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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. © 2012.

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 clinical manifestations of Plasmodium falciparum malaria occur when parasites are multiplying within erythrocytes. During this stage, some parasites develop into sexual forms (male and female gametocytes) that are transmitted to the mosquito during their feeding on blood. The development of new drugs or vaccines targeting both asexual and sexual blood stage parasites is important to strengthen existing tools for fighting against malaria, and this is an essential component of the overall strategy of malaria eradication. However, there are still many unsolved questions concerning the cellular development and cell biology of P.falciparum asexual parasites, as well as for the onset and the development of the gametocyte stage. This is partly because of the inability to obtain good workable quantities of parasite material. Routine bulking-up of cultures in most laboratories involves establishing multiple culture flasks, which is very time consuming and labour intensive. To obtain large quantities of parasite material with high consistency, we recently reported the use of the Wave Bioreactor system for the culture of asexual blood stage of P. falciparum in suspension [1]. Controlled wave-induced motion within the bioreactors provides low-shear, favourable hydrodynamic conditions and very efficient gas transfer that is ideal for parasite cultivation. We used the Wave Bioreactor™ 20/50 EHT which is composed of a single-use plastic Cellbag, the cultivation chamber, that sits on a heated and rocking platform. We established important parameters for maintaining P. falciparum cultures, such as rocking motion, temperature and gas flow. Moreover, by monitoring parasite growth along with glucose consumption, pH dynamic and lactic acid production we characterized the development of the parasites within the bioreactor. We showed that malaria parasites grow far better in these rocking cultures than in static flask cultures in terms of preserving parasite cell synchronicity and reducing the number of multiple infected erythrocytes. Finally, we established a simple and straightforward protocol for bulk cultures of up to 1L of culture of P. falciparum within five days. To take advantage of the benefits of P. falciparum culture in suspension, we have now developed a simple protocol for gametocyte production and isolation from 1L culture in the wave bioreactor. This protocol includes 2 phases; the growth of the asexual parasites to stress conditions to induce gametocytes, followed by the maturation of gametocytes. In our experiments, approximately 8-10% of the asexual parasites commit to gametocytes. We reproducibly obtained mature gametocytes within 10 to 13 days (males in 10-12 days and females in 11 to 13 days). Batches of matures gametocytes can be collected from the same wave bag culture over several days. Finally, exflagellation tests confirm that gametocytes produced in the wave bioreactor are viable and, therefore, likely infectious for mosquitoes. The use of the Wave Bioreactor is a breakthrough method that will allow large scale production of Plasmodium falciparum asexual blood stage and gametocytes for antigen or organelle isolation, high throughput screening of compound libraries, for infection of mosquitoes and for whole cell blood-stage malaria vaccine development under GMP compliant procedures.

Plasmodium falciparummerozoite 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.