Molecular characterization of the PfCSP B-cell response
We generated a collection of 88 IgG antibodies, produced by recombinant expression cloning, from RTS,S vaccinated children from Mozambique. A fraction of the antibodies recognized the NANP major repeat region (NANP) of PfCSP, a dominant target site by potent antibody responses in adults, and the remaining recognized the central repeat domain (CTD). We then performed an exhaustive analysis of the molecular attributes of the antibodies revealing that PfCSP B-cell antibodies from young children present sequence hallmarks resembling Pf-naïve adults, and experienced refinement through antigen-driven selection, presumably in germinal centers, despite low somatic mutation rates.
Antiparasitic properties of PfCSP monoclonal antibodies
We subsequently evaluated the antiparasitic properties of our collection of antibodies in a wide array of in vitro assays to measure killing capacity, the level of antibody-dependent elongation induced to the parasite, and inhibition of hepatocyte invasion. We discovered a strong lethality of NANP antibodies as compared with those targeting CTD. In addition, we identified a significant association between parasite lethality and the kinetic association rates of NANP antibodies, suggesting that rapid binding to PfCSP in the surface of the parasite is a major determinant of cytotoxicity. Global analysis of the different neutralization phenotypes revealed that highly cytotoxic NANP antibodies inhibited cell invasion at variable levels and triggered a medium/long-elongated parasite phenotype. Contrarily, weak cytotoxic NANP antibodies induced a short round elongated phenotype and weak inhibition. Thus, RTS,S induces potent polyfunctional antiparasitic antibodies in children, preferentially targeting the major repeat region. The four most potent NANP antibodies induced sterile protection against parasite challenge in a mouse model. All these findings indicate that, despite the underdeveloped immune system of pediatric populations, RTS,S induces potent PfCSP neutralizing antibodies in African children, with immunoglobulin molecular features resembling adults.
Structural analysis of protective PfCSP antibodies
The four protective NANP antibodies were analyzed by cryogenic electron microscorpy (Cryo-EM) to resolve the antigen-antibody complex models. These analyses revealed particular features of NANP antibodies including the induction of an -helix conformation around PfCSP stabilized by interFab homotypic contacts. Those features resembled that observed in protective antibodies isolated from adults.
Immunophenotyping of PfCSP+ B cells
We performed immunophenotyping of PfCSP+ B cells from a subset of RTS,S vaccinated children. Analysis of the Ig isotypes revealed that CSP-reactive memory B cells (defined as CD19+, IgD-, and/or CD27+) were predominantly IgG in most donors (range 50-83%), whereas IgM represented on average 25% (range 7-35%). Contrarily, IgA represented a small fraction of CSP18+ MBC. CSP18-reactive IgG+ B cells had frequencies ranging from 1.4 to 0.41%, and were mainly resting (CD27+CD21+) and activated (CD27+CD21-) memory B cells.
Molecular characterization of a blood-stage “off-target” signature of protection
We previously described that RTS,S-vaccinated children from different sites with varying malaria transmission intensities developed PfCSP antibodies that strongly cross-recognized unrelated Pf proteins, including erythrocytic-stage antigens. Vaccinees presenting this so called “off-target” (OT) profile experienced lower clinical malaria incidence, suggesting this previously unrecognized phenomenon as a novel correlate of protection. To gain further insights on this phenomenon, we performed an in-depth serological analysis of 60 vaccinees with different OT profiles. These investigations revealed that OT plasma antibodies were enriched in both high avidity and a superior ratio of PfCSP antibodies directed to NANP over those targeting CTD, and higher recognition of the minor and junctional repeats, which are hallmarks of potent antibodies. Immunoglobulin molecular cloning and subsequent characterization of PfCSP monoclonal antibodies from selected OT-profiled donors identified 9 NANP-specific antibodies exhibiting a strong cross-reactivity with the erythrocytic merozoite surface protein 5 (PfMSP5). Fine mapping analysis revealed antibody recognition of a minimum PfMSP5 motif resembling that recognize in the PfCSP major repeat core. We identified this motif in different Pf proteins. Thus, by integrating seroepidemiological investigations and molecular analysis, we identified a phenomenon of molecular mimicry in the malaria parasite associated with a lower clinical malaria incidence, that could be used as a novel correlate of protection.
