; Liver stages; Antibodies; T-cells

Contents Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Protective immunity to pre-erythrocytic stages of malaria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Protective antigens expressed in the pre-erythrocytic stages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Protective immunity against the pre-erythrocytic stages of malaria is multi-factorial and includes antibody and T-cell responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Induction of protective antibody responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Genetically engineered proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Chemically engineered linear peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Multiple antigen peptides (MAPs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Polyoximes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Induction of CD8+ T-cell responses using synthetic immunogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Induction of protective CD4+ T-cell responses using synthetic immunogens . . . . . . . . . . . . . . . . . . . . . . . . 7. Distinct properties of peptide-based vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Protective immunity to pre-erythrocytic stages of malaria

∗

Corresponding author. Tel.: +1-212-263-6757; fax: +1-212-263-8116. E-mail address: [email protected] (F. Zavala).

The most important finding supporting the feasibility of developing a malaria vaccine was that immunization of mice, non-human primates and humans with radiation-attenuated

0161-5890/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 1 6 1 - 5 8 9 0 ( 0 1 ) 0 0 0 7 9 - 7

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sporozoites induces an immune response that confers protection against a subsequent challenge with live sporozoites (reviewed in Nardin and Zavala (1998)). In mice, immunization with radiation-attenuated Plasmodium berghei and P. yoelii sporozoites has shown to induce sterile protective immunity, i.e. complete resistance against live parasite challenge (Nussenzweig et al., 1969; Weiss et al., 1988). Likewise, immunization of rhesus monkeys with radiation-attenuated P. knowlesi sporozoites has also shown to induce protection against parasite challenge (Gwadz et al., 1979). In humans, successful attempts since the early 1970s were made by vaccinating volunteers with bites of more than 600 (at times to thousands) of X-irradiated P. falciparum or P. vivax infected mosquitoes (Clyde, 1975; Herrington et al., 1991; Egan et al., 1993). The studies in humans further demonstrated that the protective immunity elicited by irradiated sporozoites was species-specific, as volunteers immunized and protected against P. falciparum malaria remained susceptible to the bites of P. vivax infected mosquitoes and vice versa. Importantly, immunization with a particular isolate of P. falciparum also confers protection against challenge with isolates from different geographical locations (Clyde et al., 1973; Clyde, 1975). Although promising results were obtained in volunteers immunized with irradiated malaria-infected mosquitoes, this immunization method is not practical as a vaccine. Unlike traditional viral and bacterial microorganisms that can readily be grown in vitro to provide antigens for vaccines, large amounts of sporozoite stage parasites cannot be generated using in vitro culture, which are necessary for mass vaccination. In addition, exposure to multiple bites of irradiated malaria-infected mosquitoes is unacceptable for routine immunizations. In view of this situation, the design and development of subunit vaccines based on sporozoite-derived antigens has been undertaken.

2. Protective antigens expressed in the pre-erythrocytic stages Several antigens have been identified in the pre-erythrocytic stages as targets of the immune response and some of them have been studied in detail regarding their capacity to induce protective immunity. These include the circumsporozoite (CS), the thrombospondin-related anonymous protein (TRAP)/sporozoite surface protein 2(SSP2), the P. yoelii 17 kDa hepatocyte erythrocyte protein (PyHEP17)/P. falciparum exported protein 1 (PfExp-1) and the liver stage antigen 3 (LSA-3) proteins. The CS protein was the first identified target of protective anti-malaria immune responses. It is the major surface protein of sporozoites of all species of vertebrate malaria parasites, and is also expressed in the liver stages (Nussenzweig and Nussenzweig, 1989). The CS protein of all plasmodial species has the same basic structure, but little homology at the nucleotide and amino acid levels. It displays a central

region of tandem repeated amino acid sequences, each plasmodial species having a different amino acid sequence of the repeats. This central repeat region contains the immunodominant B-cell epitopes, the target of protective antibodies. Region I, at the amino-terminal, and region II, located at the carboxyl-terminal, are highly conserved among different plasmodial species. Other regions, partially overlapping with region II, flanking the repeats, contain CD4+ and CD8+ epitopes, recognized by protective T-cells. The CS protein also contains a universal CD4+ T-cell epitope (Sinigaglia et al., 1988; Calvo-Calle et al., 1997). There is little sequence homology among different species in these regions (Nardin and Zavala, 1998). The CS protein has an important role in the biology of the parasite. This protein has been implicated in mediating sporozoite binding and invasion of salivary glands in the mosquito vector and hepatocytes in mammalian hosts (Sidjanski et al., 1997; Cerami et al., 1992). Moreover, a recent study has demonstrated that this protein is structurally required for the generation of sporozoites, that formation of sporozoites was profoundly inhibited in parasites lacking the CS protein (Ménard et al., 1997). Another protein identified on the surface of sporozoites and in liver stage parasites is the P. yoelii SSP2 protein (Rogers et al., 1992), the homologue of P. falciparum TRAP (Robson et al., 1988). This protein has also been shown to be a target of protective immune response. Some reports have indicated that CD8+ T-cells (Khusmith et al., 1994), CD4+ T-cells (Wang et al., 1996) and antibodies (Rogers et al., 1992; Scarselli et al., 1993) may be involved. This protein also has a central repeat region, and displays domains homologous to region II of the CS protein. This protein has also recently been shown to be important in the biology of the parasite. Although, genetically manipulated malaria parasites lacking TRAP could develop into sporozoites, TRAP(−/−) sporozoites lack motility and cannot invade mosquito salivary glands, failing to become infective sporozoites (Sultan et al., 1997). The third protein, PyHEP17, was identified using a monoclonal antibody generated by immunization of mice with a suspension of mouse hepatocytes infected with mature P. yoelii liver stages. It is expressed not only by liver stages, but also by rings and trophozoites of infected erythrocytes, but not by sporozoites. The gene sequence presents a high homology with PfExp-1, which has the same expression pattern in the parasites as PyHEP17 (Charoenvit et al., 1995; Doolan et al., 1996). Immunization of mice with linear synthetic peptides from PyHEP17 has been shown to induce CD4+ T-cell and ␥ interferon-dependent protection against murine malaria (Charoenvit et al., 1999). The biological function of PyHEP17/PfExp-1 in pre-erythrocytic stages of the malaria parasite remains to be elucidated. Another protein, LSA-3, was identified by screening a P. falciparum l-phage library with serum samples from humans and chimpanzees that were protected after immunization with irradiated sporozoites. LSA-3 is made up of large

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non-repeated sequences flanking three Glu-rich repeated regions. Immunization of chimpanzees with peptides derived from LSA-3 and synthesized as palmitoyl-conjugated lipopeptides induced protection against challenge with P. falciparum sporozoites. The mechanism mediating this protective immunity has yet to be elucidated and may include antibodies as well as T-cells (Daubersies et al., 2000).

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et al., 1993) and in numerous studies with subunit vaccines fully confirmed the protective role of both CD8+ and CD4+ T-cells (reviewed in Nardin and Zavala, 1998).

4. Induction of protective antibody responses 4.1. Genetically engineered proteins

3. Protective immunity against the pre-erythrocytic stages of malaria is multi-factorial and includes antibody and T-cell responses Immunization with irradiated sporozoites induces a protective humoral immune response. These antibodies bind to surface proteins of sporozoites and function either by inhibiting sporozoite motility or inhibiting invasion of hepatocytes (Nussenzweig and Nussenzweig, 1989). Direct evidence that antibodies were a major protective mechanism was obtained by passive transfer of polyclonal antibodies raised against P. berghei sporozoites, which reduced the number of liver stages after homologous challenge and protected a significant proportion of na¨ıve mice as measured by the absence, or delay in the development of blood stage infection. More importantly, it was demonstrated that viable sporozoites pre-incubated with polyclonal anti-sporozoite anti-sera were no longer infective when injected into a susceptible rodent or simian host (Nussenzweig et al., 1969; Gwadz et al., 1979). The most definitive evidence on the protective role of antibodies was obtained in studies using P. berghei, in which it was demonstrated that passive transfer of monoclonal antibodies specific for the P. berghei CS protein prevented patent malaria infection (Yoshida et al., 1980). The effect was independent of the class of antibody or other secondary events such as complement fixation, agglutination or opsonization, since Fab fragments of this antibody are also effective in vitro (Potocnjak et al., 1980). Subsequent studies also demonstrated that monoclonal antibodies against P. falciparum and P. vivax CS proteins inhibit the respective sporozoite infectivity in chimpanzees (Nardin et al., 1982). Studies on the epitope specificity of these protective monoclonal antibodies indicated that they recognize the repeat regions of the respective CS proteins (Zavala et al., 1983, 1985; Romero et al., 1987). Besides the role played by antibodies in protective immunity, it also became evident in the last 10 years that T-cells—in addition to provide help for the production of antibodies—can also exert a strong anti-parasitic activity by inhibiting the development of liver stages. It was demonstrated that treatment of immunized mice with anti-CD8 antibodies (Schofield et al., 1987; Weiss et al., 1988) or anti-CD4 antibodies (Rodrigues et al., 1993) significantly decreased the protective immunity achieved after immunization. Subsequent studies with T-cell clones (Romero et al., 1989; Tsuji et al., 1990; Rodrigues et al., 1991; Renia

The use of purified recombinant proteins for immunization of experimental animals and humans relies on the choice of formulations which contain strong adjuvant properties (Allsopp et al., 1996). Early studies using a P. berghei recombinant CS protein expressed in yeast indicated that mice immunized with this protein emulsified in complete Freund’s adjuvant (CFA) developed high antibody titers and confers sterile protection to a significant proportion of immunized mice challenged with viable sporozoites (Romero et al., 1988). Until very recently, the only adjuvant allowed in human trials had been alum, an emulsion of aluminum hydroxide and aluminum phosphate. New adjuvants for human vaccination are now being investigated and some have been used on a relatively small scale. These include, but not limited to, QS21, a purified saponin, and the 3-deacylated form of monophosphoryl lipid A (MPL) derived from Salmonella minnesota lipopolysaccharide. A recent study has demonstrated that a recombinant CS protein, fused with another protein, expressed in yeast, and combined with the right adjuvant mixture, could induce a remarkable degree of protection against the pre-erythrocytic stages of malaria parasites in humans (Stoute et al., 1997). In this study, part of the CS protein of P. falciparum, consisting of 189 amino acids (aa 207–396), was fused to the S antigen, a major surface antigen of the hepatitis B virus and expressed as a particle containing lipid moieties. This particle was initially used to immunize malaria na¨ıve volunteers, in combination with three different adjuvant formulations: (1) alum plus MPL, (2) oil-in-water emulsion, and (3) the emulsion plus MPL and QS21, called SBAS2. While the antibody titers against the repeat region of the CS protein induced after immunization of the fusion protein with the three different formulations were detectable in all groups, only subjects immunized with SBAS2, showed protection against P. falciparum sporozoite challenge, with six of seven vaccinated individuals being fully protected. The immunized individuals also developed a Th1-type cellular immune response, mapped to a single CS peptide, together with their humoral response. CD8+ T-cell responses were not detected (Lalvani et al., 1999). An important conclusion of this study was that the same recombinant protein used with different adjuvant formulations can induce very different levels of protective responses. Despite the fact the use of adjuvants alum plus MPL and oil-in-water emulsion induced nearly the same level of antibodies against sporozoites, only immunization with SBAS2 succeeded in protecting a large percentage of the immunized individuals. This suggests that

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the avidity and/or the fine specificity of these antibodies may be as important as their serum concentration. The protective role of the CD4+, and possibly CD8+ T-cells, secreting IFN-␥, found in the peripheral blood of most immunized and protected individuals remains to be determined. 4.2. Chemically engineered linear peptides The use of synthetic peptides for immunization is a very attractive strategy for antigen delivery, since they are relatively easy to obtain in large quantities, with high purity. However, free synthetic peptides are poor immunogens since they are eliminated very rapidly when administered without adjuvant to experimental animals or humans. In order to increase their immunogenicity, carrier proteins are coupled with the linear peptide, and adjuvants are included in the immunogen formulation. Evidence that synthetic vaccines capable of inducing high titers of anti-repeat antibodies could confer protection against sporozoite challenge was initially described in the rodent P. berghei malaria model. Mice immunized with a repeat peptide of the P. berghei CS protein, couple to tetanus toxoid (TT) as carrier, developed high titers of antibodies reactive with the native CS protein on P. berghei sporozoites. The magnitude of the anti-sporozoite antibody response directly correlated with protection (Zavala et al., 1987). This study demonstrated that the nature of the coupling agent as well as the orientation of the peptide was critical for the induction of protective antibody responses. Another study clearly indicated that the peptide concentration contained in the conjugate vaccine was also critical for the induction of sterile immunity (Reed et al., 1996). Immunization with P. berghei CS repeats coupled to BSA, at peptide to protein rations of 6:1, 55:1 or 170:1, protected 20, 50 and 100% of the immunized mice, respectively (Reed et al., 1996). The failure to achieve a significant degree of protection after immunization with other synthetic peptide constructs, as described in early studies (Lal et al., 1987; Egan et al., 1987), is likely to be related to the strict conformational requirements that appear to be necessary to induce an effective antibody response. A peptide-based P. falciparum conjugate malaria vaccine for human use was developed based on the immunodominant repeat sequence of the CS protein, (NANP)3 . The (NANP)3 peptide was shown to be recognized by sera of sporozoite-immunized hosts and naturally-infected individuals and was found to effectively inhibit the binding of monoclonal and polyclonal anti-sporozoite antibodies to P. falciparum sporozoites (Zavala et al., 1985). Phase I and Phase II clinical trials of (NANP)3 linked to tetanus toxoid ((NANP)3 -TT) demonstrated the safety and immunogenicity of the peptide-protein malaria vaccine, when administered adsorbed to alum as adjuvant (Herrington et al., 1987; Etlinger et al., 1988). The frequency and the magnitude of the antibody responses were dose dependent, and the majority of vaccines seroconverted after injection of the highest doses of the vaccine. Although the antibody titers

elicited were suboptimal (IFA titers 105 ) of anti-sporozoite antibodies and were protected against sporozoite challenge. Immunization with these MAPs induces a long-lasting immune response, and induces secondary antibody and T-cell responses in mice previously immunized with parasites (Chai et al., 1992). In these initial studies on the immunogenicity of MAPs, several key observations were made (Tam et al., 1990). First, it was determined that polymeric synthetic constructs containing covalently linked B- and CD4+ T-cell epitopes were able to induce high level of antibodies, while monomers with exactly the same antigenic information induce only low antibody responses. However, the size of the polymer is an important factor only up to a certain degree since 4and 8-branched peptides induced the same levels of antibody responses. Second, an important finding was that the MAP constructs need to contain equimolar amounts of Band T-epitopes. In fact, it was determined that constructs containing 4-T-cell epitopes and 1-B-cell epitopes as well as those containing 4-B-cell epitope and 1-T-cell epitopes were very poor immunogens when compared to constructs which contain equal numbers of B- and T-epitopes. Finally, it was determined that the orientation of the B- and T-epitope does not seem to have a major effect on their immunogenicity. Subsequent studies showed that protection could also be achieved using a MAP containing only a T-cell epitope derived from the N terminus of the P. berghei CS protein

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(aa 57–70), or in combination with the repeat B-cell epitope (Migliorini et al., 1993). Following challenge by the bite of malaria-infected mosquitoes, 62–66% of the MAP immunized mice developed sterile immunity. In other studies, MAPs containing B- and CD4+ T-cell epitopes from P. yoelii CS protein also elicited high titers of anti-sporozoite antibodies and a high degree of protection against parasite challenge (Wang et al., 1995; Marussig et al., 1997). The results obtained with the rodent systems stimulated studies using MAPs containing sequences of B- and T-epitopes located in P. falciparum antigens. A MAP construct, (T1B)4 consisting of four branches, each containing the B-cell epitope (NANP)3 and a highly conserved T-cell epitope of the P. falciparum CS protein, designated T1 (DPNANPNVDPNANPNV), was very immunogenic in three of four inbred strains of mice (Munesinghe et al., 1991), in three phenotypically and karyotypically different species of Aotus monkeys (Moreno et al., 1999), and in 10 of 12 volunteers expressing certain DQ and DR (Nardin et al., 2000). The addition of adjuvants, such as alum and QS21, significantly increased the level of antibody responses in mice and monkeys (Moreno et al., 1999), and more importantly in humans (Nardin et al., 2000). When T-cell epitopes derived from the parasite were used to elicit an immune response, it was determined that this response is genetically restricted to those individuals whose haplotypes were shown to be recognized in vitro by binding to certain epitopes. This restriction can be circumvented if a vaccine includes “universal” or promiscuous T-cell epitopes, recognized by a large proportion of humans. In fact, when two universal (promiscuous) T-cell helper epitopes derived from tetanus toxoid were included in a construct containing the P. falciparum B-cell epitope (NANP)6 , the immune response in strains of mice that responded poorly to the repeats was highly enhanced (Valmori et al., 1992; Grillot et al., 1993). When this MAP construct was encapsulated in liposomes with lipid A and adsorbed to alum and given three times at 4 week intervals into mice, the resultant antibody prevented 100% of sporozoites from invading and developing into liver stage infection in vitro (Le et al., 1998). Furthermore, a MAP construct containing a P. falciparum-derived universal T-cell epitope (comprising amino acids 326–345 of the CS protein), which could bind to multiple DR and DQ molecules in vitro, elicited high titers of anti-sporozoite antibodies in eight inbred mice strains (H-2a,b,d,k,p,q,r,s ), circumventing genetic restriction (Calvo-Calle et al., 1997). Using P. vivax CS protein, MAPs containing a B-cell epitope of the repeat region of the P. vivax CS protein and a universal T-cell epitope derived from either P. vivax CS protein or tetanus toxoid were constructed and shown to be highly immunogenic and induce high levels of antibodies that recognize the native CS protein in Aotus monkeys (Herrera et al., 1997). In another study, Saimiri monkeys were immunized three times with MAP containing the B-cell epitope of the P. vivax CS protein and the T-cell epitope of the tetanus toxoid formulated in different adjuvants (Yang et al.,

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1997). Animals that received a non-ionic block copolymer (P1005), as an adjuvant, elicited a high level of antibodies against CS repeat and sporozoites, which persists 7 months after the last immunization. More importantly, a significant percentage of the immunized monkeys were fully protected against sporozoite challenge 22 weeks after the last immunization (Yang et al., 1997). 4.4. Polyoximes Other than MAPs, an improved and more precise method of assembling peptides has recently been developed. Those polymers, named polyoximes, were constructed by chemoselective ligation of the repeat B-cell epitope and a universal T-cell epitope plus T1 of the CS protein of P. falciparum, and has been shown to be highly immunogenic in murine strains of diverse H-2 haplotypes (Nardin et al., 1998). In vaccines of diverse HLA haplotypes these polyoximes elicited anti-CS and anti-sporozoite antibodies in 7 out of 10 volunteers (Nardin et al., 2001). Both in the P. falciparum MAPs, as well as polyoxime vaccination, there was high reactivity not only with the polymers, but also with the native CS protein on the sporozoite surface. Based on these findings, it is clear that subsequent studies with these polyoximes and MAPs vaccines are warranted and need to be further investigated regarding the capacity to induce protective immunity in human beings.

5. Induction of CD8+ T-cell responses using synthetic immunogens Besides the induction of antibody responses, it has also been shown that T-cell priming and protective immunity can be achieved by immunization with monomeric and polymeric synthetic antigen constructs. In fact, immunization with linear 9-mer peptides representing the CTL epitopes of P. yoelii and P. berghei have been shown to induce CD8+ T-cell responses when emulsified in IFA together with a synthetic peptide or polymer representing a T helper epitope (Widmann et al., 1992; Valmori et al., 1994). Furthermore, it was shown that when peptide-induced CD8+ T-cells obtained from lymph nodes were transferred into na¨ıve mice, they exerted an efficient anti-parasitic activity and conferred a significant degree of protection in mice challenged with live parasites (Renggli et al., 1995). This indicates that immunization with this minimal antigenic information is sufficient to induce the proliferation and differentiation of na¨ıve cells into effectors. The immunogenicity of small synthetic peptides representing T-cell epitopes, particularly those recognized by CD8+ T-cells, can be significantly enhanced by linkage to lipid moieties. This has been demonstrated in several systems in which the capacity to induce epitope-specific cytotoxic cells by lipopeptide or peptide alone—both containing the same antigenic information—was compared

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(Schild et al., 1991). In malaria, lipopeptides containing P. berghei or P. falciparum epitopes and a single N-terminal mono-palmitic acid have been shown to be effective in inducing respective CD8+ T-cell responses (Romero et al., 1992; Miyahira et al., 1998; BenMohamed et al., 1997). A comparison of various formulations using the P. berghei CD8+ T-cell epitope indicated that the P3C moeity containing a Ser(Lys)4 amino acid spacer sequence provided an optimal induction of CD8+ T-cell responses (Hioe et al., 1996). Furthermore, the incorporation of these lipopeptides in microspheres prepared from poly(lactide-co-glycolide) polymers also significantly enhanced the CD8+ T-cell response to the P. berghei epitope (Nixon et al., 1996). Not only monomeric peptide constructs are capable of inducing CD8+ T-cell responses, as shown in studies using the P. yoelii system. In this case, mice were immunized with a tetra-branched MAP containing a 20-mer CD8+ T-cell eptitope peptide sequence synthesized in tandem with a tetanus toxoid epitope. It was shown that significant protection could be achieved after immunization with this immunogen (Franke et al., 1997). A key component in the induction of protective immunity in the case was the administration of MAP mixed with the cationic lipid lipofectin that based on previous work is believed to facilitate antigen processing through the intracellular MHC Class I pathway (Walker et al., 1992). In addition to branched synthetic peptide constructs, immunization with large linear recombinant or synthetic polypeptides (larger than 100-mer) representing the CS protein of human and rodent malaria have also shown to elicit CD8+ T-cell responses when administered and emulsified in adjuvants (Heppner et al., 1996; Roggero et al., 2000). In fact, it has been shown that some of these immunogens, besides inducing antibodies that react with the native protein, induce CD8+ T-cell responses very efficiently as compared to immunization with small peptides (Roggero et al., 2000). These large polypeptides, rather than being internalized by antigen-presenting cells, have been shown to be digested by extracellular proteases generating smaller peptides that directly bind to the MHC and thus presented to T-cells (Eberl et al., 1999). Another synthetic/recombinant immunogen, which has also been evaluated, are virus-like particles, which represents proteins of viral origin with the capacity to self-assemble into particles which contain lipid moieties. A variety of foreign epitopes can be inserted into the gene encoding this protein thus generating recombinant virus-like particles. These recombinant particles have been shown to be immunogenic in several systems and capable of inducing both humoral and T-cell mediated immunity. A major advantage of these synthetic recombinant constructs is that they do not require the use of strong adjuvants. In the malaria system, Ty-virus like particles have been evaluated using the P. berghei and P. yoelii systems and they have been shown to efficiently induce responses against the respective CD8+ T-cell epitopes (Allsopp et al., 1996; Oliveira-Ferreira et al., 2000).

Importantly, when mice primed with these particles are then boosted with recombinant vaccinia virus, strong memory recall responses are induced and sterile protective immunity can be achieved in a significant proportion of the immunized mice (Oliveira-Ferreira et al., 2000). As for human malaria epitopes, a P. falciparum CS peptide, which is recognized by HLA-A2, was shown to be immunogenic in transgenic mice expressing HLA-A2.1 Class I molecule (Blum-Tirouvanziam et al., 1995). In another study, CTL responses to a P. falciparum CS epitope recognized by B10.BR mice was efficiently induced using synthetic peptides coupled to a lipid tail. Interestingly, this immunization induced a memory response, which was largely expanded when immunized mice were boosted with a recombinant vaccinia virus expressing the same epitope (Miyahira et al., 1998). Most importantly, a recent study with human volunteers showed that immunization with a large synthetic polypeptide containing P. falciparum B- and T-cell epitopes elicited strong malaria-specific humoral, CD4+ and CD8+ T-cell responses (Lopez et al., 2001).

6. Induction of protective CD4+ T-cell responses using synthetic immunogens Synthetic peptides have also been used for the induction of CD4+ T-cell responses. These cells—besides being necessary for the development of antibody responses—have also been shown to be able to recognize parasite antigens and exert a direct anti-parasitic effect. The protective activity of these cells has been demonstrated for both P. berghei and P. yoelii, as evidenced by the ability of these cells to inhibit parasite development independently of the antibody response they help to induce. Initial studies in which peptides or whole parasite-derived protein extracts were used to identify CD4+ T-cell epitopes and subsequently, to generate Th1 and Th2 CD4+ T-cell clones (Romero et al., 1988; Tsuji et al., 1990; Renia et al., 1993; Takita-Sonoda et al., 1996). Adoptive transfer of these T-cell clones revealed that these cells can exert a potent anti-parasite activity regardless of their pattern of cytokine production. Furthermore, mice immunized with a tetrameric MAP containing a Class II-restricted T helper epitope of the CS protein of P. berghei was shown to induce protective immunity when mice were challenged with viable sporozoites (Migliorini et al., 1993). Similar protective activity was obtained in mice immunized with linear synthetic peptides representing a CD4+ T-cell epitope of the PyHEP17 pre-erythrocytic antigen (Charoenvit et al., 1999). Protective immunity mediated by CD4+ T-cells have also been induced by immunization with synthetic linear peptides as well as MAPs containing CD4+ T-cell epitopes from the P. yoelii TRAP antigen. It was determined that this protection was at least in part, mediated IFN-␥ (Wang et al., 1996). Finally, in humans, there is still limited experience on the induction of T-cell mediated immunity after immuniza-

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tion with subunit vaccines. Recently, it has been shown that human volunteers immunized with a recombinant protein expressing the CS protein of P. falciparum developed antigen-specific CD4+ T-cell responses of Th1-type which is believed to contribute to protection in vaccinated volunteers (Lalvani et al., 1999).

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2. For most, if not all, synthetic immunogens, powerful adjuvants are required to elicit protective immune responses and unfortunately, very few highly efficient adjuvants are currently approved for human use.

8. Concluding remarks

7. Distinct properties of peptide-based vaccines Synthetic peptide-based vaccines provide several advantages and disadvantages when compared to vaccines based on recombinant proteins, DNA constructs, attenuated bacterial or viral vectors expressing microbial antigens. The following are the most advantageous properties of synthetic peptide-based vaccines: 1. The magnitude of the antibody responses achieved with synthetic polymers is possibly the highest that can be achieved with current experimental immunogens. This is in sharp contrast to naked DNA or recombinant live vectors that in general have shown to be less efficient in inducing efficient antibody responses particularly in humans and non-human primates. 2. Another significant advantage is that all components contained in the synthetic peptide vaccines are chemically defined and readily available, and provide, at least in principle, the rapid production of an unlimited supply of immunogen. In contrast to the cold chain required for attenuated vaccines, the stability of peptides to the temperature facilitates vaccine delivery in underdeveloped areas of the world. 3. Of particular importance for the implementation of vaccines in areas affected by HIV/AIDS is that peptides are non-infectious and provide an additional safety advantage over immunogens based on bacterial or viral vectors that have the potential for eliciting pathogenic and/or reactogenic complications. 4. Finally, unlike some DNA-based construct and some of viral vectors, peptides do not integrate into the chromosome of the immunized host, and thus, they cannot cause mutations in host cells. There are also distinct disadvantages that synthetic peptide-based vaccines need to overcome to become a better alternative to currently used subunit vaccines. 1. Perhaps the most important is that due to the simplified molecular nature of these immunogens, well-defined peptides representing helper T-cell epitopes are required in order to provide help for B-cells to produce antibodies. Since T-cell epitopes are strictly restricted to different MHCs and the MHCs are quite diverse among human populations, the peptides should contain multiple T-cell epitopes to overcome the MHC restriction.

The results obtained in experimental systems are extremely encouraging and provide a rationale and experimental basis for the development of effective vaccines for human immunization. Overall, the current experience with synthetic immunogens has clearly demonstrated that they have great potentials for the development of vaccines against malaria and other infectious pathogens. They have shown to be particularly efficient in inducing strong antibody responses, while their capacity to induce T-cell mediated responses, particularly CD8+ T-cell responses, appears to be more limited although their effect can be enhanced by using a prime-boost strategy in combination with other vectors. However, although this is an area of research in which there is only a limited experience, it is also fast evolving. Further advances can be expected in the development of combinations of synthetic peptides and immunomodulatory molecules that may provide safer and more efficient new generation of subunit vaccines.

Acknowledgements We thank Julius C.R. Hafalla for his help in preparing this manuscript. Support from the National Institutes of Health, AI 44375 and AI 40656, is acknowledged.

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