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It is well known that exposure to one antigen can modulate the immune responses that develop following exposure to closely related antigens. It is also known that the composition of the repertoire can be skewed to favor epitopes shared between a current infection and a preceding one, a phenomenon referred to as “original antigenic sin.” It was of interest, therefore, to investigate the antibody response that develops following exposure to the malaria vaccine candidate homologuePlasmodium yoeliiMSP119in mice that had previously experienced malaria infection and vice versa. In this study, preexposure of mice toPlasmodium yoeliielicited native anti-MSP119antibody responses, which could be boosted by vaccination with recombinant MSP119. Likewise, infection of MSP119-primed mice withP. yoeliiled to an increase of anti-MSP119antibodies. However, this increase was at the expense of antibodies to parasite determinants other than MSP119. This change in the balance of antibody specificities significantly affected the ability of mice to withstand a subsequent infection. These data have particular relevance to the possible outcome of malaria vaccination for those situations where the vaccine response is suboptimal and suggest that suboptimal vaccination may in fact render the ultimate acquisition of natural immunity more difficult.

Apical membrane antigen 1 (AMA1), a polymorphic merozoite surface protein, is a leading blood-stage malaria vaccine candidate. A phase 1 trial was conducted with 30 malaria-naïve volunteers to assess the safety and immunogenicity of the AMA1-C1 malaria vaccine. AMA1-C1 contains an equal mixture of recombinant proteins based on sequences from the FVO and 3D7 clones of Plasmodium falciparum . The proteins were expressed in Pichia pastoris and adsorbed on Alhydrogel. Ten volunteers in each of three dose groups (5 μg, 20 μg, and 80 μg) were vaccinated in an open-label study at 0, 28, and 180 days. The vaccine was well tolerated, with pain at the injection site being the most commonly observed reaction. Anti-AMA1 immunoglobulin G (IgG) was detected by enzyme-linked immunosorbent assay (ELISA) in 15/28 (54%) volunteers after the second immunization and in 23/25 (92%) after the third immunization, with equal reactivity to both AMA1-FVO and AMA1-3D7 vaccine components. A significant dose-response relationship between antigen dose and antibody response by ELISA was observed, and the antibodies were predominantly of the IgG1 isotype. Confocal microscopic evaluation of sera from vaccinated volunteers demonstrated reactivity with P. falciparum schizonts in a pattern similar to native parasite AMA1. Antigen-specific in vitro inhibition of both FVO and 3D7 parasites was achieved with IgG purified from sera of vaccinees, demonstrating biological activity of the antibodies. To our knowledge, this is the first AMA1 vaccine candidate to elicit functional immune responses in malaria-naïve humans, and our results support the further development of this vaccine.

Montanide ISA 720 is an experimental adjuvant, formulated as water-in-oil emulsions, that induces high antibody titers in several animal species. It has been used in human vaccine trials with malaria and HIV vaccines. The heightened response is likely due, in part, to the formation of a depot at the injection site. However, post-formulation modifications were seen with seven proteins tested during storage of ISA 720 formulations at 37 degrees C for 1 week and two proteins stored longer at 4 degrees C. Potency studies in mice, in which the stored vaccines were diluted into placebo emulsions for appropriate dosing, indicated that this instability could lead to loss of immunogenicity in the post-injection depot, limiting the allowable storage time of preformed vaccines. We describe point-of-injection formulation for ISA 720 vaccines that meets the requirement for in vitro stability. For preformed vaccines, addition of glycine or glycylglycine prevented antigen modification on storage at 37 degrees C, providing a potential way of stabilizing antigen/ISA 720 formulations for in vitro storage and the post-injection depot.

Transmission-blocking vaccines target the sexual stages of the malaria parasite and prevent further development within the mosquito vector halting the transmission of the parasite. Zygote/ookinetes are potential targets of antibodies inhibiting oocyst development in the mosquito midgut and rendering mosquitoes non-infectious. DNA vaccine constructs were developed expressing Pvs25 and Pvs28 (Plasmodium vivax zygote/ookinete surface proteins) fused at the amino terminus with tissue plasminogen activator signal peptide. Antibodies produced in mice after immunization with three doses recognized respective antigens in the parasites and in an ELISA, and these antibodies when tested in membrane feeding assay were potent blockers of P. vivax transmission. Co-immunization with Pvs25 and Pvs28 DNA vaccine constructs did not affect the antigen specific antibody responses against individual antigens, and the antibodies remained effective in blocking parasite transmission demonstrating 91-99% reduction in oocyst number in the mosquito midgut. Several combinations of homologous and heterologous antigen-delivery prime boost strategy were also evaluated and the results suggested that antibody titers and transmission-blocking activities by the three prime-boost strategies (DNA prime/DNA boost, DNA prime/protein boost, and protein prime/protein boost) were comparable with slightly better immunogenicity of heterologous antigen-delivery prime/boost as compared to DNA/DNA alone. These results demonstrate potent immunogenicity of DNA vaccines encoding Pvs25 and Pvs28 and warrant further evaluation in non-human primates.

Unmethylated CpG dinucleotide motifs present in bacterial genomes or synthetic oligodeoxynucleotides (ODNs) serve as strong immunostimulatory agents in mice, monkeys and humans. We determined the adjuvant effect of murine CpG ODN 1826 on the immunogenicity and protective efficacy of theSaccharomyces cerevisiae-expressed 19-kDa C-terminal region of merozoite surface protein 1 (yMSP119) of the murine malaria parasitePlasmodium yoelii. We found that in C57BL/6 mice, following sporozoite challenge, the degree of protective immunity against malaria induced by yMSP119in a formulation of Montanide ISA 51 (ISA) plus CpG ODN 1826 was similar or superior to that conferred by yMSP119emulsified in complete Freund's adjuvant (CFA/incomplete Freund's adjuvant). In total, among mice immunized with yMSP119, 22 of 32 (68.7%) with ISA plus CpG 1826, 0 of 4 (0%) with CFA/incomplete Freund’s adjuvant, 0 of 4 (0%) with CpG 1826 mixed with ISA (no yMSP119), and 0 of 11 (0%) with CpG 1826 alone were completely protected against development of erythrocytic stage infection after sporozoite challenge. The adjuvant effect of CpG ODN 1826 was manifested as both significantly improved complete protection from malaria (defined as the absence of detectable erythrocytic form parasites) (P= 0.007, chi square) and reduced parasite burden in infected mice. In vivo depletions of interleukin-12 and gamma interferon cytokines and CD4+and CD8+T cells in vaccinated mice had no significant effect on immunity. On the other hand, immunoglobulin G (IgG) isotype levels appeared to correlate with protection. Inclusion of CpG ODN 1826 in the yMSP119plus ISA vaccine contributed towards the induction of higher levels of IgG2a and IgG2b (Th1 type) antibodies, suggesting that CpG ODN 1826 caused a shift towards a Th1 type of immune response that could be responsible for the higher degree of protective immunity. Our results indicate that this potent adjuvant formulation should be further evaluated for use in clinical trials of recombinant malarial vaccine candidates.

Protection againstPlasmodium falciparumcan be induced by vaccination in animal models with merozoite surface protein 1 (MSP1), which makes this protein an attractive vaccine candidate for malaria. In an attempt to produce a product that is easily scaleable and inexpensive, we expressed the C-terminal 42 kDa of MSP1 (MSP142) inEscherichia coli, refolded the protein to its native form from insoluble inclusion bodies, and tested its ability to elicit antibodies with in vitro and in vivo activities. Biochemical, biophysical, and immunological characterization confirmed that refoldedE. coliMSP142was homogeneous and highly immunogenic. In a formulation suitable for human use, rabbit antibodies were raised against refoldedE. coliMSP142and tested in vitro in aP. falciparumgrowth invasion assay. The antibodies inhibited the growth of parasites expressing either homologous or heterologous forms ofP. falciparumMSP142. However, the inhibitory activity was primarily a consequence of antibodies directed against the C- terminal 19 kDa of MSP1 (MSP119). Vaccination of nonhuman primates withE. coliMSP142in Freund's adjuvant protected six of sevenAotusmonkeys from virulent infection withP. falciparum. The protection correlated with antibody-dependent mechanisms. Thus, this new construct,E. coliMSP142, is a viable candidate for human vaccine trials.

The protozoan parasite Plasmodium causes malaria, with hundreds of millions of cases recorded annually. Protection against malaria infection can be conferred by antibodies against merozoite surface protein (MSP)-1, making it an attractive vaccine candidate. Here we present the structure of the C-terminal domains of MSP-1 (known as MSP-1(19)) from Plasmodium knowlesi. The structure reveals two tightly packed epidermal growth factor-like domains oriented head to tail. In domain 1, the molecule displays a histidine binding site formed primarily by a highly conserved tryptophan. The protein carries a pronounced overall negative charge primarily due to the large number of acidic groups in domain 2. To map protein binding surfaces on MSP-1(19), we have analyzed the crystal contacts in five different crystal environments, revealing that domain 1 is highly preferred in protein-protein interactions. A comparison of MSP-1(19) structures from P. knowlesi, P. cynomolgi, and P. falciparum shows that, although the overall protein folds are similar, the molecules show significant differences in charge distribution. We propose the histidine binding site in domain 1 as a target for inhibitors of protein binding to MSP-1, which might prevent invasion of the merozoite into red blood cells.

It is widely believed that subunit vaccines composed of multiple components will offer greater protection against challenge by malaria, and yet there is little experimental evidence to support this view. We set out to test this proposition in thePlasmodium yoeliichallenge system in rodents by comparing the degree of protection conferred by immunization with a mixture of merozoite surface proteins to that conferred by single proteins. We therefore examined a defined protein mixture made of the epidermal growth factor-like domains ofP. yoellimerozoite surface protein 1 (MSP1) and MSP4/5, the homologue ofP. falciparumMSP4 and MSP5. In the present study we demonstrate that this combination of recombinant proteins dramatically enhances protection against lethal malaria challenge compared to either protein administered alone. Many mice immunized with the MSP4/5 plus MSP119combination did not develop detectable parasitemia after challenge. Combined immunization with MSP119and yMSP4/5, a product characterized by lower protective efficacy, also greatly enhanced protection by reducing peak parasitemias and increasing the numbers of survivors. In some combination trials, levels of antibodies to MSP119were elevated compared to the MSP119alone group; however, improved protection occurred regardless of whether boosting of the anti-MSP119response was observed. Boosting of anti-MSP119did not appear to be due to contaminating endotoxin in the EcMSP4/5 material since enhanced protection was observed in C3H/HeJ mice, which are endotoxin insensitive. Collectively, these experiments show that multiantigen combinations offer enhanced levels of protection against asexual stage infection and suggest that combinations of MSP1, MSP4, and MSP5 should be evaluated further for use in humans.

Immunity induced by the 19-kDa fragment ofPlasmodium yoeliimerozoite surface protein 1 (MSP119) is dependent on high titers of specific antibodies present at the time of challenge and a continuing active immune response postinfection. However, the specificity of the active immune response postinfection has not been defined. In particular, it is not known whether anti-MSP119antibodies that arise following infection alone are sufficient for protection. We developed systems to investigate whether an MSP119-specific antibody response alone both prechallenge and postchallenge is sufficient for protection. We were able to exclude antibodies with other specificities, as well as any contribution of MSP119-specific CD4+T cells acting independent of antibody, and we concluded that an immune response focused solely on MSP119-specific antibodies is sufficient for protection. The data imply that the ability of natural infection to boost an MSP119-specific antibody response should greatly improve vaccine efficacy.

The budding yeast Saccharomyces cerevisiae has been used to express the recombinant protein Pvs25H, currently the only candidate transmission-blocking vaccine against Plasmodium vivax malaria. This molecule contains four epidermal growth factor-like domains and is expressed as at least two stable monomeric forms with different physicochemical properties. Pvs25H-A is apparently homogeneous and seems to have a correct disulfide bond structure. By contrast, Pvs25H-B is produced as a heterogeneous population of molecules, some of which are associated with an as yet unidentified chromophore, and it contains both internal and N-terminal cleavages. We report here a procedure for successfully separating these two forms with a process suitable for clinical production of this antigen.

A blood-stage vaccine based on Plasmodium falciparum merozoite surface protein 3 (MSP3) was tested for eff icacy in ap rimate model.Aotus nancymai monkeys were vaccinated with yeast-expressed MSP3 before a lethal challenge with Plasmodium falciparum parasites. Five of 7 control monkeys had acute infections and required treatment to control parasitemia. Only 1 of 7 monkeys vaccinated with MSP3 required this treatment. The efficacy of the MSP3 vaccination appeared to be comparable to that of MSP142, a leading asexual vaccine candidate, in response to which 2 monkeys experienced acute infections. In the MSP3-vaccinated group, protection correlated with prechallenge titers of antibody to MSP3. In the MSP1 and control groups, protection correlated with antibody to MSP3 raised by challenge infection. Malaria remains one of the world’s most burdensome infectious diseases, with mortality and morbidity at near-record levels. All pathology associated with malaria occurs as the parasite undergoes rounds of asexual multiplication in the host’s blood. The goal of malaria blood-stage vaccines is to reduce disease severity [1]. Proteins associated with the surface of merozoites (the red blood cell–invasive form of the parasite) are targets for such a vaccine, because they may induce antibodies that interrupt the parasite’s life cycle [2]. We report here an attempt to develop a blood-stage vaccine based on Plasmodium falciparum merozoite surface protein 3 (MSP3).

Vaccination of mice with yeast-secretedPlasmodium yoelii-derived 19-kilodalton merozoite surface protein 1 (yMSP119) has been shown to afford protection from challenge with a lethal strain ofP. yoelii. Sterile immunity can be achieved when MSP119is emulsified in Freund adjuvant but not when it is adsorbed to aluminum hydroxide gel (alum). Because complete Freund adjuvant is not an acceptable adjuvant for use in humans, alternative adjuvants must be identified for formulating MSP119as a vaccine for use in humans. To determine whether oligodeoxynucleotides with CpG motifs (ODN), reported to be a powerful new class of adjuvants, could enhance the immunogenicity of yMSP119, C57BL/6 mice were vaccinated either with yMSP119formulated with Freund adjuvant, with alum, or with ODN plus alum and challenged intravenously withP. yoelii17XL asexual blood-stage parasites. Adsorption of immunogen and adjuvant to alum was optimized by adjusting buffer (phosphate versus acetate) and pH. We found that the adjuvant combination of ODN plus alum with yMSP119, injected intraperitoneally (i.p.), increased immunoglobulin G (IgG) yMSP119-specific antibody production 12-fold over Freund adjuvant given i.p., 3-fold over Freund adjuvant given subcutaneously (s.c.), 300-fold over alum given i.p., and 48-fold over alum given s.c. The predominant antibody isotype in the group receiving alum-ODN-yMSP119was IgG1. Increased antibody levels correlated to protection from a challenge withP. yoelii17XL. Supernatant cytokine levels of gamma interferon in yMSP119-stimulated splenocytes were dramatically elevated in the alum-ODN-yMSP119group. Interleukin-10 (IL-10) levels were also elevated; however, no IL-5 was detected. The cytokine profile, as well as the predominant IgG1 antibody isotype, suggests the protective immune response was a mixed Th1/Th2 response.

Two strains of transgenic mice have been generated that secrete into their milk a malaria vaccine candidate, the 42-kDa C-terminal portion of Plasmodium falciparum merozoite surface protein 1 (MSP1 42 ). One strain secretes an MSP1 42 with an amino acid sequence homologous to that of the FVO parasite line, the other an MSP1 42 where two putative N-linked glycosylation sites in the FVO sequence have been removed. Both forms of MSP1 42 were purified from whole milk to greater than 91% homogeneity at high yields. Both proteins are recognized by a panel of monoclonal antibodies and have identical N termini, but are clearly distinguishable by some biochemical properties. These two antigens were each emulsified with Freund's adjuvant and used to vaccinate Aotus nancymai monkeys, before challenge with the homologous P. falciparum FVO parasite line. Vaccination with a positive control molecule, a glycosylated form of MSP1 42 produced in the baculovirus expression system, successfully protected five of six monkeys. By contrast, vaccination with the glycosylated version of milk-derived MSP1 42 conferred no protection compared with an adjuvant control. Vaccination with the nonglycosylated, milk-derived MSP1 42 successfully protected the monkeys, with 4/5 animals able to control an otherwise lethal infection with P. falciparum compared with 1/7 control animals. Analysis of the different vaccines used suggested that the differing nature of the glycosylation patterns may have played a critical role in determining efficacy. This study demonstrates the potential for producing efficacious malarial vaccines in transgenic animals.

Transmission-blocking vaccines against malaria parasites target molecules expressed by sexual stage parasites to elicit antibodies that prevent the infection of the mosquito vector. Pvs25 and Pvs28, expressed on the surface of ookinetes, are potential candidates for such a vaccine and induce antibodies that block the infectivity of Plasmodium vivax in immunized animals. To improve the ability to induce transmission-blocking antibodies, Pvs25 and Pvs28 were produced as a single fusion protein by the yeast Saccharomyces cerevisiae. Mice immunized with a low dose of the chimeric molecule (Pvs25–28) developed higher antibody responses compared with mice immunized with either Pvs25 or Pvs28. In membrane feeding assays, both anti-Pvs25–28 and anti-Pvs25 antisera had similarly potent transmission-blocking activities (and both were much greater than anti-Pvs28). Furthermore, serum from mice simultaneously immunized with both Pvs25 and Pvs28, or serum mixtures of anti-Pvs25 alone and anti-Pvs28 alone did not enhance the efficacy over anti-Pvs25 serum alone, demonstrating that there is no synergism in the ability to block transmission of P. vivax between anti-Pvs25 and anti-Pvs28 antibodies.

Immunity induced by the 19-kDa fragment of merozoite surface protein 1 is dependent on CD4+ Th cells. However, we found that adoptively transferred CFSE-labeled Th cells specific for an epitope on Plasmodium yoelii 19-kDa fragment of merozoite surface protein 1 (peptide (p)24), but not OVA-specific T cells, were deleted as a result of P. yoelii infection. As a result of infection, spleen cells recovered from infected p24-specific T cell-transfused mice demonstrated reduced response to specific Ag. A higher percentage of CFSE-labeled p24-specific T cells stained positive with annexin and anti-active caspase-3 in infected compared with uninfected mice, suggesting that apoptosis contributed to deletion of p24-specific T cells during infection. Apoptosis correlated with increased percentages of p24-specific T cells that stained positive for Fas from infected mice, suggesting that P. yoelii-induced apoptosis is, at least in part, mediated by Fas. However, bystander cells of other specificities also showed increased Fas expression during infection, suggesting that Fas expression alone is not sufficient for apoptosis. These data have implications for the development of immunity in the face of endemic parasite exposure.

The development of malaria blood-stage vaccines is gathering momentum: there are several new funding initiatives, one multiantigen formulation is currently being tested and at least one other blood-stage vaccine is expected to begin trials in 2001. However, there is no consensus over the best way to select which form of an antigen to take into clinical testing. There is thus a danger that less-effective vaccines might be tested in the field in the order of their availability, rather than merit. Here, we argue that first proving efficacy in the New World monkey challenge model would accelerate development.

The gene encoding the Plasmodium yoelii homologue of P. falciparum merozoite surface proteins 4 (MSP4) and 5 (MSP5) has been expressed in Escherichia coli and Saccharomyces cerevisiae. The protein contains a single epidermal growth factor (EGF)-like domain and is expressed in a form lacking the predicted N-terminal signal and glycosyl phosphatidylinositol (GPI) attachment sequences. The recombinant protein derived from E. coli (EcMSP4/5) was highly effective at protecting mice against lethal challenge with 10(5) parasites of the P. yoelii YM strain. In contrast, the protective efficacy of yeast-derived MSP4/5 (yMSP4/5) was considerably less. The antibody titres in both groups were significantly different with mice immunised with yeast-derived protein showing significantly lower pre-challenge antibody responses. There was a significant inverse correlation between antibody levels as measured by ELISA and peak parasitaemia. Mice immunised with EcMSP4/5 produced anti-PyMSP4/5 antibodies predominantly of the IgG2a and IgG2b isotypes, whereas, mice immunised with yMSP4/5 mainly produced antibodies of the IgG1 isotype. The differences in antibody titres and subtype distribution may account for the observed differences in protective efficacy of these protein preparations. Levels of protective efficacy of MSP4/5 were compared with that obtained using P. yoelii MSP1 produced in S. cerevisiae. Levels of protection induced by E. coli derived MSP4/5 were superior to those induced by MSP1 which in turn were better than those induced by yeast-derived MSP4/5.

Malaria is still a leading cause of morbidity and mortality in human populations. Problems, including drug-resistant parasites and insecticide resistant mosquitoes, ensure the continued hold of malaria in the tropics and sub-tropics. Each year around 100 million cases of malaria result in at least 50,000 deaths outside of sub-Saharan Africa; within sub-Saharan Africa itself, malaria causes around one million child deaths per year. New approaches for malaria control are badly needed and much effort has gone to develop malaria vaccines. In addition to giving personal protection, most such vaccines would also tend to reduce the transmission of malaria. One class of vaccine is being developed specifically for this purpose--the malaria transmission-blocking vaccines (TBV). TBVs are based upon antigens expressed on the surface of the sexual and mosquito mid-gut stages of malaria parasites. These antigens are the targets of antibodies induced by vaccination of the host and ingested with the parasites in a mosquito blood meal. The antibodies act by inhibiting the parasite's development within the mosquito itself and they thereby prevent the onward transmission of the parasites. TBVs could contribute to the total interruption of malaria transmission in many locations with relatively low transmission rates, mostly outside sub-Saharan Africa. Under almost all transmission rates, however, TBVs would help reduce malaria incidence and malaria-related morbidity and mortality. Promising recombinant TBV candidate antigens for the two main human malaria parasite species, Plasmodium falciparum and Plasmodium vivax, have been produced and tested in the laboratory; one has undergone early clinical trials.

Merozoite surface protein 4 (MSP4) ofPlasmodium falciparumis a glycosylphosphatidylinositol-anchored integral membrane protein that is being developed as a component of a subunit vaccine against malaria. We report here the measurement of naturally acquired antibodies to MSP4 in a population of individuals living in the Khanh-Hoa region of Vietnam, an area where malaria is highly endemic. Antibodies to MSP4 were detected in 94% of the study population at titers of 1:5,000 or greater. Two forms of recombinant MSP4 produced in eitherEscherichia coliorSaccharomyces cerevisiaewere compared as substrates in the enzyme-linked immunosorbent assay. There was an excellent correlation between reactivity measured to either, although the yeast substrate was recognized by a higher percentage of sera. Four different regions of MSP4 were recognized by human antibodies, demonstrating that there are at least four distinct epitopes in this protein. In the carboxyl terminus, where the single epidermal growth factor-like domain is located, the reactive epitope(s) was shown to be conformation dependent, as disruption of the disulfide bonds almost completely abolished reactivity with human antibodies. The anti-MSP4 antibodies were mainly of the immunoglobulin G1 (IgG1) and IgG3 subclasses, suggesting that such antibodies may play a role in opsonization and complement-mediated lysis of free merozoites. Individuals in the study population were drug-cured and followed up for 6 months; no significant correlation was observed between the anti-MSP4 antibodies and the absence of parasitemia during the surveillance period. As a comparison, antibodies to MSP119, a leading vaccine candidate, were measured, and no correlation with protection was observed in these individuals. The anti-MSP119antibodies were predominantly of the IgG1 isotype, in contrast to the IgG3 predominance noted for MSP4.

In an attempt to produce a more defined, clinical-grade version of a vaccine based onPlasmodium falciparummerozoite surface protein 1 (MSP1), we evaluated the efficacy of two recombinant forms of MSP1 in anAotus nancymaichallenge model system. One recombinant vaccine, bvMSP142, based on the 42-kDa C-terminal portion of MSP1, was expressed as a secreted protein in baculovirus-infected insect cells. A highly pure baculovirus product could be reproducibly expressed and purified at yields in excess of 8 mg of pure protein per liter of culture. This protein, when tested for efficacy in theAotuschallenge model, gave significant protection, with only one of seven monkeys requiring treatment for uncontrolled parasitemia after challenge withP. falciparum. The second recombinant protein, P30P2MSP119, has been used in previous studies and is based on the smaller, C-terminal 19-kDa portion of MSP1 expressed inSaccharomyces cerevisiae. Substantial changes were made in its production process to optimize expression. The optimum form of this vaccine antigen (as judged by in vitro and in vivo indicators) was then evaluated, along with bvMSP142, for efficacy in theA. nancymaisystem. The new formulation of P30P3MSP119performed significantly worse than bvMSP142and appeared to be less efficacious than we have found in the past, with four of seven monkeys in the vaccinated group requiring treatment for uncontrolled parasitemia. With both antigens, protection was seen only when high antibody levels were obtained by formulation of the vaccines in Freund's adjuvant. Vaccine formulation in an alternate adjuvant, MF59, resulted in significantly lower antibody titers and no protection.

A recombinant protein expression system based on Saccharomyces cerevisiae has been used to express malarial vaccine candidate antigens. The antigens so produced have been used in three Phase 1 clinical trials and numerous preclinical non-human primate trials. Further Phase I trials are planned using these candidate vaccine antigens. These molecules were identified as attractive candidates for antimalarial vaccines, as they are all surface-exposed at some stage in the parasite's life cycle. They all share an unusual structural feature: epidermal growth factor (EGF)-like motifs. When these proteins are expressed in our S. cerevisiae expression system, they are produced as a series of stable structural conformers, each with a different disulphide bonding pattern. This leads to both biochemical and, more importantly, antigenic differences between the conformers (e.g. presence or absence of an antibody B cell epitope). These findings have important ramifications for other EGF-domain-containing proteins expressed in S. cerevisiae, or for proteins which contain other cysteine-folding motifs not normally expressed by this organism, both for vaccine production or for research/reagent purposes.

Transmission-blocking vaccines are one strategy for controlling malaria, whereby sexual-stage parasites are inhibited from infecting mosquitoes by human antibodies. To evaluate whether the recently clonedPlasmodium vivaxproteins Pvs25 and Pvs28 are candidates for a transmission-blocking vaccine, the molecules were expressed in yeast as secreted recombinant proteins. Mice vaccinated with these proteins adsorbed to aluminum hydroxide developed strong antibody responses against the immunogens, although for Pvs28, this response was genetically restricted. Antisera against both recombinant Pvs25 and Pvs28 recognized the corresponding molecules expressed by cultured sexual-stage parasites isolated from patients withP. vivaxmalaria. The development of malaria parasites in mosquitoes was completely inhibited when these antisera were ingested with the infected blood meal. Pvs25 and Pvs28, expressed inSaccharomyces cerevisiae, are as yet the only fully characterized transmission-blocking vaccine candidates againstP. vivaxthat induce such a potent antiparasite response.

Each of the four epidermal growth factor (EGF)-like domains of thePlasmodium falciparumsexual-stage antigen Pfs25 has been individually expressed as a yeast-secreted recombinant protein (yEGF1 through yEGF4). All four are recognized by the immune sera of animals and humans vaccinated with TBV25H (the corresponding yeast-secreted full-length recombinant form of Pfs25), with antibody titers to yEGF1 and yEGF2 weakly correlating with the ability of the sera to block the transmission of parasites to the mosquito host. All four proteins are poorly immunogenic in mice vaccinated with aluminum hydroxide-absorbed formulations. However, all four successfully primed the mice to mount an effective secondary antibody response after a single boost with TBV25H. Sera from mice vaccinated with yEGF2-TBV25H completely block the development of oocysts in mosquito midguts in membrane-feeding assays. Further, of the four proteins, only the depletion of antibodies to yEGF2 from the sera of rabbits vaccinated with TBV25H consistently abolished the ability of those sera to block oocyst development. Thus, antibodies to the second EGF-like domain of Pfs25 appear to mediate a very potent blocking activity, even at low titers. Vaccination strategies that target antibody response towards this domain may improve the efficacy of future transmission-blocking vaccines.

The merozoite surface protein 2 of P. falciparum is highly polymorphic in nature, but has regions of almost complete conservation at its N- and C-termini. We produced a chimeric recombinant protein comprising these regions only (hereafter termed NC). Mice immunized with the NC antigen produce antibodies at levels comparable to those immunized with 1624, a full-length recombinant protein representing MSP2 from P. falciparum. Antisera raised against NC recognized P. falciparum schizonts by IFA and a P. falciparum protein of M-r 45 kDa by Western blot. However, antibody specificities were observed to differ between anti-NC and anti-1624 sera, and this resulted in differences in parasite recognition, despite similar levels of antibodies having been produced. The response to the NC antigen was also shown to be restricted in some mice (H2-d), but this was overcome by including appropriate T-cell help, which was accomplished by creating recombinant protein chimeras that contained NC and T-helper epitopes from Tetanus toxoid, or MSP1(19) from P. berghei.

ThePlasmodium falciparumrhoptry-associated proteins 1 and 2 (RAP1 and RAP2) are candidate antigens for a subunit malaria vaccine. The design of the study, which looks at the acquisition of immunity to malaria from childhood to old age, has allowed us to document the interaction of HLA and age on levels of antibody to specific malarial antigens. Antibodies reach maximum levels to RAP1 after the age of 15 but to RAP2 only after the age of 30. The effect of HLA-DRB1 and -DQB1 and age on levels of antibody to rRAP1 and rRAP2 was analyzed with a multiple regression model in which all HLA alleles and age were independent variables. DQB1*0301 and -*03032 showed an age-dependent association with levels of antibody to rRAP1, being significant in children 5 to 15 years (P< 0.001) but not in individuals over 15 years of age. DRB1*03011 showed an age-dependent association with antibody levels to rRAP2; however, this association was in adults over the age of 30 years (P< 0.01) but not in individuals under the age of 30 years. No associations were detected between DRB1 alleles and RAP1 antibody levels or between DQB1 alleles and RAP2 antibody levels. Thus, not only the HLA allele but also the age at which an interaction is manifested varies for different malarial antigens. The interaction may influence either the rate of acquisition of antibody or the final level of antibody acquired by adults.

An electrophoretic procedure based on isotachophoresis has been developed for protein purification on a preparative scale in the 10 to 500 mg range. The system is simple, uses well understood physical properties, does not need ampholyte spacers and is able to produce sterile products of clinical grade. We demonstrate the applicability of this apparatus for the purification of denatured recombinant proteins and complex mixtures of proteins. The system may also be used for both cationic and anionic purification of proteins in their native form. The system is scalable from analytical to preparative protein loads at consistently high protein yields and purity levels. Total protein loads may vary as much as 1000 fold with the use of interchangeable columns of varying diameter and constant length. At both preparative and analytical scales concentration of products at greater than 20 mg/ml are obtainable. Toxicological considerations are addressed with assays for endotoxin, acrylamide and SDS concentrations, as well as the prevention of covalent protein modification.

Mice and rabbits immunized with recombinant forms of malaria vaccine candidate antigens rhoptry-associated proteins 1 and 2 (RAP-1, RAP-2 and rRAP-1, rRAP-2) produce antibodies at titres equivalent to monoclonal antibody ascites fluid raised against the native proteins. Sera from animals immunized with rRAP-1 contain antibodies which recognize the native protein by indirect immunofluorescence and immunoblotting, partially inhibit erythrocyte invasion in vitro and are long lasting. Epitope mapping shows these antibodies predominantly recognize epitopes in the N-terminal third of rRAP-1, some of which coincide with the targets of inhibitory monoclonal antibodies. By contrast, sera from animals immunized with rRAP-2 contain antibodies which recognize the recombinant but not the native protein.

P25 and P28 proteins are essential for Plasmodium parasites to infect mosquitoes and are leading candidates for a transmission-blocking malaria vaccine. The Plasmodium vivax P25 is a triangular prism that could tile the parasite surface. The residues forming the triangle are conserved in P25 and P28 from all Plasmodium species. A cocrystal structure shows that a transmission-blocking antibody uses only its heavy chain to bind Pvs25 at a vertex of the triangle.

Plasmodium vivax is responsible for the majority of malaria cases outside of Africa, and results in substantial morbidity. Transmission blocking vaccines are a potentially powerful component of a multi-faceted public health approach to controlling or eliminating malaria. We report the first phase 1 clinical trial of a P. vivax transmission blocking vaccine in humans. The Pvs25H vaccine is a recombinant protein derived from the Pvs25 surface antigen of P. vivax ookinetes. The protein was expressed in Saccharomyces cerevisiae, purified, and adsorbed onto Alhydrogel. Ten volunteers in each of three dose groups (5, 20, or 80 microg) were vaccinated by intramuscular injection in an open-label study at 0, 28 and 180 days. No vaccine-related serious adverse events were observed. The majority of adverse events causally related to vaccination were mild or moderate in severity. Injection site tenderness was the most commonly observed adverse event. Anti-Pvs25H antibody levels measured by ELISA peaked after the third vaccination. Vaccine-induced antibody is functionally active as evidenced by significant transmission blocking activity in the membrane feeding assay. Correlation between antibody concentration and degree of inhibition was observed. Pvs25H generates transmission blocking immunity in humans against P. vivax demonstrating the potential of this antigen as a component of a transmission blocking vaccine.

The apical membrane antigen 1 (AMA1), merozoite surface antigen 2 (MSA2), and merozoite surface protein 1 (MSP1) are asexual-stage proteins currently being evaluated for inclusion in a vaccine forPlasmodium falciparum. Accordingly, it is important to understand factors that control antibody responses to these antigens. Antibody levels in plasma from residents of Etoa, Cameroon, between the ages of 5 and 70 years, were determined using recombinant AMA1, MSA2, and the N-terminal region of MSP1 (MSP1-190L). In addition, antibody responses to four variants of the C-terminal region of MSP1 (MSP119) were assessed. Results showed that all individuals produced antibodies to AMA1, MSA2, and MSP1-190L; however, a proportion of individuals never produced antibodies to the MSP119variants, although the percentage of nonresponders decreased with age. The influence of age and human leukocyte antigen (HLA)-DRB1/DQB1 alleles on antibody levels was evaluated using two-way analysis of variance. Age was correlated with levels of antibodies to AMA1 and MSP119but not with levels of antibodies to MSA2 and MSP1-190L. No association was found between a single HLA allele and levels of antibodies to MSA2, MSP1-190L, or any of the MSP119variants. However, individuals positive for DRB1*1201 had higher levels of antibodies to the variant of recombinant AMA1 tested than did individuals of all other HLA types. Since the effect was seen across all age groups, HLA influenced the level but not the rate of antibody acquisition. This association for AMA1, combined with the previously reported association between HLA class II alleles and levels of antibodies to rhoptry-associated protein 1 (RAP1) and RAP2, indicates that HLA influences the levels of antibodies to three of the five vaccine candidate antigens that we have evaluated.

Antibodies directed against Pfs25, a protein present on the surface of zygotes and ookinetes of Plasmodium falciparum , completely block pathogen transmission. We evaluated the immunomodulatory effect of CpG oligodeoxynucleotides (ODN) on the immunogenicity of recombinant Pfs25 (rPfs25) formulated in alum (Al). Immunization of mice with rPfs25 plus CpG ODN improved both the antibody titer (a 30-fold-higher antibody response than that with rPfs25-Al alone) and avidity. Coadministration of CpG ODN dramatically enhanced the titer of immunoglobulin G2A (IgG2a) compared to the titer of the IgG1-dominant response caused by rPfs25-Al alone, and the sera from the CpG ODN-coadministered group completely blocked the transmission of P. falciparum parasites to mosquitoes, as determined by membrane feeding assays. However, transmission-blocking experiments revealed that blocking efficacy was dependent on high-titer antibody levels, independent of isotypes. These results suggest that CpG ODN can be used as an adjuvant to enhance the immunogenicity of rPfs25 as a malaria transmission-blocking vaccine.

Transmission-blocking vaccines (TBVs) targeting ookinete surface proteins expressed on sexual-stage malaria parasites are considered one promising strategy for malaria control. To evaluate the prospect of developing non-invasive and easy-to-administer mucosal malaria transmission-blocking vaccines, mice were immunized intranasally with a Plasmodium vivax ookinete surface protein, Pvs25 with a mucosal adjuvant cholera toxin (CT). Immunization induced significant serum IgG with high IgG1/IgG2a ratio (indicative of Th-2 type immune response). Feeding Anopheles dirus mosquitoes with mixtures of immune sera and gametocytemic blood derived from vivax-infected volunteer patients in Thailand significantly reduced both the number of midgut oocysts as well as the percentage of infected mosquitoes. The observed transmission-blocking effect was dependent on immune sera dilution. This study demonstrates for the first time that the mucosally induced mouse immune sera against a human malaria ookinete surface protein can completely block parasite transmission to vector mosquitoes, suggesting the possibility of non-invasive mucosal vaccines against mucosa-unrelated important pathogens like malaria.

A major challenge facing malaria vaccine development programs is identifying efficacious combinations of antigens. To date, merozoite surface protein 1 (MSP1) is regarded as the leading asexual vaccine candidate. Apical membrane antigen 1 (AMA1) has been identified as another leading candidate for an asexual malaria vaccine, but without any direct in vivo evidence that a recombinant form ofPlasmodium falciparumAMA1 would have efficacy. We evaluated the efficacy of a form ofP. falciparumAMA1, produced inPichia pastoris, by vaccinatingAotus vociferansmonkeys and then challenging them withP. falciparumparasites. Significant protection from this otherwise lethal challenge withP. falciparumwas observed. Five of six animals had delayed patency; two of these remained subpatent for the course of the infection, and two controlled parasite growth at

We have constructed a DNA plasmid vaccine encoding the C-terminal 42-kDa region of the merozoite surface protein1 (pMSP1 42 ) from the 3D7 strain of Plasmodium falciparum (Pf3D7). This plasmid expressed recombinant MSP1 42 after in vitro transfection in mouse VM92 cells. Rhesus monkeys immunized with pMSP1 42 produced antibodies reactive with Pf3D7 infected erythrocytes by IFAT, and by ELISA against yeast produced MSP1 19 (yMSP1 19 ). Immunization also induced antigen specific T cell responses as measured by interferon-γ production, and by classical CTL chromium release assays. In addition, immunization with pMSP1 42 primed animals for an enhanced antibody response to a subsequent boost with the recombinant yMSP1 19 . We also evaluated Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) as an adjuvant for pMSP1 42. We tested both rhesus GM-CSF expressed from a DNA plasmid, and E. coli produced recombinant human GM-CSF. Plasmids encoding rhesus GM-CSF (prhGM-CSF) and human GM-CSF (phuGM-CSF) were constructed; these plasmids expressed bio-active recombinant GMCSF. Co-immunization with a mixture of prhGM-CSF and pMSP1 42 induced higher specific antibody responses after the first dose of plasmid, but after three doses of DNA monkeys immunized with or without prhGM-CSF had the same final antibody titers and T cell responses. In comparison, rhuGM-CSF protein did not lead to accelerated antibody production after the first DNA dose. However, antibody titers were maintained at a slightly higher level in monkeys receiving GM-CSF protein, and they had a higher response to boosting with recombinant MSP1 19 . The GM-CSF plasmid or protein appears to be less potent as an adjuvant in rhesus monkeys than each is in mice, and more work is needed to determine if GM-CSF can be a useful adjuvant in DNA vaccination of primates.

A vaccine trial was conducted with rhoptry-associated proteins 1 and 2 (RAP1 and RAP2) of Plasmodium falciparum in Saimiri boliviensis monkeys to compare the ability of parasite-derived (PfRAP1 and 2) and recombinant proteins (rRAP1 and 2) to induce protective immune responses and to find adjuvants suitable for use in humans. Eight groups of 6 monkeys each were immunized with parasite-derived or recombinant RAP1 and 2 with Freund's complete adjuvant (FCA) followed by Freund's incomplete adjuvant (FIA), Montanide ISA720 adjuvant, or CRL1005 adjuvant. Recombinant RAP1 and RAP2 were also administered separately, with Montanide ISA720. After 3 immunizations, monkeys were challenged by iv inoculation of 50,000 parasites of the Uganda Palo Alto strain of P. falciparum. Of the animals vaccinated using FCA/FIA, 1 of 6 control monkeys, 3 of 6 immunized with PfRAP1 and 2, and 2 of 6 with rRAP1 and 2 did not require drug treatment. Of the monkeys vaccinated with Montanide ISA720 adjuvant, 0 of the 6 control monkeys, 2 of 6 immunized with RAP1 and 2, 1 of 6 immunized with rRAP1, and 4 of 6 immunized with RAP2 did not require drug treatment. Two of 6 monkeys immunized with PfRAP1 and 2 with CRL1005 did not require treatment. All groups receiving RAP1, RAP2, or both had a significant decrease in initial parasite multiplication rates and there was a significant negative correlation between anti-RAP2 antibody and multiplication rates. Animals were rechallenged with the homologous parasite 126 days after the first challenge. Of the monkeys that did not require drug treatment after the first challenge, none developed detectable parasitemia following rechallenge.

Plasmodium falciparum merozoite surface protein 4 (MSP4) is being developed as a component of a subunit vaccine against asexual stages of malaria. Three DNA constructs were produced that induced expression of MSP4 either in the cytoplasm of transfected cells or secreted from cells under the control of the human tissue plasminogen activator (TPA) signal or the native P. falciparum MSP4 signal. Only the construct containing the TPA signal induced detectable antibodies in mice, although gene expression was demonstrated in all constructs and MSP4 was shown to be secreted using either signal by in vitro transient transfection of COS cells. Two recombinant MSP4 proteins that encoded the same sequence as the plasmid DNA were produced in E. coli (EcMSP4-His) and S. cerevisiae (yMSP4-His) and used to raise antibodies in mice. Comparison of the antibodies elicited by these various antigen formulations showed differences in titer, isotype and epitope recognition. The titer of antibodies induced by DNA vaccination was lower than that induced by yMSP4-His, which in turn was lower than that induced by EcMSP4-His. The isotype profiles of the antibodies were also different, the plasmid DNA induced predominantly IgG 2a responses whereas the two proteins induced predominantly IgG 1 responses. The antibodies induced by DNA and yMSP4-His recognized predominantly the C-terminal epidermal growth factor (EGF)-like domain of the protein, whereas EcMSP4-His induced antibodies recognizing all domains of the protein equally. The antibodies induced by DNA vaccination were directed almost extensively to conformational epitopes so that reactivity with native MSP4 was abolished after disulfide bonds in the protein were disrupted. Antibodies induced by recombinant proteins recognized linear epitopes as well and reactivity to native MSP4 was preserved after reduction and alkylation of parasite proteins.

A placebo controlled, randomised, double blind trial was conducted in human volunteers to test a mixture of three recombinant Plasmodium falciparum blood stage antigens for its ability to reduce the initial growth rates of parasites. The vaccine contained recombinant MSP2 (3D7 allele), a portion of MSP1 (190LCS.T3) and part of the RESA antigen (C terminal 771 amino acids) in the Montanide ISA 720 adjuvant (SEPPIC). Twelve volunteers received two doses of the vaccine, 6 weeks apart. The five participants in the placebo group received an equivalent volume of the adjuvant emulsion using the same schedule. Antibody responses were low, as has been reported in earlier studies with this combination, while T cell responses were stronger. All the volunteers were challenged with approximately 140 ring infected red cells of the 3D7 cloned line, 4 weeks after the second dose. Parasitaemia was determined once daily from day 4 using a sensitive and quantitative PCR assay. All the volunteers were infected and were treated on day 8, before any developed symptoms. There was no significant difference in initial parasite growth rates between the verum and placebo groups, nor was there any significant correlation between parasite growth rates and any of the measured immunological responses. These results suggest that the formulation tested in this trial did not generate immune responses that were strong enough to reduce parasite growth in naive volunteers.

Two phase I vaccine trials were conducted to test the immunogenicity and safety of a vaccine containing three recombinant malaria antigens from the asexual stage of Plasmodium falciparum. The three antigens are a fragment of MSP1 (190LCS.T3); MSP2 and a portion of RESA and were formulated in Montanide ISA720 adjuvant. These trials investigated the dose response of each antigen for eliciting both antibody and T-cell responses and the immunogenicity of a mixture of the antigens compared with the antigens injected separately. All three antigens elicited both antibody and T-cell responses. Strong T-cell responses were observed with 190LCS.T3 and RESA with stimulation indices exceeding 100 for peripheral blood leucocytes in some individuals. The antibody responses were generally weak. The human antibody responses observed with MSP2 in Montanide ISA720 were not significantly different from those obtained in an earlier trial which used MSP2 with alum as the adjuvant. No antigenic competition was observed: volunteers receiving a mixture of antigens had similar responses to those receiving the three antigens at separate sites. Tenderness and pain at the injection site were common over the first few days following immunization. In some volunteers, especially those receiving the highest doses tested, there was a delayed reaction at the injection site with pain and swelling occurring approximately 10 days after injection.

Naturally occurring antibody responses to Plasmodium falciparum rhoptry-associated proteins 1 and 2 (RAP-1 and RAP-2) were measured with recombinant and parasite-derived forms of the antigens. For comparative purposes, responses to multiple forms of three other malarial antigens were also examined. The sera of 100 Papua New Guineans were screened for antibodies. Eighty-six and 82% of individuals over 30 years of age had antibodies that recognized parasite-derived RAP-1 and RAP-2, respectively. Importantly, we found that recombinant and native antigens share linear epitopes seen by the human immune system; thus, the recombinant proteins may be adequate human immunogens. However, antibodies affinity purified on recombinant RAP-1 reacted with other antigens in addition to parasite-derived RAP-1. Thus, the antigenicity of RAP-1 may have been overestimated previously. The recognition of RAP-1 and RAP-2 correlated with age and with the recognition of recombinant forms of the ring-infected erythrocyte surface antigen, merozoite surface protein 1, and merozoite surface antigen 2 (MSA2) antigens. Antibodies to these antigens appear to be generated in response to the total exposure to malaria of the host. Antibodies to conserved regions of MSA2 had stronger correlations with both age and the recognition of other antigens than did the full-length recombinant MSA2 molecule. In contrast to results with the other antigens, there was no significant difference in the ages of individuals with a certain antibody titer to the full-length recombinant or parasite-derived MSA2 molecule, but antibodies to these two antigens did correlate with parasitemia. For all antigens tested, antibody levels after two infections can approach the peak levels of antibodies obtained in immune individuals.

Mice immunised with a recombinant form of malarial antigen rhoptry-associated protein 2 (RAP2) produce antisera which recognise the native protein by indirect immunofluorescence and immunoblotting. Purified IgG components of the antisera partially inhibit erythrocyte invasion in vitro. This response was obtained only if the recombinant immunogen was presented to the mice in the presence of reducing and denaturing agents. An 8-mer epitope in RAP2 was recognised by antibodies in three of the antisera: E25TEFSKLY32. Immunisation with this octapeptide raised antibodies which strongly recognised reduced RAP2 in seven out of eight mice. However, this antisera either failed to recognise (five out of eight mice), or only weakly (three out of eight mice) recognised nonreduced RAP2. Examination of disulphide bonds in native RAP2 showed that the 4 cysteines of RAP2 form two disulphide bridges: Cys24-Cys88, and Cys277-Cys376. One of these (Cys24-Cys88) is adjacent to the octapeptide in the native protein. Surprisingly, seven out of eight mice immunised with the octapeptide also raised antibodies against native rhoptry-associated protein 1 (RAP1). The raising of antibodies which recognise RAP1 was induced specifically by the RAP2 octapeptide rather than the carrier protein used for immunisation. The epitope in RAP1 recognised by the antibodies was identified and shown not to be the result of any shared contiguous homologous sequence between the two proteins, but to shared homologous amino acids in critical positions within the epitope. Purified IgG components from the antisera of mice immunised with the octapeptide gave partial inhibition of erythrocyte invasion in vitro.

The kinetics of the gamma delta T-cell response was analysed in the context of the overall haematological response in subjects experimentally infected with sporozoites of Plasmodium falciparum. Numbers of gamma delta and alpha beta T cells and NK cells declined markedly during infection to reach minimum values 12-13 days post-infection when the patients were ill. This decline commenced from the beginning of the erythrocytic cycle and well before parasites could be detected microscopically and clinical symptoms developed. Platelet numbers also declined. In vivo activation of gamma delta T cells was evident with sequential up-regulation of the activation markers CD69 and HLA-DR. gamma delta T cell numbers were highest after treatment with the majority being CD4-CD8-, HLA-DR+ and showing reduced CD45RA expression. Contrary to some published observations gamma delta T-cell percentages remained within the normal range. Little evidence of upregulation of activation or memory markers was observed in the alpha beta T-cell population. In vitro proliferative responses to malaria antigen which involve gamma delta T cells were lost as the infection progressed and the lymphocyte count declined but these could be restored with the addition of exogenous IL-2 to cultures. The authors findings are consistent with a protective and/or immunomodulatory role for gamma delta T cells in malaria.

The genetic diversity of Solomon Island Plasmodium falciparum isolates was examined using MSA-2 as a single locus marker. Amplification of MSA-2 gene fragments showed size polymorphism and the presence of mixed infections. Sequence analysis indicated a global representation of MSA-2 alleles with representatives of 3D7/CAMP allelic subfamilies and the FCQ-27 allelic family being identified. A simplified method of characterisation, utilising PCR-RFLPs of MSA-2 gene fragments, was developed. The RFLPs allowed identification of allelic families and further distinction within the 3D7/CAMP family. The amplification of MSA-2 gene fragments from culture derived lines revealed a loss of diversity for a number of Solomon Island isolates. Genomic diversity was confirmed for Solomon Island lines, along with Papua New Guinean and Thai lines, by the generation of 7H8/6 fingerprints. All lines were distinct and band sharing frequencies and Wagner tree construction failed to identify any geographic clustering.

The gene coding for a 42-kDa rhoptry protein of Plasmodium falciparum has been cloned. On the basis of prior monkey vaccination studies, this protein is regarded as an important vaccine candidate, but its identity has been the subject of considerable uncertainty. Analysis of the cloned sequence shows that it is a basic, hydrophobic protein, without repetitive elements, unrelated to any of the previously postulated gene products and shows minimal sequence diversity. The availability of the corresponding recombinant protein will enable studies of its efficacy in human vaccine trials to be undertaken.

Objectives: To assess the safety and immunogenicity of two vaccines, MSP142-FVO/Alhydrogel and MSP142-3D7/Alhydrogel, targeting blood-stage Plasmodium falciparum parasites. Design: A Phase 1 open-label, dose-escalating study. Setting: Quintiles Phase 1 Services, Lenexa, Kansas between July 2004 and November 2005. Participants: Sixty healthy malaria-naïve volunteers 18–48 y of age. Interventions: The C-terminal 42-kDa region of merozoite surface protein 1 (MSP142) corresponding to the two allelic forms present in FVO and 3D7 P. falciparum lines were expressed in Escherichia coli, refolded, purified, and formulated on Alhydrogel (aluminum hydroxide). For each vaccine, volunteers in each of three dose cohorts (5, 20, and 80 μg) were vaccinated at 0, 28, and 180 d. Volunteers were followed for 1 y. Outcome Measures: The safety of MSP142-FVO/Alhydrogel and MSP142-3D7/Alhydrogel was assessed. The antibody response to each vaccine was measured by reactivity to homologous and heterologous MSP142, MSP119, and MSP133 recombinant proteins and recognition of FVO and 3D7 parasites. Results: Anti-MSP142 antibodies were detected by ELISA in 20/27 (74%) and 22/27 (81%) volunteers receiving three vaccinations of MSP142-FVO/Alhydrogel or MSP142-3D7/Alhydrogel, respectively. Regardless of the vaccine, the antibodies were cross-reactive to both MSP142-FVO and MSP142-3D7 proteins. The majority of the antibody response targeted the C-terminal 19-kDa domain of MSP142, although low-level antibodies to the N-terminal 33-kDa domain of MSP142 were also detected. Immunofluorescence microscopy of sera from the volunteers demonstrated reactivity with both FVO and 3D7 P. falciparum schizonts and free merozoites. Minimal in vitro growth inhibition of FVO or 3D7 parasites by purified IgG from the sera of the vaccinees was observed. Conclusions: The MSP142/Alhydrogel vaccines were safe and well tolerated but not sufficiently immunogenic to generate a biologic effect in vitro. Addition of immunostimulants to the Alhydrogel formulation to elicit higher vaccine-induced responses in humans may be required for an effective vaccine., Editorial Commentary Background: Generally, adults living in parts of the world where malaria is common develop protective immunity against the parasite. This means they may get infected but not become ill as a result. However, there are individuals, such as pregnant women and children under the age of five, who are more likely to develop symptoms of malaria due to no (or reduced) natural immunity. A successful malaria vaccine would stimulate an individual's immune system to respond to the malaria parasite and prevent serious clinical disease. Many different groups are currently developing potential vaccines. Several candidates are based on a protein called MSP1 (merozoite surface protein 1) which is found on the surface of the blood-stage form of the malaria parasite. However, in nature parasites carry different versions of the MSP1 protein, and ideally a successful vaccine would bring about immune responses against these different versions. The researchers carrying out this trial wanted to compare the safety and immune responses against candidate vaccines representing two different MSP1 proteins, which covered many different parasite lines. As a phase 1 trial, the study was carried out in healthy adult volunteers. Sixty individuals were assigned to receive an injection of the vaccines, either containing a recombinant protein analogous to the FVO parasite line (termed MSP142-FVO) or the 3D7 parasite line (termed MSP142-3D7) at three different dose levels. The trial's primary objective was to assess safety, which was done by collecting data on any abnormal signs or symptoms up to 14 d after each of three vaccinations. These outcomes were graded and then defined as related to the vaccine or not. The researchers also looked at antibody levels in participants' blood against different variants of the MSP1 protein, as well as using in vitro tests to see whether antibodies from vaccinated individuals could prevent malaria parasites from growing in lab culture. What the trial shows: The safety outcomes of the trial showed that the most common type of side effect experienced by the volunteers was pain at the injection site. The vast majority of such events were graded as mild, although there was one single case of a severe event (high levels of pain experienced by one volunteer at the injection site). There was no significant association between the chance of side effects and the vaccine dosage that an individual received. Following vaccination, antibody levels against the protein on which the vaccine was based were detected, although these levels dropped over time. The researchers did not see a strong association between the vaccine dosage that individuals received and the level of antibody response. However, the two vaccines when compared seemed to be equally good at raising an immune response and both caused antibodies to be raised corresponding to different variants of the MSP1 protein. However, the antibodies raised did not seem to be particularly effective at preventing malaria parasites from growing in lab culture. Strengths and limitations: Strengths of this study include a comparison of three different dosage levels of the vaccines under study, as well as a comparison of two vaccines based on the same protein, representing different parasite lines. Limitations to the study include the small number of participants, which makes the trial underpowered to detect all but large differences in side effects between the groups being compared. A placebo arm was not included in the trial, so it is not possible to be sure that the numbers of side effects observed here can be attributed to the vaccines or not. Finally, the procedure for assigning individuals to the two different vaccines involved alternation, rather than true randomization, which could have minimized the risk of bias. Contribution to the evidence: The trial reported here is an essential step in vaccine development. The results provide the first evidence relating to safety for these two vaccines, and do not raise any safety concerns at this stage. Although the vaccines raised an immune response, the antibodies raised did not seem to have much of an effect on malaria parasites in vitro. While these vaccines are safe, alternative MSP1 vaccine formulations anticipated to bring about a greater immune response will likely be studied before proceeding to field studies.

Several prototype vaccines against the asexual blood stage of malaria are undergoing preclinical and phase I testing. Although these vaccines have been chosen for their ability to elicit an anti-parasite response, no practical and sensitive clinical trial procedure has been available for measuring their impact on parasite growth. We describe a system that allows parasite growth rates to be measured in volunteers through the incubation period. Two necessary elements of this system are developed: suitable blood-stage Plasmodium falciparum inocula, and a highly sensitive and quantitative assay to measure parasite growth during the incubation period. We infected five nonimmune volunteers with an inoculum as small as 300 parasites and demonstrated that the resultant in vivo asexual parasite growth rates were reproducible at 12-15-fold per cycle. The system allowed the infection to be followed for eight days before treatment without symptoms developing. These findings suggest that it is feasible to directly measure the anti-parasite efficacy of a prototype malaria vaccine in human volunteers without subjecting them to the risk of disease.

We have previously demonstrated that mouse antisera against yeast-produced recombinant forms of the ookinete surface proteins of Plasmodium vivax (Pvs25 and Pvs28) blocks transmission of the homologous P. vivax (Sal I strain). In this study, we developed mouse and rabbit antisera against Pvs25 and Pvs28 and evaluated the efficacy of these vaccine candidates against natural isolates of P. vivax in Thailand. Although both Pvs25 and Pvs28 genes are polymorphic, sera from mice immunized using alum adjuvant completely inhibited oocyst development for most human isolates, whereas sera from rabbits immunized with either alum or Freund's adjuvant were partially inhibitory. All inhibition occurred in an antibody dose dependent fashion. Data from this study clearly demonstrates that antibodies raised against Sal I-based vaccines overcome the genetic polymorphism of Pvs25 and Pvs28 present in natural isolates of P. vivax, suggesting the wide range applicability of Sal I based vaccines.