Abstract
BackgroundSeveral pre-erythrocytic malaria vaccines based on the circumsporozoite protein (CSP) antigen of Plasmodium falciparum are in clinical development. Vaccine immunogenicity is commonly evaluated by the determination of anti-CSP antibody levels using IgG-based assays, but no standard assay is available to allow comparison of the different vaccines.MethodsThe validation of an anti-CSP repeat region enzyme-linked immunosorbent assay (ELISA) is described. This assay is based on the binding of serum antibodies to R32LR, a recombinant protein composed of the repeat region of P. falciparum CSP. In addition to the original recombinant R32LR, an easy to purify recombinant His-tagged R32LR protein has been constructed to be used as solid phase antigen in the assay. Also, hybridoma cell lines have been generated producing human anti-R32LR monoclonal antibodies to be used as a potential inexhaustible source of anti-CSP repeats standard, instead of a reference serum.ResultsThe anti-CSP repeats ELISA was shown to be robust, specific and linear within the analytical range, and adequately fulfilled all validation criteria as defined in the ICH guidelines. Furthermore, the coefficient of variation for repeatability and intermediate precision did not exceed 23%. Non-interference was demonstrated for R32LR-binding sera, and the assay was shown to be stable over time.ConclusionsThis ELISA, specific for antibodies directed against the CSP repeat region, can be used as a standard assay for the determination of humoral immunogenicity in the development of any CSP-based P. falciparum malaria vaccine.
Highlights
Several pre-erythrocytic malaria vaccines based on the circumsporozoite protein (CSP) antigen of Plasmodium falciparum are in clinical development
Specific anti-CSP IgG levels are a relevant parameter in CSP-based malaria vaccine projects, as there is evidence from preclinical models that anti-CSP antibodies contribute to protection against malaria during the pre-erythrocytic stage of the disease [7,8,9,10,11]
Assay characteristics The limit of detection (LOD) was estimated at 0.17 ELISA units per ml (EU/ml), rounded off to 0.2 EU/ml and the limit of quantification (LOQ) was estimated at 0.31 EU/ml rounded off to 0.3 EU/ml
Summary
Several pre-erythrocytic malaria vaccines based on the circumsporozoite protein (CSP) antigen of Plasmodium falciparum are in clinical development. Specific anti-CSP IgG levels are a relevant parameter in CSP-based malaria vaccine projects, as there is evidence from preclinical models that anti-CSP antibodies contribute to protection against malaria during the pre-erythrocytic stage of the disease [7,8,9,10,11]. An association between anti-CSP antibody levels and protection against P. falciparum infection or clinical malaria disease has been observed in humans participating to RTS,S-based vaccine trials [12,13,14,15,16,17,18,19,20]. Recombinant proteins composed of 15 NANP repeats and an Asn-Val-Asp-Pro (NVDP) oligopeptide, NVDP(NANP) NANP repeats, [NVDP(NANP)15]2, or 45 NANP repeats, [NVDP(NANP)15]3, were shown to induce antibodies that bind to the natural CSP on P. falciparum sporozoites and to block the invasion of human hepatocytes by the parasite [27,28]
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