Toward the rational design of a malaria vaccine construct using the MSP3 family as an example: contribution of antigenicity studies in humans.

Plasmodium falciparum merozoite surface protein (MSP3) is a main target of protective immunity against malaria that is currently undergoing vaccine development. It was shown recently to belong, together with MSP6, to a new multigene family whose C-terminal regions have a similar organization, contain both homologous and divergent regions, and are highly conserved across isolates. In an attempt to rationally design novel vaccine constructs, we extended the analysis of antigenicity and function of region-specific antibodies, previously performed with MSP3 and MSP6, to the remaining four proteins of the MSP3 family using four recombinant proteins and 24 synthetic peptides. Antibodies to each MSP3 family antigen were found to be highly prevalent among malaria-exposed individuals from the village of Dielmo (Senegal). Each of the 24 peptides was antigenic, defining at least one epitope mimicking that of the native proteins, with a distinct IgG isotype pattern for each, although with an overall predominance of the IgG3 subclass. Human antibodies affinity purified upon each of the 24 peptides exerted an antiparasite antibody-dependent cellular inhibition effect, which in most cases was as strong as that of IgG from protected African adults. The two regions with high homology were found to generate a broad network of cross-reactive antibodies with various avidities. A first multigenic construct was designed using these findings and those from related immunogenicity studies in mice and demonstrated valuable immunological properties. These results indicate that numerous regions from the MSP3 family play a role in protection and provide a rationale for the tailoring of new MSP3-derived malaria vaccines.

Toward the rational design of a malaria vaccine construct using the MSP3 family as an example: contribution of immunogenicity studies in models.

Plasmodium falciparum merozoite surface protein 3 (MSP3), the target of antibodies that mediate parasite killing in cooperation with blood monocytes and are associated with protection in exposed populations, is a vaccine candidate under development. It belongs to a family of six structurally related genes. To optimize immunogenicity, we attempted to improve its design based on knowledge of antigenicity of various regions from the conserved C terminus of the six proteins and an analysis of the immunogenicity of "tailored" constructs. The immunogenicity studies were conducted in BALB/c and C57BL/6J mice, using MSP3 (referred to here as MSP3-1) as a model. Four constructs were designed in order to assess the effect of sequences flanking the 69-amino-acid region of MSP3-1 previously shown to be the target of biologically active antibodies. The results indicate major beneficial effects of removing (i) the subregion downstream from the 69-amino-acid sequence, since antibody titers increased by 2 orders of magnitude, and (ii) the upstream subregion which, although it defines a T-helper cell epitope, is not the target of antibodies. The construct, excluding both flanking sequences, was able to induce Th1-like responses, with a dominance of cytophilic antibodies. This led to design a multigenic construct based on these results, combining the six members of the MSP3 family. This new construction was immunogenic in mice, induced antibodies that recognized the parasite native proteins, and inhibited parasite growth in the functional antibody-dependent cellular inhibition assay, thus satisfying the preclinical criteria for a valuable vaccine candidate.

The development of sexual stage malaria gametocytes in a Wave Bioreactor.

BACKGROUND: Blocking malaria gametocyte development in RBCs or their fertilization in the mosquito gut can prevent infection of the mosquito vector and passage of disease to the human host. A 'transmission blocking' strategy is a component of future malaria control. However, the lack of robust culture systems for producing large amounts of Plasmodium falciparum gametocytes has limited our understanding of sexual-stage malaria biology and made vaccine or chemotherapeutic discoveries more difficult. METHODS: The Wave BioreactorTM 20/50 EHT culture system was used to develop a convenient and low-maintenance protocol for inducing commitment of P. falciparum parasites to gametocytogenesis. Culture conditions were optimised to obtain mature stage V gametocytes within 2 weeks in a large-scale culture of up to a 1 l. RESULTS: We report a simple method for the induction of gametocytogenesis with N-acetylglucosamine (10 mM) within a Wave Bioreactor. By maintaining the culture for 14-16 days as many as 100 million gametocytes (stage V) were produced in a 1 l culture. Gametocytes isolated using magnetic activated cell sorting (MACS) columns were frozen in aliquots for storage. These were revitalised by thawing and shown to retain their ability to exflagellate and infect mosquitoes (Anopheles stephansi). CONCLUSIONS: The production of gametocytes in the Wave Bioreactor under GMP-compliant conditions will not only facilitate cellular, developmental and molecular studies of gametocytes, but also the high-throughput screening for new anti-malarial drugs and, possibly, the development of whole-cell gametocyte or sporozoite-based vaccines.

Large-scale growth of the Plasmodium falciparum malaria parasite in a wave bioreactor.

We describe methods for the large-scale in vitro culturing of synchronous and asynchronous blood-stage Plasmodium falciparum parasites in sterile disposable plastic bioreactors controlled by wave-induced motion (wave bioreactor). These cultures perform better than static flask cultures in terms of preserving parasite cell cycle synchronicity and reducing the number of multiple-infected erythrocytes. The straight-forward methods described here will facilitate the large scale production of malaria parasites for antigen and organelle isolation and characterisation, for the high throughput screening of compound libraries with whole cells or extracts, and the development of live- or whole-cell malaria vaccines under good manufacturing practice compliant standards.