Abstract
The Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) has recently emerged as a leading candidate antigen against the blood-stage human malaria parasite. However it has proved challenging to identify a heterologous expression platform that can produce a soluble protein-based vaccine in a manner compliant with current Good Manufacturing Practice (cGMP). Here we report the production of full-length PfRH5 protein using a cGMP-compliant platform called ExpreS2, based on a Drosophila melanogaster Schneider 2 (S2) stable cell line system. Five sequence variants of PfRH5 were expressed that differed in terms of mutagenesis strategies to remove potential N-linked glycans. All variants bound the PfRH5 receptor basigin and were recognized by a panel of monoclonal antibodies. Analysis following immunization of rabbits identified quantitative and qualitative differences in terms of the functional IgG antibody response against the P. falciparum parasite. The antibodies induced by one protein variant were shown to be qualitatively similar to responses induced by other vaccine platforms. This work identifies Drosophila S2 cells as a clinically-relevant platform suited for the production of ‘difficult-to-make’ proteins from Plasmodium parasites, and identifies a PfRH5 sequence variant that can be used for clinical production of a non-glycosylated, soluble full-length protein vaccine immunogen.
Highlights
Plasmodium falciparum parasites are the causative agent of the most severe form of human malaria, and the development of an effective vaccine remains a key strategic goal to aid the control, local elimination and eventual eradication of this disease
In the context of natural infection, PfRH5 appears to be a non-dominant target of naturally-acquired immune responses[7,26], and the relatively high degree of PfRH5 sequence conservation is associated with low-level natural immune pressure, and functional constraints linked to basigin binding and host red blood cells (RBC) tropism[22,27,28]
Protein versions 1.0 and 2.0 were based on the 7G8 laboratory-adapted parasite line and the 3D7 clone of P. falciparum respectively, and all four putative N-linked glycosylation sequons (N-X-S/T) were mutated Thr to Ala–as performed for a previous PfRH5 protein vaccine produced in mammalian HEK293 cells and tested in rabbits[17] and Aotus monkeys[13]
Summary
Plasmodium falciparum parasites are the causative agent of the most severe form of human malaria, and the development of an effective vaccine remains a key strategic goal to aid the control, local elimination and eventual eradication of this disease. The kinetic constraints imposed by such rapid erythrocyte invasion mean that extremely high concentrations of functional antibody are required to neutralize the parasite[6] Progress in this arena, is being made with a new generation of merozoite antigen targets identified in recent years that exhibit relatively low levels of polymorphism and against which functional neutralizing antibodies can be raised by vaccination. Preclinical studies using the assay of GIA have shown that antibodies raised by PfRH5 vaccination can cross-inhibit all P. falciparum lines and field isolates tested to-date[7,16,17,18] and, secondly, that they can do so with higher efficiency than other historical target antigens (lower EC50 in terms of antigen-specific antibody concentration[16]) These studies have all used antibodies raised by full-length PfRH5 immunogens, given earlier reports using fragments of PfRH5 made in Escherichia coli that failed to induce functional antibodies[19,20]. Each of these reported approaches faces significant challenges for production of a clinically-compatible immunogen–including extremely low yield, the inclusion of C-terminal tags such as rat CD4 domains 3 and 4, production of insoluble protein within inclusion bodies, or lack of a scalable or compliant process for currrent Good Manufacturing Practice (cGMP) production of a clinical vaccine batch
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