Isolation and light chain shuffling of a Plasmodium falciparum AMA1-specific human monoclonal antibody with growth inhibitory activity.

BACKGROUND: Plasmodium falciparum, the parasite causing malaria, affects populations in many endemic countries threatening mainly individuals with low malaria immunity, especially children. Despite the approval of the first malaria vaccine Mosquirix™ and very promising data using cryopreserved P. falciparum sporozoites (PfSPZ), further research is needed to elucidate the mechanisms of humoral immunity for the development of next-generation vaccines and alternative malaria therapies including antibody therapy. A high prevalence of antibodies against AMA1 in immune individuals has made this antigen one of the major blood-stage vaccine candidates. MATERIAL AND METHODS: Using antibody phage display, an AMA1-specific growth inhibitory human monoclonal antibody from a malaria-immune Fab library using a set of three AMA1 diversity covering variants (DiCo 1-3), which represents a wide range of AMA1 antigen sequences, was selected. The functionality of the selected clone was tested in vitro using a growth inhibition assay with P. falciparum strain 3D7. To potentially improve affinity and functional activity of the isolated antibody, a phage display mediated light chain shuffling was employed. The parental light chain was replaced with a light chain repertoire derived from the same population of human V genes, these selected antibodies were tested in binding tests and in functionality assays. RESULTS: The selected parental antibody achieved a 50% effective concentration (EC50) of 1.25 mg/mL. The subsequent light chain shuffling led to the generation of four derivatives of the parental clone with higher expression levels, similar or increased affinity and improved EC50 against 3D7 of 0.29 mg/mL. Pairwise epitope mapping gave evidence for binding to AMA1 domain II without competing with RON2. CONCLUSION: We have thus shown that a compact immune human phage display library is sufficient for the isolation of potent inhibitory monoclonal antibodies and that minor sequence mutations dramatically increase expression levels in Nicotiana benthamiana. Interestingly, the antibody blocks parasite inhibition independently of binding to RON2, thus having a yet undescribed mode of action.

Acquired immune responses to three malaria vaccine candidates and their relationship to invasion inhibition in two populations naturally exposed to malaria.

BACKGROUND: Malaria still represents a major cause of morbidity and mortality predominantly in several developing countries, and remains a priority in many public health programmes. Despite the enormous gains made in control and prevention the development of an effective vaccine represents a persisting challenge. Although several parasite antigens including pre-erythrocytic antigens and blood stage antigens have been thoroughly investigated, the identification of solid immune correlates of protection against infection by Plasmodium falciparum or clinical malaria remains a major hurdle. In this study, an immuno-epidemiological survey was carried out between two populations naturally exposed to P. falciparum malaria to determine the immune correlates of protection. METHODS: Plasma samples of immune adults from two countries (Ghana and Madagascar) were tested for their reactivity against the merozoite surface proteins MSP1-19, MSP3 and AMA1 by ELISA. The antigens had been selected on the basis of cumulative evidence of their role in anti-malarial immunity. Additionally, reactivity against crude P. falciparum lysate was investigated. Purified IgG from these samples were furthermore tested in an invasion inhibition assay for their antiparasitic activity. RESULTS: Significant intra- and inter- population variation of the reactivity of the samples to the tested antigens were found, as well as a significant positive correlation between MSP1-19 reactivity and invasion inhibition (p < 0.05). Interestingly, male donors showed a significantly higher antibody response to all tested antigens than their female counterparts. In vitro invasion inhibition assays comparing the purified antibodies from the donors from Ghana and Madagascar did not show any statistically significant difference. Although in vitro invasion inhibition increased with breadth of antibody response, the increase was not statistically significant. CONCLUSIONS: The findings support the fact that the development of semi-immunity to malaria is probably contingent on the development of antibodies to not only one, but a range of antigens and that invasion inhibition in immune adults may be a function of antibodies to various antigens. This supports strategies of vaccination including multicomponent vaccines as well as passive vaccination strategies with antibody cocktails.