Periodic Reporting for period 1 - HUMALCOR (Identifying molecular features of potent antibody and memory B-cell responses associated with RTS,S malaria vaccine efficacy)
Periodo di rendicontazione: 2024-01-01 al 2025-12-31
Malaria is a major global health concern, jeopardizing the future of childhood, equity and economic development of endemic countries, particularly in the African continent. Hence, the progress towards an effective vaccine is a global priority to protect vulnerable populations and achieve elimination goals. Although RTS,S/AS01 (RTS,S) is the first malaria vaccine approved for use in African children, its efficacy during phase 3 clinical trials was partial and its duration modest. Understanding the factors behind limited vaccine efficacy and identifying the determinants of protective immunity is required to support improved next-generation vaccines aiming to provide maximum coverage. We previously reported that high antibody levels to the circumsporozoite (CSP) RTS,S immunogen are associated with vaccine efficacy. However, the molecular features supporting optimal or suboptimal antibody responses have not been fully deciphered in pediatric populations. To fill these knowledge gaps, this project envisions to perform an in-depth humoral immunoprofiling of RTS,S vaccine-induced responses in African young children. By combining high-throughput technologies including methods to directly clone and characterize large antibody collections from single antigen-specific B cells, immunophenotyping of lymphocyte populations and functional and biophysical characterization, we will elucidate the hallmarks of potent long-lasting antibody responses, and identify the mechanisms compromising them. The main goal is to generate basic scientific knowledge by studying a vulnerable group previously unexplored, to aid future development of vaccines and immunotherapeutics, and improve human health using next-generation antibody science.
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.
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.
The integration of our results provided a novel framework of knowledge on the molecular evolution of the antibody response to RTS,S in an understudied population that suffers the most severe consequences of malaria. By understanding how potent PfCSP antibodies are generated and selected as well as their antiparasitic modes of action, our research established that a strong, highly efficient humoral response can be elicited by RTS,S in pediatric groups under 1 year. The unprecedent molecular dissection of the RTS,S-induced off-target phenomenon provided new insights not only on highly potent PfCSP antibody features, but also for the selection of erythrocytic-stage antigens for multivalent next-generation vaccines with cross-stage activity. Most importantly, we provided a mechanistic explanation for the association of the off-target signature with lower clinical malaria incidence, reinforcing this phenomenon as a novel correlate of protection. By employing a broad spectrum of in vitro assays, we have extended beyond those focused predominantly on hepatocyte invasion, to comprehensively disentangle the antiparasitic mechanisms of action of PfCSP antibodies. Potent antibodies naturally occurring in humans reveal antigenic sites and epitopes that could serve as components of preventative vaccines. By identifying highly protective antibodies in vivo we will enable the rational design of next-generation immunogens aiming to elicit similar, highly effective antibodies, establishing a critical foundation for malaria vaccine development. In addition, the identification of protective PfCSP mAbs will aid current field efforts on immunotherapeutic and combination strategies with vaccine candidates. Overall, the research derived from the HUMALCOR project will open new avenues in pediatric vaccinology and immunogen refinement for next-generation malaria vaccines for the most vulnerable populations. While addressing a major challenge in the malaria field, our research also provided fundamental insights that could be applied to diverse infections in infants, such as respiratory syncytial virus (RSV) or measles.