Malaria vaccines Pedro Romero Ludwig Institute for Cancer Research, Lausanne, Switzerland The development of an effective malaria vaccine is a feasible goal. Most of the vaccines being developed today are subunit vaccines derived from selected parasite antigens or their immunologically active fragments. The precise characterization of protective immune responses against Plasmodium parasites remains a fundamental part of present research aimed at obtaining a malaria vaccine(s). Current Opinion in Immunology 1992, 4:432-441

Introduction Current efforts to control malaria involve vector control, development of chemotherapy and the formulation of vaccines. The search for malaria vaccines dates back to the 1940s but the major advances in this field have not occurred until recently [1~176 The understanding of some of the mechanisms of protective immune responses and cloning of Plasmodium genes coding for stage specific antigens have accelerated the pace of malaria vaccine development. The scope of this review will be limited to two types of malaria vaccines, namely those targeted to the pre-erythrocytic and blood stages, which are aimed at blocking the development of incoming parasites or to limit the development of already established parasites. Two additional types of malaria vaccines have been proposed that would either neutralize factors responsible for the pathology associated with malaria infection (anti-disease vaccine) or limit transmission of malaria by immune interference with the parasite's cycle of sexual development in the mosquito. The latter two types of vaccines will not be discussed in this review.

Vaccines against the pre-erythrocytic stages of malaria As protective immune responses in malaria are largely stage-specific, there is a need to identify appropriate targets at each stage. The initial impetus for a sporozoitebased vaccine was provided by the observation that complete protection could be achieved by immunizing humans with attenuated sporozoites delivered by the bite of infected and irradiated mosquitoes [3]. Thus, vaccination with irradiated sporozoites, although not feasible on a large scale, is a useful approach to dissect the mechanisms involved in protection [4,5~176 In a recent carefully conducted trial, complete protection against Plasmodium falciparum malaria was induced in

volunteers receiving a large dose of sporozoites inoculated by multiple exposures to irradiated and infected mosquitoes [9"]. Protected individuals had high levels of anti-sporozoite antibodies as well as peripheral blood T cells responding to in vitro stimulation with a recombinant P. falciparum circumsporozoite (CS) protein. The immunizing strain of P. falciparum was different to the strain from which the recombinant CS protein was de rived; this suggests that the responding T cells either recognized non polymorphic CS epitopes or cross reacted with polymorphic epitopes. A detailed specificity analysis would help in assessing the extent to which CS polymorphism may influence specific human T cell responses. At present, experiments involving the use of sporozoites are only feasible in laboratories that have access to mosquito-born sporozoites through cumbersome and expensive procedures. This bottleneck might be overcome in the future thanks to advances in culture t e c b niques for Plasmodium sexual stages [10"~ In animal models of malaria, it has been firmly established that antibodies to a repeat region present in the CS protein mediate protection against sporozoite-induced malaria [3]. Therefore, the first generation of subunit malaria vaccines to be tried in humans was based on CS-repeats. The two initial human vaccination trials in volved the use of either the synthetic dodecapeptide (Asn-Ala-Asn-Pro)3, which represents the immunodominant repeat B cell epitope in the P. falciparum CS protein, conjugated to tetanus toxoid, or of a non-conjugated recombinant polypeptide made of a stretch of 32 P. falciparum CS repeat units, [(Asn-Ala-Asn-Pro)15 Asn-ValAsp-Pro]2 , fused to a stretch of 32 amino acid residues encoded by the tetracyclin resistance gene of the plas mid vector (this vaccine formulation has been called R32tet32). In both trials the administration of the vaccines adsorbed onto alum resulted in the induction of low levels of antibodies in most of the volunteers [11,12]. Protection correlated with the presence of the highest levels

Abbreviations CS~circumsporozoite; CTL--cytotoxic T lymphocytes; GB~glycophorin binding protein; HBsAg--hepatitis B surface antigen; IFN--interferon; MSA--merozoite surface antigen; RAP--rhoptry associated protein; RESA--ring-infected erythrocyte surface antigen.

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Malaria vaccines Romero

of anti repeat antibodies. Interestingly, the administration of the recombinant vaccine R32tet32 to volunteers living in an area where malaria is endemic resulted in significant boosting of the naturally acquired antibody response [13"]. Poor immunogenicity of a recombinant P. vivax CS protein, also administered in alum, was reported in a human trial involving 30 non-exposed volunteers [14]. In view of these results, the goal now is to design a vaccination protocol that would result in the induction of high levels of antibodies to the repeat sequence with the appropriate specificity. In this direction, a different construct of the P. falciparum CS repeats inserted into the Hepatitis B surface antigen (HBsAg) has been tested for safety and immunogenicity [15]. All volunteers receiving this vaccine absorbed onto alum developed anti-CS protein antibodies. However, no assesment of protection was carried out in this trial. Clearly, adjuvants more potent than alum are required. (Asn-AlaAsn-Pro)32 fused to a portion of an influenza protein was administered with monophosphoryl lipid A, the cell-wall skeleton of mycobacteria and squalene [16o] or encapsulated in monophosphoryl lipid A-containing llposomes [17"]. The levels of antibody obtained to the repeat sequence in both trials were one order of magnitude higher than those induced by the same recombinant peptide adsorbed to alum. The fine specificity of the induced antibodies to the repeat sequence may be critical for protection [18"], and initial attempts to manipulate the specificity of these antibodies have been made using sequence variants of the repeat peptide [19]. The inclusion of defined T helper cell epitopes in the synthetic repeat immunogen may replace more complex protein carriers [20, 21]. Cell mediated immunity may also be critical in protection against sporozoite-induced malaria. In certain combinations of mouse strains and Plasmodium species, pro tection is largely mediated by the CD8 + subset of T lymphocytes. Adoptive transfer experiments have demonstrated that cloned CD8 + CTLs specific for the CS protein conferred high levels of protection [22]. Recent transfer experiments with two sets of CTLs, specific for either P. berghei or P. yoelii CS proteins, have provided evidence that CTL mediated protection involved in vivo activation of the clones by antigen and that the CTL targets were the developing liver stages of the parasite [23"]. CTLs appear to exert the destruction of intrahepatocTtic parasites by a mechanism which involves close cell-tocell contact. In a recent study, the ability of CTL clones exhibiting the same antigen specificity to transfer protection was found to correlate with the expression of high levels of certain adhesion molecules [24"]. Thus, it is likely that protection by transferred CTLs depends upon the ability of CTLs to migrate into the vicinity of the developing liver stage of Plasmodium. From these results, an important consideration in vaccine development would be whether the heterogeneity in the level of expression of adhesion molecules observed in CTLs maintained in vitro also occurs in ongoing CTL responses in viva Protection against sporozoite challenge has been ob tained by oral immunization with a Salmonella recombinant expressing the P. berghei CS gene. Protection in

this model is mediated by CTLs directed against an epitope in the CS protein [25]. The choice of the vector may be critical for the induction of protective immunity. This is illustrated by the failure of a recombinant vaccinia expressing the P. berghei CS gene to induce protection in spite of the presence of anti-CS antibodies and the induction of a mild CTL response [26]. CTL-mediated protection against sporozoite induced malaria has now been achieved in mice immunized with large numbers of tumor cells transfected with either the CS gene or a novel sporozoite surface antigen gene, SSP2 [27"]. Remarkably, these experiments suggest that protection mediated by CTLs exhibiting two independent antigen specificities can be additive. It may be that similar additive effects on protection could be obtained by combining different effector mechanisms, for example, protective CTL and neutralizing antibodies. The first epitope(s) recognized by human CTLs directed against the CS protein of P. falciparum has been located in a polymorphic CS region [8.,28.]. No specific CTL clones have been reported yet, and the presenting HLA molecule(s) have yet to be identified. In the absence of this basic information, it is premature to speculate on the influence of CS polymorphism on human CTL recognition. On the other hand, the polymorphism occurring in the CS protein of wild isolates of P. falciparum has been found to be limited [29,30"]. Detailed analysis of the sequence polymorphism of the P. falciparum CS gene indicates the presence of at least two types of polymorphism [31"]. Other sporozoite or liver stage antigens, i.e. the P. falcip a r u m LSA-1 [32] or the equivalent of the P. yoelii SSP2 in the human malaria parasites, should be examined for the presence of human CTL epitopes. In the meantime, evidence from epidemiological studies in the Gambia suggests a link between a human MHC class I molecule (HLA- Bw53) and protection from severe malaria [33"]. Thus, it is tempting to speculate that HLA-Bw53 may be involved in the presentation of a Plasmodium CTL epitope. It would be desirable to efficiently induCe a targeted CTL response by using recombinant or synthetic peptide vaccines, tt is now clear that CTLs can be induced in mice by immunization with synthetic peptides emulsified in Freund's adjuvant [34] or lipopeptides [35]. Using this approach, it was found that the specificity of the CTL response to P. berghei CS peptides or a lipopeptide was diverse and appeared to require a concomitant T helper response for optimal induction [36"]. The T-cell receptor repertoire in the CTL response to the P. berghei CS epitope was likewise extremely diverse [37"]. The ability of CTL responses elicited by immunization with peptides to induce protection against malaria infection should now be tested. The possible protective role of effector CD4 + T cells has been demonstrated by adoptive transfer of a P. bergheispecific CD4 + T-cell clone that displays cytolytic activity in vitro [38]. Another study has suggested that both CD4 + and CD8 + T cells from mice inoculated with a CS peptide could eliminate parasite-infected hepatocytes in vitro [39]. Immunization of mice with a polymeric pep-

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Immunity to infection tide representing a defined CS T helper epitope induced significant protection against sporozoite challenge in the absence of detectable anti-CS antibodies (P Migliorini, B Betschart, G Corradin, unpublished data). Together, these observations indicate that effector CD4 + T cells may constitute a distinct anti-sporozoite immune mechanism that probably acts against the Ever stages.

Blood stage based vaccines The feasibility of an efficient blood stage based vaccine has been clearly demonstrated. A high degree of protection, which was stage- and species-specific, was induced in monkeys by immunization with enriched P. falciparum merozoite preparations in Freund's adjuvant [40]. Monkeys could also be protected by vaccination with highly purified P. falciparum schizonts in a synthetic adjuvant [41]. In the case of blood stage vaccines, like sporozoite vaccines, an attenuated or killed parasite vaccine is ridden with major difficulties that render it impractical at the present time. Hence the need to obtain a detailed picture of the immune mechanisms of protection operating in clinical immunity and in vaccinated nonhuman primates, and to identify potential parasite target antigens. A large number of blood stage antigens, in contrast to sporozoite antigens, have been characterized at the molecular level due to the efforts of many research groups during the past decade. Recently, the results of several vaccination trials using recombinant or synthetic blood stage products have become available. As detailed below, these are for the most part disappointing. However, valuable information regarding the safety and immunogenicity of a number of candidate vaccines has been obtained.

Ring-infected erythrocyte surface antigen, Pf155/RESA The antigen Pf155/ring-infected erythrocyte surface antigen (RESA) [42] is a non-polymorphic P. falciparum polypeptide of 155kD that is deposited on the erythro cyte membrane during merozoite invasion [42]. It contains two repeat regions. The carboxyl-terminal region of Pf155/RESA contains a region of tandemly repeated sequences (five copies of Glu-Glu-Asn-Val-Glu-His-Asp-Ala and 30 copies of Glu-Glu-Asn-Val). Both repeat regions contain B- and T cell epitopes in humans [43-45]. However, a study of the T-cell response to this antigen in West Africans failed to reveal defined MHC class II associations [46.]. This antigen, as well as other related cross-reactive antigens, appears to be involved in the process of P. falciparum merozoite invasion in vitro and is the target of antibodies blocking parasite invasion [47]. A number of vaccination trials have included P f155/RESArelated immunogens. Affinity-purified human IgO specific for the (Glu-Glu-Asn-Val)2 or the Glu-Glu-Asn-Val-Glu His-Asp-Ala repeats was passively transferred into monkeys and a partial protective effect was observed [48]. Previous vaccination trials of Aotua monkeys using [3galactosidase fused Pf155 fragments in Freund's adjuvant induced protection [49]. The protective effect correlated with antibody responses to some of the Pf155/RESA re-

peats [50]. However, immunization of Aotus monkeys with synthetic Pf155/RESA repeat peptides conjugated to diphtheria toxoid failed to induce protection [51]. Muramyl dipeptide was the adjuvant used in the second trial. Furthermore, the recombinant Pf155/RESA fusion polypeptide administered in Freund's adjuvant that gave protection in the earlier trial failed to induce a protective response when inoculated with muramyl dipeptide adjuvant [51]. However, the observed inverse correlation between antibody titers to the repeat sequence and parasitemia supports the interpretation that protection in this system is largely antibody-mediated and that a high antibody level is required. Moreover, differences among the species of monkeys or the strains of parasites used may explain the discrepancies observed in these trials.

Saimiri monkeys were immunized with recombinant vaccinia expressing either the full length Pf155/RESA or a hybrid S antigen in which Pf155/RESA repeats were inserted, but no protection was detected with either vaccine [52]. In the same trial, a group of monkeys was injected with recombinant vaccinia virus expressing merozoite surface antigens MSA 1, MSA 2 (see below) or AMA-1 antigen in addition to Pf155/RESA. Again, p o o r immunogenicity (in terms of antibody responses) and the lack of protection to challenge with P. falciparum blood stages cast doubts on the usefulness of vaccinia virus as a delivery system. In spite of these negative results, it was encouraging that the antibody response to some of the test antigens could be boosted by a challenge with parasites. Although previous exposure to a wild type vaccinia virus was not documented in this study, such exposure has been shown to inhibit antibody responses to recombinant vaccinia viruses [53]. This difficulty may be overcome, however, by multiple inoculations with the recombinant vaccinia [53]. Finally, a recombinant protein containing (Glu-Glu-Asn-Val)8, one of the Pf155/RESA re: peats, covalently coupled to preformed influenza virus envelope glycoprotein liposomes, has been found to be a good immunogen in rabbits [54]. Merozoite surface antigens MSA 1 One of the most promising antigens for a blood stage candidate vaccine is the major merozoite surface antigen1 (MSA 1) [55]. Three related processing products of the MSA 1 polypeptide have been identified on the surface of merozoites. The MSA 1 of P. falciparum laboratory strains and isolates collected in geographically diverse endemic areas contains both highly polymorphic and highly conserved domains [56~50]. The mouse MSA 1 homologue has been shown to be a target of protective antibodies. Immunization of Aotus monkeys with purified P. falcip a r u m MSA 1 like material induced protective immunity against a challenge with infected red blood cells. These results have now been confirmed and extended to a different species of monkey, Saimiri; these monkeys were protected against challenge with a heterologous strain of P. falciparum by immunization with affinity purified native MSA 1 antigen [61"]. In the same study, recombinant potypeptides representing the MSA 1 conserved regions

Malaria vaccines Romero induced partial protection in Saimiri monkeys; however, the degree of protection was lower than that obtained with the native MSA 1. In another study, the degree of protection induced by immunization of Aotus monkeys with a conserved MSA 1 recombinant polypeptide (190 L) was significantly enhanced by the splicing of that region to a well defined P. falciparum CS T helper cell epitope [62"]. This epitope, CS.T3 [63], in fact interacts with most of the HLA-DR molecules tested and corresponds to a conserved region in the P. falciparum CS protein. The enhancing effect on protection induced by the 190 L-CS.T3 recombinant fusion protein cannot readily be explained in terms of a helper effect mediated by the addition of the CS.T3 epitope for two reasons: first, the recombinant MSA 1 fragment (190 L) itself contains multiple human T-cell epitopes, some of them binding to several HLA-DR molecules [64]; and second, the antibody levels measured in monkeys vaccinated with 190 L-CS3.T3 were similar to the antibody levels induced by immunization with 190 L. Moreover, protection did not correlate with the antibody response to either the immunogens or the par asites. Interestingly, enhanced protection correlated with elevated levels of circulating interferon (IFN)-7 at the time of challenge. The authors consider it likely that protection was T-cell-mediated through the release of IFN-7. If IFN-7 is mediating the protective effect observed with this vaccine one may expect the antiparasitic effect to be rather non-specific. This could be tested by challenge with P. vivax blood stages. It has been demonstrated that the liver stages of Plasmodia are inhibited by exposure to small amounts of IFN-7 [65]. A vaccine inducing high levels of circulating IFN-y would, in principle, limit the hepatic phase of development of sporozoites inoculated by infected mosquitoes in an endemic area. This possibility could also be tested experimentally in the monkey model. The influence of different adjuvants on the specificity of the antibody response to MSA 1 was explored in mice [66]. One group used a hybrid HBsAg, the major immunogenic domain of which was substituted with MSA 1 sequences. This hybrid HBsAg, which was still able to form particles, was delivered as a recombinant vac cinia virus vaccine. The construct was immunogenic and the antibodies elicited recognized parasite-derived MSA 1 [67]. The gene corresponding to the P. vivax MSA 1 has now been fully sequenced [68"]; thus, the way is clear for MSA 1 vaccine development against P. vivax malaria. MSA 2

The second well characterized merozoite surface antigen (MSA 2) varies in size between 45 and 55 kD and contains a highly diverse central region of approximately 160 amino acids. This region is made up of variable repetitive amino acid sequences which can be assigned to two main types [59,69-71]. Flanking the variable region there are two invariable domains. Diphtheria toxoid conjugates, containing synthetic peptides representing these highly conserved MSA 2 stretches, were used to immunize mice. The elicited anti-peptide antibodies also recognized parasites and significant protection was observed

after challenge with P. chabaudL Protection correlated with antibody levels [72~ Recombinant MSA 2 proteins were capable of inducing proliferative and T helper cell responses in mice and defined T cell epitopes have been mappped to both amino- and carboxyl-terminal conserved MSA 2 regions [73]. The antibody response induced against an allellc form of MSA 2 in Saimiri monkeys vaccinated with recombinant vaccinia was efficiently boosted by parasites carrying the alternative MSA 2 allele [52]. Taken together, these results underline the enormous potential of MSA 2, in particular its conserved sequences, for vaccine development. Trials with other blood stage antigens The so-called glycophorin binding protein (GBP)130, a soluble protein present in the parasitophorous vacuole, contains a highly conserved amino-terminal region and a stretch of 11 tandem repeat units of 50 amino acids each. A recombinant polypeptide containing three repeat units was immunogenic in experimental animals but failed to induce protection in monkeys. However, immunoglobulin fractions of immune serum from monkeys that recognize GBP 130 failed to react with the immunizing recombinant polypeptide [74]. Thus, it is possible that failure to induce protection resulted from the lack of production of antibodies with the appropriate specificity, as was shown to be the case for a P. vivax CS vaccine [18"]. Antigen 5.1 (also known as Exp-1 or QF 116) is expressed in the parasitophorous vacuole membrane and in the cytoplasm of infected erythrocytes. Immunization of Saimirimonkeys with recombinant 5.1 resulted in partial protection from challenge with P. falciparum blood stages [75]. In the same study, a second recombinant 5.1 antigen was designed to contain a (Asn-Ala-Asn-Pro)50 (representing a P. falciparum CS repeat) polypeptide. The new protein was immunogenic in mice and monkeys but failed to induce protection against challenge with P. falciparum sporozoites in a small group of volunteers. Two protective blood stage antigens, P. falciparum serine-rich protein and histidine-rich protein II, have been incorporated in a novel delivery system. The antigens are expressed on the surface of Salmonella phimurium as Escherichia coli outer membrane protein II (omp A) fusion proteins [76~ Oral immunization of mice resulted in the induction of specific IgM and IgG antibodies. The P. falciparum rhoptry complex has been shown to protect Saimiri monkeys [77]. The molecular identity of three of the components of this complex has now been clarified through gene cloning and sequencing. Two unrelated genes encode the rhoptry associated proteins 1 (p80) and 2 (p42) (RAP-1 and RAP-2), and the third molecular species (p65) arises from the proteolytic cleavage of the RAP-1 gene product [78",79"]. Future analyses should determine which of these molecules induces protective immunity. The P. falciparum synthetic vaccine, SPf 66 As pointed out by others [1"], much attention has been focused on a synthetic polymeric blood stage vaccine

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which induced significant protection in a small group of volunteers [80]. This is the first malaria vaccine that has undergone extensive field trials, most of them in Colombia. The relative lack of information regarding these trials, which has been criticized by others [ 1",81], is now being rectified [82,83,84..,85~ The synthetic vaccine SPf 66 is a polymer built from a unit containing three short peptides representing sequences from three P. falciparum blood stage antigens. The peptides SPf 35.1 and SPf 55.1 were based on partial sequences obtained from 2 as yet unidentified P. falciparum molecules. The third peptide, SPf 83.1, corresponds to P. falciparum MSA 1 region 43-53, a highly conserved region according to Tanabe et al. [90]. The m o n o m e r unit of the SPf 66 is synthesized as shown in Fig. 1. Polymerization is achieved by oxidation under a stream of oxygen, but a lot of arguments have been put forward concerning the reproducibility of this procedure. SPf 35.1

Tyr-Gly-Gly-Pro-Ala-Asn-Lys-Lys-Asn-Ala-Gly SPf 55.1

Asp-Glu-Leu-Glu-Ala-Glu-Thr-Gln-Asn-VaI-Tyr-Ala-Ala

were detected in most of the individuals after receiving a third inoculation. Selected sera were shown to recognize several parasite-derived polypeptides and, strikingly, to inhibit parasite growth in vitro. These results indicate that the SPf 66 vaccine is safe and immunogenic in humans. HLA typing in a subgroup of 115 volunteers from the same two trials revealed a correlation between a low antibody (IgG) responder phenotype group and HLADR4 suggesting that the immune response to SPf 66 is under genetic control [85"]. In the initial human vaccination trial with SPf 66 [80], specific proliferative responses from the peripheral blood lymphocytes of some volunteers were recorded. More recently, a panel of T-cell clones from vaccinees has been analyzed for the expression of the T-cell receptor V[3 genes [86]. From these and two other trials involving 352 additional volunteers subjected to different immunization protocols, the authors conclude that the best immunization schedule consists of three 2 mg doses of SPf 66 in alum administered at days 0, 30 and 180 [87]. Attempts to document protection from natural exposure to malaria after immunization with this SPf 66 vaccine [89] are as yet unconvincing and await the results of new trials currently underway in Colombia and possibly future independent trials elsewhere.

SPf 83.1

Tyr-Ser-teu-Phe-GIn-Lys-Gtu-Lys-Met-Val-Leu Structure of monomer unit in the polymer synthetic vaccine SPf 66

Cys-Gly - (SPf 55.1) - Pro-Asn-Ala~Asn-Pro (SPf83.1) - Pro-AsnAla-Asn-Pro - (*SPf 35.1) - Cys

Fig.1. Amino acid sequences of peptides from P. falciparum blood stage antigens and the construction of the synthetic vaccine SPf 66. Peptide SPf 35.1 corresponds to the 11 amino-termial amino acids of a 35 kD protein and peptide SPf 55.1 to the 13 amino-terminal amino acids of a 55 kD protein [80]. Peptide SPf 83.1 is an 11-amino acid synthetic peptide corresponding to residues 43-53 of the MSA 1 antigen. The building unit of the SPf 66 polymer vaccine is a 45 amino acid residue long hybrid synthetic peptide that includes the sequences of peptide SPf 55.1, of peptide SPf 83.1 and the 8 carboxyl-terminal residues of peptide SPf 35.1. These sequences are spaced by two pentapeptides (Pro-Asn-AlaAsh-Pro). One Cys residue is added at each end of the hybrid peptide. Upon oxidation, a variety of polymers is generated in which individual hybrid peptides would be joined through the disulphide bridges formed between pairs of Cys residues. *The first three amino terminal residues of the peptide 5Pf 35.1 are omitted.

Immunization with a cocktail of the three SPf peptides, conjugated independently to bovine serum albumin, or with SPf 66, induced protection in Aotus monkeys [82]. However, no protection was detected with either product in an independent trial although the observation that the peptides were immunogenic was partially reproduced [83]. The crucial test for the vaccine is for it to work in humans. The magnitude of the humoral immune response induced in a total of 185 volunteers who received two immunizations of SPf 66 (each consisted of 2 mg adsorbed onto alum) was found to be variable [84~176However, significant antipeptide serum IgG levels

The same group has also tried to address the mechanism involved in the antiparasite effect of the SPf 66 vaccine. They have now found that several of a large panel of P. falciparum blood stage-derived synthetic peptides bind to human red blood cells and that their ability to bind appears to correlate with the presence of certain peptide motifs [88"~ Of note, two of the three peptides built into the SPf 66 polymer (SPf 55.1 and SPf 83.1) bind to red blood cells and, in addition, the peptide equivalent to SPf 55.1 inhibited parasite invasion of red blood cells in vitro. The relevant question now is whether this phenomenon relates in any way to parasite invasion of, and/or parasite survival inside, the red blood cell. The fine specificity of some anti-SPf 83.1 polyclonal antibodies raised in volunteers vaccinated with SPf 66 has also been characterized. Analyses with single amino acid-substituted SPf 83.1 peptides suggest that a pair of charged amino acid residues in the middle of the peptide sequence, Lys-Glu, may be a critical part of the epitope(s) [89]. Since peptide SPf 83.1 binds to human red blood cells, it would be of interest to determine the relative red blood cell binding activities of the SPf 83.1 variant peptides utilized for the analysis of antibody fine specificity. It also remains to be determined whether the antibody fraction specifically recognizing peptide SPf 83.1 may affect parasite growth in vitro. In this regard, the existence of a parasitophorous duct that connects the parasitophorous vacuole with the red blood cell membrane has been noted. Macromolecules can reach the parasitophorous vacuole through the duct and then the trophozoite can import them by endocytosis [91"]. The existence of this substructure may provide not only an explanation for the inhibitory effect of certain antibodies but also a new target for immunological attack against Plasmodium blood stages.

Malaria vaccines Romero

Conclusion The field of malaria vaccines is very active with a large number of vaccine candidates now at different stages of development. There are probably many more in the pipeline. Improvement of the existing candidate vaccines may be feasible through the use of more potent adjuvants and efficient delivery systems. Attenuated live vectors including Salmonella or Mycobacterium BCG are among the promising delivery systems. The pace of malaria vaccine development has been accelerated by advances in understanding the mechanisms underlying naturally acquired immunity in humans and experimentally induced immunity in animal models. It is not surprising that cell-mediated immunity may play a major role in protective responses. In a relatively short time, it has become clear that sporozoite-induced CTL responses are protective. Furthermore, the targets of this CTL response are not sporozoites themselves but the next stage of development, the liver stage of the parasite. Induction of sporozoite-induced CTL responses may prove to be an efficient strategy to eliminate the parasite for a number of reasons. First, many more sporozoite antigens, surface or internal, may be good CTL targets in the liver stages. Second, specific liver stage antigens may induce CTL responses as well. And finally, several blood stage antigens, such as MSA 1, are expressed during the liver stage of parasite development; both antibody and Tcell responses directed against these antigens might be as efficient, or even more efficient, against liver stages than against blood stages. There is still a long way to go before an accurate picture of the mechanisms of protective immunity against blood stage of the parasite is obtained. For example, the issue of the role of the spleen has been recendy raised and it is the subject of intense study [92"']. It is likely that while antibodies play a major role in protection, other non-antibody pathways will be unveiled.

Acknowledgements I would like to gratefully acknowledge JL Maryanski and J-C Cerottini for helpful discussions and critical reading of the manuscript mad RS Nussenzweig for her constant support and encouragement.

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MORENO A, CLAVIJO P, EDELMAN R, DAVIS J, SZTEIN M, HERRINGTOND, NARDIN E: Cytotoxic CD4 + T Cells from a Sporozoite-immunized Volunteer Recognize the Plasmodi u m f a l c i p a r u m CS Protein. Int Immunol 1991, 3:99~1003. Reports the isolation of human CEM + CTL clones from a volunteer immunized with P. falciparum irradiated sporozoites. The epitope corresponds to the CS protein and is defined with synthetic peptides that are recognized in the context of HLA-DR7. 6.

NARDINE, CLAVIJOP, MONS B, VAN BELKUM A, PONNUDURAIT, NUSSENZWEIG RS: T Cell Epitopes of the C i r c u m s p o r o z o i t e Protein of P l a s m o d i u m vivax. Recognition by Lymphocytes of a Sporozoite-immunized Chimpanzee. J Immunol 1991, 146:1674-1678.

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MILLETP, COLLINS WE, BRODERSON JR, BATHURST I, NARDIN EH, NUSSENZWE1GRS: Inhibitory Activity against Plasmodium v i v a x Sporozoites Induced by Plasma from Saimiri Monkeys Immunized with Circumsporozoite Recombinant Proteins or Irradiated Sporozoites. A m J Trop Med Hyg 1991, 45:44-48.

8. .

MALIK A, EGAN JE, HOUGHTEN RA~ SADOFFJC, HOFFMAN SL: Human Cytotoxic T Lymphocytes Against the P l a s m o d i u m f a l c i p a r u m Circumsporozoite Protein. Proc Natl Acad Sci USA 1991, 88:3300-3304. Characterization of the first human CTL epitope(s) on the/~ falcgoarum CS protein. 9. ~

HERRINGTOND, DAVISJ, NARDINE, BE1ER M, CORTESEJ, EDDY H, LOSONSKYG, HOLLINGDALEM, SZTEIN M, LEVINE M, ET AL: Successful Immunization of Humans with Irradiated Malaria Sporozoites: H u m o r a l and Cellular R e s p o n s e s of the Protected Individuals. Am J Trop Med Hyg 1991, 45:539-547. This study confirms classical experiments conducted in the 1970s. Induction of complete protection appeared to require frequent exposure to large numbers of infected and irradiated mosquitoes. 10.

WARBURGA, MILLERLH: Sporogonic Development of a Malaria Parasite in Vitro. Science 1992, 255:448-450. T~is is the first time that the complete development of the sporogonic cycle of a Plasmodium parasite is achieved in vitro. This study opens the possibility of studying the factors important for sexual development of the parasite in the mosquito. It might also allow the regular production of sporozoites in culture. 11.

HERRINGTONDA, CLYDEDF, LOSONSKYG, CORTESIAM, MURPHY JR, DAVISJ, BAQAR S, FELIX AM, HEIMEREP, ET AL: Safety and Immunogenicity in Man of a Synthetic Peptide Malaria Vaccine against P l a s m o d i u m f a l c i p a r u m Sporozoites. Nature 1987, 328:257-259.

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BALLOUWR, HOFFMAN SL, SHERWOOD JA, HOLLINGDALE MR, NEVA FA, HOCKMEYERWT, GORDON DM, SCHNEIDER I, WIRTZ R& ET AL.: Safety and Efficacy of a Recombinant DNA P l a s m o d i u m f a l c i p a r u m Sporozoite Vaccine. Lancet 1987, 1:1277-1281.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: 9 of special interest 99 of outstanding interest 1. 9

HOMMELM: Steps Toward a Malaria Vaccine. Res Immunol 1991, 142:618-738.

see [2-]. 2. 9

HOFFMANSL, NUSSENZWEIG V, SADOFF JC, NUSSENZWEIG RS: Progress Toward Malaria Preerythrocytic Vaccines. Science 1991, 252:520-521. This together with [1% 3] is an excellent review of the topic. 3.

NUSSENZWEIGV, NUSSENZWEIG R: Rationale for the Development of an Engineered Sporozoite Malaria Vaccine. Adv lmmunol 1989, 45:283-334.

NARDINEH, HERRINGTON DA, DAVISJ, LEONE M, STUBER D, TAKACS B, GASPERS P, BARR P, ALTSZULER R, CLAVIJO P, ET ~.: Conserved Repetitive Epitope Recognized by CD4 + Clones from a Malaria Immunized Volunteer. Science 1989, 246:1603 1606.

13. 9

SHERWOODJA, OSTER CN, ADOYO-ADOYOM, BEIERJC, GACHIHI GS, NYAKUNDI PM, BALLOU WR, BRANDLING-BENNETT AD, SCHWARTZ IK, WERE JBO, ET AL.: Safety and Immunogenicity of a P l a s m o d i u m f a l c i p a r u m Sporozoite Vaccine: Boosting of Antibody Response in a Population with Prior Natural Exposure to Malaria. Trans R Soc Trop Med Hyg 1991, 85:336-340. Demonstrates that administration of the recombinant vaccine R32tet32 boosts the anti-CS antibody response present in volunteers living in an area where malaria is endemic. 14.

HERRINGTONDA, NARD1NEH, LOSONSKYG, BATHURSTIC, BARR PJ, HOLL1NGDALEMR, EDELMANR, LEVINE MM: Safety and Immunogenieity of a Recombinant Sporozoite Malaria Vaccine Against P l a s m o d i u m vivax. Am J Trop Med Hyg 1991, 45:695-701.

437

438

I m m u n i t y to i n f e c t i o n 15.

VREDEN SGS, VERHAVE JP, OETTINGER T, SAUERWEIN RW, MEUW1SSEN JHET: Phase I Clinical Trial of a Recombinant Malaria Vaccine Consisiting of the Circumsporozoite Repeat Region of P l a s m o d i u m f a l c i p a r u m Coupled to Hepatitis B Surface Antigen. Am J Trop Med Hyg 1991, 45:533~538.

16. 9

PaCKMANiS, GORDON DM, W[STARJR R, KRZYCH U, GROSS M, HOLLINGDALEMR, EGAN JE, CHULAY JD, HOFFMAN SL: Use of Adjuvant Containing Mycobacterial Cell-wall Skeleton, Monophosphoryl Lipid A, and Squalane in Malaria Circumsporozoite Protein Vaccine. Lancet 1991, 337:998-1001. See [17"]. 17. 9

FRIESLF, GORDON DM, PdCHARDS RL, EGAN JE, HOLLINGDALE MR, GROSS M, SILVERMANC, ALVING CR: Liposomal Malaria Vaccine in Humans: A Safe and Potent Adjuvant Strategy. Proc Natl Acad Sci USA 1992, 89:358-362. This together with [16"] shows that adjuvants more potent than alum greatly improve the immunogenicity, in terms of the antibody response, of a recombinant P. falc~arum CS vaccine. 18. 9

CHAROENVITY, COLLINS WE, JONES TR, MILLET P, YUAN L, CAMPBELLGH, BEAUDOINRL, BRODERSONJR, HOFFMAN SL: Inability of Malaria Vaccine to Induce Antibodies to a Protective Epitope within Its Sequence. Science 1991, 251:6684571. This study indicates that the fine specificity of the antibody response to a recombinant P. vivax CS vaccine may be critical for protection. 19.

ETLINGERHM, REN1A L, MATtLE H, MANNEBERG M, MAZIER D, TRZECIAK A, GILLESSEN D: Antibody Responses to a Synthetic Peptide-based Malaria Vaccine Candidate: Influence of Sequence Variants of the Peptide. E u r J lmmunol 1991, 21:1505-1511.

20.

KIRONDEFAS, RAO KVS, SHAH S, KUMARA, SAHOO N: Towards the Design of Heterovalent Anti-malaria Vaccines: a Hybrid Immunogen Capable of Eliciting Immune Responses to Epitopes of Circumsporozoite Antigens from Two Different Species of the Malaria Parasite, Plasmodium. Immunol 1991, 74:323-328.

21.

VALMORID, PESSI A, B1ANCHI E, CORRADING: Use of Human Universally Antigenic Tetanus Toxin T Cell Epitopes as Carriers for Human Vaccination. J Immunol 1992, in press.

22.

ROMERO P, MARYANSK] JL, CORRADIN G, NUSSENZWEIG RS, NUSSENZWEIG V, ZAVALAF: Cloned Cytotoxic T Cells Recognize an Epitope in the Circumsporozoite Protein and Protect Against Malaria. Nature 1989, 341:323-326.

23. 9

RODRIGUESMM, CORDEYA-S, ARREAZAG, CORRAD1NG, ROMERO P, MARYANSK/JL, NUSSENZWEIGRS, ZAVALAF: CD8 + Cytolytic T Cell Clones Derived Against the P l a s m o d i u m yoelii Circumsporozoite Protein Protect Against Malaria. Int Immunol 1991, 3:579-585. A study of the species-specificity of the protective effect against sporozoite challenge transferred by cloned CTL specific for the CS protein. The evidence indicates that CTL inhibition of liver stage development requires in vivo activation by antigen. 24. 9.

RODRIGUESM, NUSSENZWEIGRS, ROMERO P, ZAVALAF: The in Vivo Cytotoxic Activity of CD8 + T Cell Clones Correlates with Their Levels of Expression of Adhesion Molecules. J Exp Med 1992, 175:895-905. Exploiting the same system of adoptive transfer of protection by certain CS-specific CTLs [22, 23"], this study shows that the expression of high levels of adhesion molecule CD44 on CTLs correlates with abil ity to protect against malaria. This study has important implications for immunotherapy. 25.

AGGARWALA, KUMARS, JAFFE R, HONE D, GROSS M, SADOFFJ: Oral Salmonella. Malaria Circumsporozoite Recombinants Induce Specific CD8 + Cytotoxic T Cells. J Exp Med 1990, 172:1083-1090.

26.

SATCHIDANANDAM V, ZAVALAF, MOSS B: Studies Using a Recombinant Vaccinia Virus Expressing the Cirmsporozoite Protein of P l a s m o d i u m berghei. Mol B i ~ m Parasitol 1991, 48:89-100.

27. ..

KHUSMITHS, CHAROENVITY, KUMARS, SEDEGAH M, BEAUDOIN RIO HOFFMANSL: Protection Against Malaria by Vaccination with Sporozoite Surface Protein 2 plus CS Protein. Science 1991, 252:715~18. Reports that a second sporozoite surface antigen can be the target of protective CTLs. The authors also demonstrate that two CTL responses to diffferent antigens have an additive effect on protection against malaria. 28. .

DOOLANDL, HOUGHTEN RA, GOOD MF: Location of Human Cytotoxic T Cell Epitopes within a Polymorphic Domain of the P l a s m o d i u m f a l c i p a r u m Circnmsporozoite Protein. Int Immunol 1991, 3:511-516. First location of an epitope recognized by human CTLs within a polymorphic CS region. See also [8"]. 29.

YOSHIDA N, DI SANTI SM, DUTRA AP, NUSSENZWEIG RS, NUSSENZWEIG V, ENEA V: P l a s m o d i u m falciparur~. Restricted Polymorphism of T Cell Epitopes of the Circumsporozoite Protein in Brazil. Exp Parasitol 1990, 71:386-392.

30. .

DOOLANDL, SAUL AJ, GOOD MF: Geographically Restricted Heterogeneity of the P l a s m o d i u m f a l c i p a r u m Circumsporozoite Protein: Relevance for Vaccine Development. Infect I m m u n 1992, 60:675~682. See [31"]. 31. 9

MCCUTCHANTF, LALAA, DO ROSAmOV, WATERSAP: Two Types of Sequence Polymorphism in the Circumsporozoite Gene of P l a s m o d i u m falciparum. Mol Biochem Parasitol 1992, 50:37-46. This paper together with [29] and [30*] reports that the polymorphism in the P. falciparum CS gene is restricted. Polymorphism, therefore, may not represent an insurmountable obstacle in the development of a CS based malaria vaccine. 32.

ZHU J, HOLLINGDALE MR: Structure of P l a s m o d i u m falcip a r u m Liver Stage Antigen-1. Mol Biochem Parasitol 1991, 48: 223-226.

33. .

HILL AVS, ALLSOPP CEM, KWIATKOWSKID, ANSTEYNM, TWUMASl P, ROWE PA, BENNETT S, BREWSTER D, McMICHAEL AJ, GREENWOOD BM: C o m m o n West African HLA Antigens Are Associated with Protection from Severe Malaria. Nature 1991, 352:595400. This is an epidemiological survey, conducted in an area with high malaria transmission, showing that the human leukocyte class I antigen, HLA-Bw53, and an HLA class II haplotype (DRB 1"1302-DQBl*0501 ) are independently associated with protection from severe malaria. 34.

AICHELEP, HENGARTNERH, ZINKERNAGELRM, SCHULZ M: Antiviral Cytotoxic T Cell Response Induced by in Vivo Priming with a Free Synthetic Peptide. J Exp #led 1990, 171:1815-1820.

35.

DERESK, SCHILDH, WIESMIJLLERK-H, JUNG G, RAMMENSEEH-G: In Vivo Priming of Virus-specific Cytotoxic T Lymphocytes with Synthetic Lipopeptide Vaccine. Nature 1989, 342:561-564.

36.

ROMEROP, EBERL G, CASANOVAJ-L, CORDEY A-S, W[DMANNC, LUESCHERIF, CORRADING, MARYANSKIJL: Immunization with Synthetic Peptides Containing a Defined Malaria Epitope Induces a Highly Diverse CTL Response. Evidence that Two Peptide Residues are Buried in the MHC Molecule. J Immunol 1992, 148:1871-1878. See [37"]. 9

37. 9

CASANOVA J-L, ROMERO P, WIDMANNC, KOUmtSKYP, MARYANSVa JL: T Cell Receptor Genes in a Series of Class I Major Histocompatibility Complex-restricted Cytotoxic T Lymphocyte Clones Specific for a P l a s m o d i u m berghei Nonapeptide: Implications for T Cell Allelic Exclusion and Antigen-specific Repertoire. J Exp Med 1991, 174:1371 1383. This study and [36*] constitute the first detailed analysis of the T-cell repertoire for a parasite antigen. The CTL response against a P. berghei CS epitope was found to display a high degree of diversity, both in terms of fine specificity of recognition and of the structure of the T cell receptor.

Malaria vaccines R o m e r o 38.

VK, ET AL: Immunization of Owl Monkeys with the Ringinfected Erythrocyte Surface Antigen of Piasmodium falciparum. Am J Trop Med Hyg 1991, 44:34-41.

Tsuji M, ROMERO P, NUSSENZWEIGRS, ZAVALAF: CD4 + Cytolytic T Cell Clone Confers Protection against Murine Malaria. J Exp Med 1990, 172:1353-1357.

39.

RENIAI. MARUSSIGMS, GRILLOTD, PIED S, CORRADING, MILTGEN E, DEL GIUDICE G, MAZIER D: In vitro Activity of CO4 + and CD8 + T Lymphocytes from Mice Immunized with a Synthetic Malaria Peptide. Proc Natl Acad Sci USA 1991, 88:7963-7967.

40.

MITCHELLGH, PdCHARDSWHG, BUTCHER GA, COHEN S: Merozoite Vaccination of Douroucouli Monkeys against Falciparum Malaria. Lancet 1977, I:1335-1338

41.

SIDDIQUIWA, KAN S~C, KRAMERK, CASE S, PALMERK, NIBLACK .IF: Use of a Synthetic Adjuvant in an Effective Vaccination of Monkeys Against Malaria. Nature 1981, 289:64-66.

42.

PEREMANN P, BERZINS K, PERLMANN H, TROYE-BLOMBERG M, WAHLGREN M, WAHIaN B: Malaria Vaccines:lmmunogen Selec tion and Epitope Mapping. Vaccine 1988, 6:183-187.

43.

RZEPCZYKCM, RAMASAMYR, HO P, MUTCH DA, ANDERSONKL, DUGGELBY RG, DORAN TJ, MURRAYBJ, IRVINGDO, WOODROW GC, PARKINSOND, ETAL: Identification of T Epitopes within a Potential Plasmodium f a l c i p a r u m Vaccine Antigen. J Immunol 1988, 141:3197-3202.

44.

45.

TROYE-BLOMBERGM, RILEY EM, PERLMANN H, ANDERSSON G, IAIZSSON A, SNOW RW, ALLEN SJ, HOUGHTEN RA, OLERUP O, GREENWOOD BM, ET AL.: T and B cell Responses of Plasmodium f a l c i p a r u m Malaria-immune Individuals to Synthetic Peptides Corresponding to Sequences in Different Regions of the Plasmodium f a l c i p a r u m Antigen Pf155/RESA. J Immunol 1989, 143:3043-3048. CHOUGNETC, TROYE-BLOMBERG M, DELORON P, KABILAN L, LEPERSJP, SAVELJ, PERLMANN P: Human Immune Response in Plasmodium f a l c i p a r u m Malaria. Synthetic Peptides Corresponding to Known Epitopes of the Pf155/RESA Antigen Induce Production of Parasite-specific Antibodies in Vitro. J Immunol 1991, 147:2295-2301.

TROYE-BLOMBERG M, OLERUP O, LARSSON /g SJOBERG K, PERLMANNH, RILEY E, LEPERS J-P, PERLMANN P: Failure to Detect MHC Class II Associations of the Human Immune Response Induced by Repeated Malaria Infections to the Plasmodium f a l c i p a r u m Antigen Pf155/RESA. Int Immunol 1991, 3:1043-1051. This study suggests that the expected impact of MHC class II gene prod ucts in the human immune response to defined Pf155/RESA epitopes is not apparent in outbred populations. Data from twin pairs clearly indicate that other non-HLA coded factors may regulate the specific immune responses.

52.

PYE D, EDWARDS SJ, ANDERS RE, O'BRIEN CM, FRANCHINA P, CORCORAN LN, MONGER C, PETERSON MG, VANDERBERG KL, SMYTHE JA, ET ,1l.: Failure of Recombinant Vaccinia Viruses Expressing Plasmodium f a l c i p a r u m Antigens to Protect Saimiri Monkeys Against Malaria. I n f l m m u n 1991, 59:2403-2411.

53.

ETLINGERHM, ALTENBURGERW: Overcoming Inhibition of Antibody Responses to a Malaria Recombinant Vaccinia Virus Caused by Prior Exposure to Wild Type Virus. Vaccine 1991, 9:470-472.

54.

SJOLANDERA, LOVGREN K, STAHL S, ~LUND L, HANSSON M, NYGRENP-A, LARSSONM, HAGSTEDTM, W.~d-ILINB, BERZINSK, ET AL.: High Antibody Responses in Rabbits Immunized with Influenza Virus ISCOMs Containing a Repeated Sequence of the Plasmodium f a l c i p a r u m Antigen Pf155/RESA. Vaccine 1991, 9:443~i50.

55.

HOLDERA& The Precursor to Major Merozoite Surface Antigens: Structure and Role in Immunity. Prog Allergy 1988, 41:72-97.

56.

MCBRIDEJS, NEWBOLD CI, ANAND R: Polymorphism of a High Molecular Weight Schizont Antigen of the Human Malaria Parasite Plasmodium falciparum. J Exp Med 1985, 161:160-180.

57.

SCHERFA, MATrEI D, SARTHOUJ-L: Multiple Infections and Unusual Distribution of Block 2 of the MSA 1 Gene of Plasmodium f a l c i p a r u m Detected in West African Clinical Isolates by Polymerase Chain Reaction. Mol Biochem Parasitol 1991, 44: 29~300.

58.

BABIKERHA, CREASEYAM, FENTONB, BAYOUMIRAL, ARNOTDE, WALLtKER D: Genetic Diversity of Plasmodium f a l c i p a r u m in a Village in Eastern Sudan. 1. Diversity of Enzymes, 2DPAGE Proteins and Antigens. Tram R Soc Trop Med Hyg 1991, 85: 572-577.

59.

SNEWINVA, HERRERA M, SANCHEZ G, SCHERF A, LANGSLEYG, HERRERA S: Polymorphism of the Alleles of the Merozoite Surface Antigens MSA1 and MSA2 in Plasmodium falcg p a r u m Wild Isolates from Colombia. Mol Biochem Parasitol 1991, 49:265~276.

60.

CONWAYDJ, ROSARIOV, ODUOLAAMJ, SALAKOLA, GREENWOOD BM, MCBRIDE JS: Plasmodium falciparum. Intragenic Recombination and Nonrandom Associations between Polymorphic Domains of the Precursor to the Major Merozoite Surface Antigens. Exp Parasitol 1991, 73:469-480.

46. 9

47.

W~LIN B, SJOLANDER A, AHLBORG N, UDOMSANGPETCH R, SCHERF A, MATnlI D, BERZINS K, PERLMANN P: Involvement of Pf155/RESA and Cross-reactive Antigens in Plasmodium falciparum Merozoite Invasion in Vitro. Inf lmmun 1992, 60:443-449.

48.

BERZINS K, PERLMANN H, WAHLIN B, EKRE H-P, HOGH B, PETERSEN E, WELLDE B, SCHOENBECHLERM, WILLIAMSJ, CHULAY J, PEREAMNNP: Passive Immunization of Aotus Monkeys with Human Antibodies to the Plasmodium f a l c i p a r u m Antigen Pf155/RESA. I n f l m m u n 1991, 59:1500-1506.

49.

COLHNSWE, ANDERSRE, PAPPAIOANOUM, CAMPBELLGH, BROWN GV, KEMP DJ, COPPEL RL, SKINNERJC, ANDRYSIAKPM, FAVALORO JM, ET AL.: Immunization of Aotus Monkeys with Recombinant Proteins of an Erythrocyte Surface Antigen of Plasmodium falciparum. Nature 1986, 323:259-262.

50.

COLLINSWE, PAPPAIOANOUM, ANDERSRE, CAMPBELLGH, BROWN GV, KEMP DJ, BRODERSONJR, COPPEL RL, SKINNERJC, PROCELL PM, ET AL: Immunization Trials with the Ring-infected Erythrocyte Surface Antigen of Plasmodium f a l c i p a r u m in Owl Monkeys (Aotus vociferans). Am J Trop Meal Hyg 1988, 38:26~282.

51.

COLLINSWE, ANDERS RE, RUEBUSHII TK, KEMP DJ, WOODROW GC, CAMPBELLGH, BROWN GV, IRVING DO, GOSS N, F1L1PSKI

61. .

ETL1NGERHM, CASPERS P, MATILEH, SCHOENFELDH-J, STUEBER D, TAKACS B: Ability of Recombinant or Native Proteins to Protect Monkeys against Heterologous Challenge with Plasmodium falciparum. Inf Immun 1991, 59:3498-3503. This study confirms that immunization of monkeys with native P. falciparum MSA 1 protein induces a high degree of protection. Recombinant MSA 1 fragments carrying conserved sequences were also protective; however, protection was less efficient than that induced by native MSA 1. Protection correlated with levels of anti-parasite antibody elicited by vaccination with different MSA 1 antigen preparations. 62.

HERRERAMA, ROSERO F, HERRERA S, CASPERS P, ROTMANN D, S1N1GAGLIAF, CERTA U: Protection Against Malaria in Aotus Monkeys Immunized with a Recombinant Blood-stage Antigen Fused to a Universal T-cell Epitope: Correlation of Serum Gamma Interferon Levels with Protection. l n f Immun 1992, 60:154-158. The partial protection induced in monkeys by immunization with a re combinant conserved MSA 1 fragment is augmented by fusing the MSA 1 fragment to a universal /3. falciparum CS T-cell epitope. Protection did not correlate with antibody responses. Instead, it correlated with the levels of IFN-y present in the serum on the day of challenge with parasite. 9

439

440

Immunity to infection 63.

SINIGAGLIAF, GuTrINGER M, KILGUSJ, DORAN DM, MATILE H, ETLINGERH, TP,ZECL~KA, GILLESEND, PINKJRL: A Malaria T-cell Epitope Recognized in Association with Most Mouse and Human MHC Class II Molecules. Nature 1988, 336:778-780.

64.

GUTTINGERM, ROMAGNOLIP, VANDELL, MELOEN R, TAKACSB, PINKJRL, SINIGAGLIAF: HLA Polymorphism and T Cell Recognition of a Conserved Region of p190, a Malaria Vaccine Candidate. Int I m m u n o l 1991, 3:899-906.

65.

FERREIRAA, SCHOFIELD L, ENEA V, SCHELLEKENS H, VAN DER MEIDE P, COLLINS WE, NUSSENZWEIG RS, NUSSENZWE1G V: Inhibition of Development of Exoerythrocytic Forms of Malaria Parasites by Gamma Interferon. Science 1986, 232:881-884.

66.

67.

HUI GSN, CHANGSP, GIBSONH, HASHIMOTOA, HASHIROC, BARR PJ, KOTANIS: Influence of Adjuvants on the Antibody Specificity to the P l a s m o d i u m f a l c i p a r u m Major Merozoite Surface Protein, gp195. J I m m u n o l 1991, 147:3935-3941. VON BRUNN A, FRUH K, MOILER H-M, ZENTGRAF H-W, BUJARD H: Epitopes of the Human Malaria Parasite P. f a l c i p a r u m Carried on the Surface of HBsAg Particles Elicit an Immune Response against the Parasite. Vaccine 1991, 9:477~i84.

68. 9

DEL PORT1LLO HA, LONGACRES, KHOURI E, DAVID PH: Primary Structure of the Merozoite Surface Antigen 1 of Plasmodi u m v i v a x Reveals Sequences Conserved between Different P l a s m o d i u m Species. Proc Natl Acad Sci USA 1991, 88:4030-4034. Reports the complete primary structure of the gene encoding the /~ vivax MSA 1 antigen. This achievement opens the way for the development of a MSA 1 vaccine against P. vivax, the most prevalent human malaria parasite. 69.

SMYTHEJA, COPPEL RL, DAY KP, MARTIN RK, ODUOLA AMJ, KEMP DJ, ANDERSRF: Structural Diversity in the P l a s m o d i u m f a l c i p a r u m Merozoite Surface Antigen 2. Proc Natl Acad Sci USA 1991, 88:1751-1755.

70,

MARSHALLVIVI, COPPEL RL, MARTIN RK, ODUOLA AMJ, ANDERS RF, KEMP DJ: A P l a $ m o d i u m f a l c i p a r u m MSA-2 Gene Apparently Generated by Intragenic Recombination between the Two Allelic Families. Mol Biochem Parasitol 1991, 45:349-352.

71.

FENTONB, CLARKJT, KHAN CMA, ROBINSONJV, WALLiKER D, RIDLEY R, SCAIFEJG, MCBRIDE JS: Structural and Antigenic Polymorphism of the 35- to 48-kilodalton Merozoite Surface Antigen (MSA-2) of the Malaria Parasite P l a s m o d i u m f a l c i p a r u m . Mol Cell Biol 1991, 11:963471.

72. .

SAUL A, LORD R, JONES GL, SPENCER L: Protective Iramunization with Invariant Peptides of the P l a s m o d i u m f a l c i p a r u m Antigen MSA2. J l m m u n o l 1992, 148:208-211. This study documents the protective value conserved sequences of MSA 2. Mice immunized with diphtheria toxoid conjugates that con mined synthetic peptides representing the conserved regions of MSA 2 afforded protection that correlated with antibody levels. 73.

RZEPCZYKCM, CSUP,HES PA, SAULAJ, JONES GL, DYER S, CHEE D, GOSS N, IRVING DO: Comparative Study of the T Cell Response to Two Allelic Forms of a Malarial Vaccine Candidate Protein. J I m m u n o l 1992, 148:1197-1204.

74.

ARONSONNE, S1LVERMANC, WASSERMAN GF, KOCHAN J, HALL BT, ESSER K, YOUNG JE, CHULAYJD: Immunization of Owl Monkeys with a Recombinant Protein Containing Repeated Epitopes of a P l a s m o d i u m f a l c i p a r u m Glycophorin-binding Protein. Am J Trop Med Hyg 1991, 45:548-559.

75.

76. 9

CASPERSP, ETL1NGERH, MATILE H, PINKJR, ST~BER D, TAKACS B: A P l a s m o d i u m f a l c i p a r u m Malaria Vaccine Candidate which Contains Epitopes from the Circumsporozoite Protein and a Blood Stage Antigen, 5.1. Mol Biochem Parasitol 1991, 47:143 150. SCHORRJ, KNAPP B, HUNDT E, KUPPER HA, AMANN E: Surface Expression of Malarial Antigens in S a l m o n e l l a tYp h i m u r i u m . Induction of Serum Antibody Response u p o n Oral Vaccination of Mice. Vaccine 1991, 9:675-681.

This study shows that a novel recombinant carrier, the E coli OmpA system, can accommodate relatively large sequences. Interestingly, expression is obtained on the surface of attenuated S. O/phimurium and oral immunization of mice with recombinant Salmonella, expressing t~ falciparum serine-rich protein or histidine-rich protein as fusion proteins with OmpA, resulted in the induction of a specific humoral immune response. 77.

RIDLEYRG, TAKACSB, ETLINGERH, SCAIFEJG: Malaria Vaccine: a Rhoptry Antigen from P l a s m o d i u m f a l c t p a r u m is Protective in S a i m i r i Monkeys. Parasitology 1990, 101:187-192.

78. 9

RIDLEYRG, LAHM H-W, TAKACS B, SCAIFE JG: Genetic and Structural Relationships b e t w e e n Components of a Protective Rhoptry Antigen Complex from P l a s m o d i u m falcip a r u m . Mol Biocbem Parasitol 1992, 47:245-246. See [79"]. 79. 9

SAULA, COOPERJ, HAUQUITZD, IRVINGD, CHENGQ, STOWERSA, LIMPAIBOON T: The 42-kilodalton Rhoptry-associated Protein of P l a s m o d i u m f a l c i p a r u m . Mol Biochem Parasitol 1992, 50:139-150. This paper, together with [789 further characterizes a protective rhoptry complex at the molecular level. As a consequence, vaccination studies with recombinant rhoptry components should be possible. 80.

PATARROYOME, AMADOR R, CLAV1JO P, MORENO A, GUZMAN F, ROMERO P, TASCON R, FRANCO A, MUPdLLO LA, PONTON G, TRUJILLO G: A Synthetic Vaccine Protects Humans against Challenge with Asexual Blood Stages of P l a s m o d i u m falc i p a r u m Malaria. Nature 1988, 332: 158-161.

81.

MITCHELLGH: Progress Toward Malaria Vaccination. Curr Opin Infect Dis 1990, 3:387-392.

82.

RODRIGUEZR, MORENO A, GUZMAN F, CALVO M, PATARROYO ME: Studies in Owl Monkeys Leading to the Development of a Synthetic Vaccine Against the Asexual Blood Stages of P l a s m o d i u m f a l c i p a r u ~ Am J Trop Med Hyg 1990, 43:339-354.

83.

RUEBUSHII TK, CAMPBELL GH, MORENO A, PATARROYO ME, COLUNS WE: Immunization of Owl Monkeys with a Combination of P l a s m o d i u m f a l c i p a r u m Asexual Blood-stage Synthetic Peptide Antigens. A m J Trop Med Hyg 1990, 43:355-366.

84. 99

SALCEDOM, BARRETO L, ROJAS M, MOYA R, COTE J, PATARROYO ME: Studies on the Humoral Immune Response to a Synthetic Vaccine against P l a s m o d i u m f a l c i p a r u m Malaria. Clin Exp l m m u n o l 1991, 84:122-128. Reports the first detailed analysis of the humoral immune response to vaccination of a large group of volunteers with the synthetic polymer SPf 66. These results indicate that the SPf 66 product is safe and immunogenic in humans. 85. 9

PATARROYOME, VINASCO J, AMADOR R, ESPEJO F, SILVA Y, MORENOA, ROJAS M, MORA AL, SALCEDO M, VALERO V, ET AL: Genetic Control of the Immune Response to a Synthetic Vaccine against P l a s m o d i u m f a l c i p a r u m . Parasite Immunol 1991, 13: 509-516. An extension of the previous study [ 8 5 " ] , this report shows that a small group of the volunteers immunized with SPf 66 had low or no antibody responses and a significantly high proportion of these individuals were HLA-DR4+. 86.

MuPaLLOI ~ TENJO FA, CLAVIJO OP, OROZCO MA, SAMPAIO S, KALILJ, PATARROYOME: A Specific T-cell Receptor Genotype Preference in the Immune Response to a Synthetic Plasm o d i u m f a l c i p a r u m Malaria Vaccine. Parasite l m m u n o l 1992, 14:87 94.

87.

ROCHACL, MUPdLLO LA, MORA AL, ROJAS M, FRANCO L, COTE J, VALERO MV, MORENO A, AMADORR, NUNEZ F, ET AL: Determination of the Immunization Schedule for Field Trials with the Synthetic Malaria Vaccine SPf 66. Parasite l m m u n o l 1992, 14:95-109.

88. 99

CALVO M, GUZMAN F, PEREZ E, SEGURA CH, MOLANO A, PATARROYOME: Specific Interactions of Synthetic Peptides

Malaria vaccines Romero Derived from P. f a l c i p a r u m Merozoite Proteins with Human Red Blood Cells. Pept Res 1991, 4:324-333. This study reveals that an unusually high proportion of blood stage antigen derived peptides binds to red blood cells from humans and from some other mammmals. The authors argue that the presence of certain peptide motifs made of short consensus sequences is critical for peptide binding to the red blood cell. 89.

MOLANOA, SEGURAC, GUZMANF, LOZADAD, PATARROYOME: In Human Malaria Protective Antibodies are Directed Mainly Against the Lys-Glu Ion Pair Within the Lys-Glu-Lys Motif of the Synthetic Vaccine SPf 66. Parasite Immunol 1992, 14:111-124.

90.

TANABE K, MACKAYM, GOMAN M, SCAIFEJG: Allelic Dimorphism in a Surface Antigen Gene of the Malaria Parasite Plasmodium falciparum. J Mol Biol 1987, 195:273-287.

91. ,D.

POUVELLEB, SPIEGEL R, HSL~O L, HOWARD RJ, MORRIS RL, THOMASA.P, TARASCHI TF: Direct Access to Serum Macro-

molecules by Intraerythrocytic Malaria Parasites. Nature 1991, 353:73-75. This study provides evidence for the existence of a duct that connects the plasma membrane of the red blood cell with the membrane lining the parasitophorous vacuole. This finding has important implications for understanding the cell biology of the blood stage form and for devising new antiparasite strategies. 92. 9.

GOODMF: The Implications for Malaria Vaccine Programs if Memory T Cells from Non-exposed Humans Can Respond to Malaria Antigens. Curr Opin Immunol 1991, 3:496-502. In this provocative review, the author raises important issues concern ing cell-mediated immunity against Plasmodium blood stages. Of particular interest, it is suggested that an attenuated parasite vaccine is an alternative to subunit malaria vaccines that should be re-examined.

P Romero, Ludwig Institute for Cancer Research, lausanne Branch, Chemin des Boveresses, CH1066, Epalinges, Switzerland.

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