Safety and immunogenicity of multi-antigen AMA1-based vaccines formulated with CoVaccine HT™ and Montanide ISA 51 in rhesus macaques

<p>Abstract</p> <p>Background</p> <p>Increasing the breadth of the functional antibody response through immunization with <it>Plasmodium falciparum </it>apical membrane antigen 1 (<it>Pf</it>AMA1) multi-allele vaccine formulations has been demonstrated in several rodent and rabbit studies. This study assesses the safety and immunogenicity of three <it>Pf</it>AMA1 Diversity-Covering (DiCo) vaccine candidates formulated as an equimolar mixture (DiCo mix) in CoVaccine HT™ or Montanide ISA 51, as well as that of a <it>Pf</it>AMA1-MSP1₁₉fusion protein formulated in Montanide ISA 51.</p> <p>Methods</p> <p>Vaccine safety in rhesus macaques was monitored by animal behaviour observation and assessment of organ and systemic functions through clinical chemistry and haematology measurements. The immunogenicity of vaccine formulations was assessed by enzyme-linked immunosorbent assays and <it>in vitro </it>parasite growth inhibition assays with three culture-adapted <it>P. falciparum </it>strains.</p> <p>Results</p> <p>These data show that both adjuvants were well tolerated with only transient changes in a few of the chemical and haematological parameters measured. DiCo mix formulated in CoVaccine HT™ proved immunologically and functionally superior to the same candidate formulated in Montanide ISA 51. Immunological data from the fusion protein candidate was however difficult to interpret as four out of six immunized animals were non-responsive for unknown reasons.</p> <p>Conclusions</p> <p>The study highlights the safety and immunological benefits of DiCo mix as a potential human vaccine against blood stage malaria, especially when formulated in CoVaccine HT™, and adds to the accumulating data on the specificity broadening effects of DiCo mix.</p

Immunization with different PfAMA1 alleles in sequence induces clonal imprint humoral responses that are similar to responses induced by the same alleles as a vaccine cocktail in rabbits

<p>Abstract</p> <p>Background</p> <p>Antibodies to key <it>Plasmodium falciparum </it>surface antigens have been shown to be important effectors that mediate clinical immunity to malaria. The cross-strain fraction of anti-malarial antibodies may however be required to achieve</p> <p>strain-transcending immunity. Such antibody responses against <it>Plasmodium falciparum </it>apical membrane antigen 1 (<it>Pf</it>AMA1), a vaccine target molecule that is expressed in both liver and blood stages of the parasite, can be elicited through immunization with a mixture of allelic variants of the parasite molecule. Cross-strain antibodies are most likely elicited against epitopes that are shared by the allelic antigens in the vaccine cocktail.</p> <p>Methods</p> <p>A standard competition ELISA was used to address whether the antibody response can be further focused on shared epitopes by exclusively boosting these common determinants through immunization of rabbits with different <it>Pf</it>AMA1 alleles in sequence. Th<it>e in vitro </it>parasite growth inhibition assay was used to further evaluate the functional effects of the broadened antibody response that is characteristic of multi-allele vaccine strategies.</p> <p>Results</p> <p>A mixed antigen immunization protocol elicited humoral responses that were functionally similar to those elicited by a sequential immunization protocol (p > 0.05). Sequential exposure to the different <it>Pf</it>AMA1 allelic variants induced immunological recall of responses to previous alleles and yielded functional cross-strain antibodies that would be capable of optimal growth inhibition of variant parasites at high enough concentrations.</p> <p>Conclusions</p> <p>These findings may have implications for the current understanding of the natural acquisition of clinical immunity to malaria as well as for rational vaccine design.</p

Malaria infection by sporozoite challenge induces high functional antibody titres against blood stage antigens after a DNA prime, poxvirus boost vaccination strategy in Rhesus macaques

<p>Abstract</p> <p>Background</p> <p>A DNA prime, poxvirus (COPAK) boost vaccination regime with four antigens, i.e. a combination of two <it>Plasmodium knowlesi </it>sporozoite (<it>csp/ssp2</it>) and two blood stage (<it>ama1/msp1</it>_<it>42</it>) genes, leads to self-limited parasitaemia in 60% of rhesus monkeys and survival from an otherwise lethal infection with <it>P. knowlesi</it>. In the present study, the role of the blood stage antigens in protection was studied in depth, focusing on antibody formation against the blood stage antigens and the functionality thereof.</p> <p>Methods</p> <p>Rhesus macaques were immunized with the four-component vaccine and subsequently challenged i.v. with 100 <it>P. knowlesi </it>sporozoites. During immunization and challenge, antibody titres against the two blood stage antigens were determined, as well as the <it>in vitro </it>growth inhibition capacity of those antibodies. Antigen reversal experiments were performed to determine the relative contribution of antibodies against each of the two blood stage antigens to the inhibition.</p> <p>Results</p> <p>After vaccination, PkAMA1 and PkMSP1₁₉antibody titres in vaccinated animals were low, which was reflected in low levels of inhibition by these antibodies as determined by <it>in vitro </it>inhibition assays. Interestingly, after sporozoite challenge antibody titres against blood stage antigens were boosted over 30-fold in both protected and not protected animals. The <it>in vitro </it>inhibition levels increased to high levels (median inhibitions of 59% and 56% at 6 mg/mL total IgG, respectively). As growth inhibition levels were not significantly different between protected and not protected animals, the ability to control infection appeared cannot be explained by GIA levels. Judged by <it>in vitro </it>antigen reversal growth inhibition assays, over 85% of the inhibitory activity of these antibodies was directed against PkAMA1.</p> <p>Conclusions</p> <p>This is the first report that demonstrates that a DNA prime/poxvirus boost vaccination regimen induces low levels of malaria parasite growth inhibitory antibodies, which are boosted to high levels upon challenge. No association could, however, be established between the levels of inhibitory capacity <it>in vitro </it>and protection, either after vaccination or after challenge.</p

Susceptibility of human Plasmodium knowlesi infections to anti-malarials

Background: Evidence suggests that Plasmodium knowlesi malaria in Sarawak, Malaysian Borneo remains zoonotic, meaning anti-malarial drug resistance is unlikely to have developed in the absence of drug selection pressure. Therefore, adequate response to available anti-malarial treatments is assumed. Methods: Here the ex vivo sensitivity of human P. knowlesi isolates in Malaysian Borneo were studied, using a WHO schizont maturation assay modified to accommodate the quotidian life cycle of this parasite. The in vitro sensitivities of P. knowlesi H strain adapted from a primate infection to in vitro culture (by measuring the production of Plasmodium lactate dehydrogenase) were also examined together with some assays using Plasmodium falciparum and Plasmodium vivax. Results: Plasmodium knowlesi is uniformly highly sensitive to artemisinins, variably and moderately sensitive to chloroquine, and less sensitive to mefloquine. Conclusions: Taken together with reports of clinical failures when P. knowlesi is treated with mefloquine, the data suggest that caution is required if using mefloquine in prevention or treatment of P. knowlesi infections, until further studies are undertaken

KAI407, a potent non-8-aminoquinoline compound that kills Plasmodium cynomolgi early dormant liver stage parasites in vitro.

Preventing relapses of Plasmodium vivax malaria through a radical cure depends on use of the 8-aminoquinoline primaquine, which is associated with safety and compliance issues. For future malaria eradication strategies, new, safer radical curative compounds that efficiently kill dormant liver stages (hypnozoites) will be essential. A new compound with potential radical cure activity was identified using a low-throughput assay of in vitro-cultured hypnozoite forms of Plasmodium cynomolgi (an excellent and accessible model for Plasmodium vivax). In this assay, primary rhesus hepatocytes are infected with P. cynomolgi sporozoites, and exoerythrocytic development is monitored in the presence of compounds. Liver stage cultures are fixed after 6 days and stained with anti-Hsp70 antibodies, and the relative proportions of small (hypnozoite) and large (schizont) forms relative to the untreated controls are determined. This assay was used to screen a series of 18 known antimalarials and 14 new non-8-aminoquinolines (preselected for blood and/or liver stage activity) in three-point 10-fold dilutions (0.1, 1, and 10 μM final concentrations). A novel compound, designated KAI407 showed an activity profile similar to that of primaquine (PQ), efficiently killing the earliest stages of the parasites that become either primary hepatic schizonts or hypnozoites (50% inhibitory concentration [IC50] for hypnozoites, KAI407, 0.69 μM, and PQ, 0.84 μM; for developing liver stages, KAI407, 0.64 μM, and PQ, 0.37 μM). When given as causal prophylaxis, a single oral dose of 100 mg/kg of body weight prevented blood stage parasitemia in mice. From these results, we conclude that KAI407 may represent a new compound class for P. vivax malaria prophylaxis and potentially a radical cure

Measurement of the plasma levels of antibodies against the polymorphic vaccine candidate apical membrane antigen 1 in a malaria-exposed population

<p>Abstract</p> <p>Background</p> <p>Establishing antibody correlates of protection against malaria in human field studies and clinical trials requires, amongst others, an accurate estimation of antibody levels. For polymorphic antigens such as apical membrane antigen 1 (AMA1), this may be confounded by the occurrence of a large number of allelic variants in nature.</p> <p>Methods</p> <p>To test this hypothesis, plasma antibody levels in an age-stratified cohort of naturally exposed children from a malaria-endemic area in Southern Ghana were determined by indirect ELISA. Titres against four single <it>Pf</it>AMA1 alleles were compared with those against three different allele mixtures presumed to have a wider repertoire of epitope specificities. Associations of antibody levels with the incidence of clinical malaria as well as with previous exposure to parasites were also examined.</p> <p>Results</p> <p>Antibody titres against <it>Pf</it>AMA1 alleles generally increased with age/exposure while antibody specificity for <it>Pf</it>AMA1 variants decreased, implying that younger children (≤ 5 years) elicit a more strain-specific antibody response compared to older children. Antibody titre measurements against the FVO and 3D7 AMA1 alleles gave the best titre estimates as these varied least in pair-wise comparisons with titres against all <it>Pf</it>AMA1 allele mixtures. There was no association between antibody levels against any capture antigen and either clinical malaria incidence or parasite density.</p> <p>Conclusions</p> <p>The current data shows that levels of naturally acquired antigen-specific antibodies, especially in infants and young children, are dependent on the antigenic allele used for measurement. This may be relevant to the interpretation of antibody titre data from measurements against single <it>Pf</it>AMA1 alleles, especially in studies involving infants and young children who have experienced fewer infections.</p

The Novel bis-1,2,4-Triazine MIPS-0004373 Demonstrates Rapid and Potent Activity against All Blood Stages of the Malaria Parasite

Novel bis-1,2,4-triazine compounds with potent in vitro activity against Plasmodium falciparum parasites were recently identified. The bis-1,2,4-triazines represent a unique antimalarial pharmacophore and are proposed to act by a novel but as-yet-unknown mechanism of action. This study investigated the activity of the bis-1,2,4-triazine MIPS-0004373 across the mammalian life cycle stages of the parasite and profiled the kinetics of activity against blood and transmission stage parasites in vitro and in vivo. MIPS-0004373 demonstrated rapid and potent activity against P. falciparum, with excellent in vitro activity against all asexual blood stages. Prolonged in vitro drug exposure failed to generate stable resistance de novo, suggesting a low propensity for the emergence of resistance. Excellent activity was observed against sexually committed ring stage parasites, but activity against mature gametocytes was limited to inhibiting male gametogenesis. Assessment of liver stage activity demonstrated good activity in an in vitro P. berghei model but no activity against Plasmodium cynomolgi hypnozoites or liver schizonts. The bis-1,2,4-triazine MIPS-0004373 efficiently cleared an established P. berghei infection in vivo, with efficacy similar to that of artesunate and chloroquine and a recrudescence profile comparable to that of chloroquine. This study demonstrates the suitability of bis-1,2,4-triazines for further development toward a novel treatment for acute malaria

Correction: Variant Exported Blood-Stage Proteins Encoded by Plasmodium Multigene Families Are Expressed in Liver Stages Where They Are Exported into the Parasitophorous Vacuole.

[This corrects the article DOI: 10.1371/journal.ppat.1005917.]