Malaria ookinete surface protein-based vaccination via the intranasal route completely blocks parasite transmission in both passive and active vaccination regimens in a rodent model of malaria infection.

Malaria vaccines based on ookinete surface proteins (OSPs) of the malaria parasites block oocyst development in feeding mosquitoes and hence disrupt the parasite life cycle and prevent the disease from being transmitted to other individuals. To investigate whether a noninvasive mucosal vaccination regimen effectively blocks parasite transmission in vivo, Plasmodium yoelii Pys25, a homolog of the Pfs25 and Pvs25 OSPs of Plasmodium falciparum and Plasmodium vivax, respectively, was intranasally (i.n.) administered using a complement-deficient DBA/2 mouse malaria infection model, in which a highly elevated level of oocysts develops in feeding mosquitoes. Vaccinated mice developed a robust antibody response when the vaccine antigen was given together with cholera toxin adjuvant. The induced immune serum was passively transferred to DBA/2 mice 3 days after infection with P. yoelii 17XL, and Anopheles stephensi mosquitoes were allowed to feed on the infected mice before or after serum transfusion. This passive immunization completely blocked oocyst development; however, immune serum induced by the antigen or adjuvant alone did not have such a profound antiparasite effect. Further, when i.n. vaccinated mice were infected with the parasite and then mosquitoes were allowed to directly feed on the infected mice, complete blockage of transmission was again observed. To our knowledge, this is the first time that mucosal vaccination has been demonstrated to be efficacious for directly preventing parasite transmission from vaccinated animals to mosquitoes, and the results may provide important insight into rational design of nonparenteral vaccines for use against human malaria.

Effect of Plasmodium yoelii exposure on vaccination with the 19-kilodalton carboxyl terminus of merozoite surface protein 1 and vice versa and implications for the application of a human malaria vaccine.

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 homologue Plasmodium yoelii MSP1(19) in mice that had previously experienced malaria infection and vice versa. In this study, preexposure of mice to Plasmodium yoelii elicited native anti-MSP1(19) antibody responses, which could be boosted by vaccination with recombinant MSP1(19). Likewise, infection of MSP1(19)-primed mice with P. yoelii led to an increase of anti-MSP1(19) antibodies. However, this increase was at the expense of antibodies to parasite determinants other than MSP1(19). 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.

Phase 1 study of two merozoite surface protein 1 (MSP1(42)) vaccines for Plasmodium falciparum malaria.

OBJECTIVES: To assess the safety and immunogenicity of two vaccines, MSP1(42)-FVO/Alhydrogel and MSP1(42)-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 (MSP1(42)) 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 microg) were vaccinated at 0, 28, and 180 d. Volunteers were followed for 1 y. OUTCOME MEASURES: The safety of MSP1(42)-FVO/Alhydrogel and MSP1(42)-3D7/Alhydrogel was assessed. The antibody response to each vaccine was measured by reactivity to homologous and heterologous MSP1(42), MSP1(19), and MSP1(33) recombinant proteins and recognition of FVO and 3D7 parasites. RESULTS: Anti-MSP1(42) antibodies were detected by ELISA in 20/27 (74%) and 22/27 (81%) volunteers receiving three vaccinations of MSP1(42)-FVO/Alhydrogel or MSP1(42)-3D7/Alhydrogel, respectively. Regardless of the vaccine, the antibodies were cross-reactive to both MSP1(42)-FVO and MSP1(42)-3D7 proteins. The majority of the antibody response targeted the C-terminal 19-kDa domain of MSP1(42), although low-level antibodies to the N-terminal 33-kDa domain of MSP1(42) 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 MSP1(42)/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.

The essential mosquito-stage P25 and P28 proteins from Plasmodium form tile-like triangular prisms.

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.

posttranslational modification of recombinant Plasmodium falciparum apical membrane antigen 1: impact on functional immune responses to a malaria vaccine candidate.

Recombinant apical membrane antigen 1 (AMA1) is a leading vaccine candidate for Plasmodium falciparum malaria, as antibodies against recombinant P. falciparum AMA1 (PfAMA1) interrupt merozoite invasion into erythrocytes. In order to investigate the role of posttranslational modification in modulating the functional immune response to recombinant AMA1, two separate alleles of PfAMA1 (FVO and 3D7), in which native N-glycosylation sites have been mutated, were produced using Escherichia coli and a Pichia pastoris expression system. Recombinant Pichia pastoris AMA1-FVO (PpAMA1-FVO) and PpAMA1-3D7 are O-linked glycosylated, and 45% of PpAMA1-3D7 is nicked, though all four recombinant molecules react with conformation-specific monoclonal antibodies. To address the immunological effect of O-linked glycosylation, we compared the immunogenicity of E. coli AMA1-FVO (EcAMA1-FVO) and PpAMA1-FVO antigens, since both molecules are intact. The effect of antigen nicking was then investigated by comparing the immunogenicity of EcAMA1-3D7 and PpAMA1-3D7. Our data demonstrate that there is no significant difference in the rabbit antibody titer elicited towards EcAMA1-FVO and PpAMA1-FVO or to EcAMA1-3D7 and PpAMA1-3D7. Furthermore, we have demonstrated that recombinant AMA1 (FVO or 3D7), whether expressed and refolded from E. coli or produced from the Pichia expression system, is equivalent and mimics the functionality of the native protein in in vitro growth inhibition assay experiments. We conclude that in the case of recombinant AMA1, the E. coli- and P. pastoris-derived antigens are immunologically and functionally equivalent and are unaffected by the posttranslational modification resulting from expression in these two systems.

Phase 1 clinical trial of apical membrane antigen 1: an asexual blood-stage vaccine for Plasmodium falciparum malaria.

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-naive 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 mug, 20 mug, and 80 mug) 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-naive humans, and our results support the further development of this vaccine.

Phase 1 vaccine trial of Pvs25H: a transmission blocking vaccine for Plasmodium vivax malaria.

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.

Montanide ISA 720 vaccines: quality control of emulsions, stability of formulated antigens, and comparative immunogenicity of vaccine formulations.

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.

Potent immunogenicity of DNA vaccines encoding Plasmodium vivax transmission-blocking vaccine candidates Pvs25 and Pvs28-evaluation of homologous and heterologous antigen-delivery prime-boost strategy.

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.

Human leukocyte antigen class II alleles influence levels of antibodies to the Plasmodium falciparum asexual-stage apical membrane antigen 1 but not to merozoite surface antigen 2 and merozoite surface protein 1.

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 for Plasmodium 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 (MSP1(19)) 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 MSP1(19) variants, 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 MSP1(19) but 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 MSP1(19) variants. 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.

CpG oligodeoxynucleotide and Montanide ISA 51 adjuvant combination enhanced the protective efficacy of a subunit malaria vaccine.

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 the Saccharomyces cerevisiae-expressed 19-kDa C-terminal region of merozoite surface protein 1 (yMSP1(19)) of the murine malaria parasite Plasmodium yoelii. We found that in C57BL/6 mice, following sporozoite challenge, the degree of protective immunity against malaria induced by yMSP1(19) in a formulation of Montanide ISA 51 (ISA) plus CpG ODN 1826 was similar or superior to that conferred by yMSP1(19) emulsified in complete Freund's adjuvant (CFA/incomplete Freund's adjuvant). In total, among mice immunized with yMSP1(19), 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 yMSP1(19)), 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 yMSP1(19) plus 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.

Effect of CpG oligodeoxynucleotides on the immunogenicity of Pfs25, a Plasmodium falciparum transmission-blocking vaccine antigen.

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.

Blocking of transmission to mosquitoes by antibody to Plasmodium vivax malaria vaccine candidates Pvs25 and Pvs28 despite antigenic polymorphism in field isolates.

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.

Biochemical and immunological characterization of bacterially expressed and refolded Plasmodium falciparum 42-kilodalton C-terminal merozoite surface protein 1.

Protection against Plasmodium falciparum can 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 (MSP1(42)) in Escherichia 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 refolded E. coli MSP1(42) was homogeneous and highly immunogenic. In a formulation suitable for human use, rabbit antibodies were raised against refolded E. coli MSP1(42) and tested in vitro in a P. falciparum growth invasion assay. The antibodies inhibited the growth of parasites expressing either homologous or heterologous forms of P. falciparum MSP1(42). However, the inhibitory activity was primarily a consequence of antibodies directed against the C- terminal 19 kDa of MSP1 (MSP1(19)). Vaccination of nonhuman primates with E. coli MSP1(42) in Freund's adjuvant protected six of seven Aotus monkeys from virulent infection with P. falciparum. The protection correlated with antibody-dependent mechanisms. Thus, this new construct, E. coli MSP1(42), is a viable candidate for human vaccine trials.

Serum antibodies induced by intranasal immunization of mice with Plasmodium vivax Pvs25 co-administered with cholera toxin completely block parasite transmission to mosquitoes.

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.

Expression of malaria transmission-blocking vaccine antigen Pfs25 in Pichia pastoris for use in human clinical trials.

In previously published studies, Saccharomyces cerevisiae recombinant protein expression systems have been employed to express the malaria parasite antigen Pfs25, a candidate transmission-blocking vaccine antigen against Plasmodium falciparum malaria. However, despite having been in two Phase 1 trials, the recombinant Pfs25 so produced (previously called TBV25H) exists as a mixture of two monomeric protein conformational forms, Pfs25H-A and Pfs25H-B. In this study, we optimized the expression and purification of the two Pfs25H conformers in S. cerevisiae, and characterized their biochemical and antigenic properties, immunogenicities, and transmission-blocking activities. Pfs25H-A is apparently homogeneous, and has the correct conformation as measured by monoclonal antibody recognition. It is, however, expressed at a low yield of only 0.19mg/l. By contrast, Pfs25H-B is produced as a heterogeneous population of molecules that do not seem to have the correct conformation. Nonetheless, both forms appear equally effective in their ability to produce transmission-blocking antibodies in mice. To address the low yield seen with S. cerevisiae, we also expressed Pfs25 in Pichia pastoris. P. pastoris is apparently superior to S. cerevisiae in producing higher yield, immunologically more potent, biologically more active Pfs25H-A.

Structure of the C-terminal domains of merozoite surface protein-1 from Plasmodium knowlesi reveals a novel histidine binding site.

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.

Immunization with a combination of merozoite surface proteins 4/5 and 1 enhances protection against lethal challenge with Plasmodium yoelii.

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 the Plasmodium yoelii challenge 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 of P. yoelli merozoite surface protein 1 (MSP1) and MSP4/5, the homologue of P. falciparum MSP4 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 MSP1(19) combination did not develop detectable parasitemia after challenge. Combined immunization with MSP1(19) and 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 MSP1(19) were elevated compared to the MSP1(19) alone group; however, improved protection occurred regardless of whether boosting of the anti-MSP1(19) response was observed. Boosting of anti-MSP1(19) did 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.

In vitro studies with recombinant Plasmodium falciparum apical membrane antigen 1 (AMA1): production and activity of an AMA1 vaccine and generation of a multiallelic response.

Apical membrane antigen 1 (AMA1) is regarded as a leading malaria blood-stage vaccine candidate. While the overall structure of AMA1 is conserved in Plasmodium spp., numerous AMA1 allelic variants of P. falciparum have been described. The effect of AMA1 allelic diversity on the ability of a recombinant AMA1 vaccine to protect against human infection by different P. falciparum strains is unknown. We characterize two allelic forms of AMA1 that were both produced in Pichia pastoris at a sufficient economy of scale to be usable for clinical vaccine studies. Both proteins were used to immunize rabbits, singly and in combination, in order to evaluate their immunogenicity and the ability of elicited antibodies to block the growth of different P. falciparum clones. Both antigens, when used alone, elicited high homologous anti-AMA1 titers, with reduced strain cross-reactivity. Similarly, sera from rabbits immunized with a single antigen were capable of blocking the growth of homologous parasite strains at levels theoretically sufficient to clear parasite infections. However, heterologous inhibition was significantly reduced, providing experimental evidence that AMA1 allelic diversity is a result of immune pressure. Encouragingly, rabbits immunized with a combination of both antigens exhibited titers and levels of parasite inhibition as good as those of the single-antigen-immunized rabbits for each of the homologous parasite lines, and consequently exhibited a broadening of allelic diversity coverage.