Search

Abstract The generation of high-quality antibody responses to Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP), the primary surface antigen of Pf sporozoites, is paramount to the development of an effective malaria vaccine. Here we present an in-depth structural and functional analysis of a panel of potent antibodies encoded by the immunoglobulin heavy chain variable (IGHV) gene IGHV3-33, which is among the most prevalent and potent antibody families induced in the anti-PfCSP immune response and targets the Asn-Ala-Asn-Pro (NANP) repeat region. Cryo-electron microscopy (cryo-EM) reveals a remarkable spectrum of helical antibody-PfCSP structures stabilized by homotypic interactions between tightly packed fragments antigen binding (Fabs), many of which correlate with somatic hypermutation. We demonstrate a key role of these mutated homotypic contacts for high avidity binding to PfCSP and in protection from Pf malaria infection. Together, these data emphasize the importance of anti-homotypic affinity maturation in the frequent selection of IGHV3–33 antibodies and highlight key features underlying the potent protection of this antibody family.

Abstract The symptoms of malaria occur during the blood stage of infection, when the parasite replicates within human red blood cells. The human malaria parasite, Plasmodium vivax, selectively invades reticulocytes in a process which requires an interaction between the ectodomain of the human DARC receptor and the Plasmodium vivax Duffy-binding protein, PvDBP. Previous studies have revealed that a small helical peptide from DARC binds to region II of PvDBP (PvDBP-RII). However, it is also known that sulphation of tyrosine residues on DARC affects its binding to PvDBP and these residues were not observed in previous structures. We therefore present the structure of PvDBP-RII bound to sulphated DARC peptide, showing that a sulphate on tyrosine 41 binds to a charged pocket on PvDBP-RII. We use molecular dynamics simulations, affinity measurements and growth-inhibition experiments in parasites to confirm the importance of this interaction. We also reveal the epitope for vaccine-elicited growth-inhibitory antibody DB1. This provides a complete understanding of the binding of PvDBP-RII to DARC and will guide the design of vaccines and therapeutics to target this essential interaction.

Abstract A primary objective in malaria vaccine design is the generation of high-quality antibody responses against the circumsporozoite protein of the malaria parasite, Plasmodium falciparum (PfCSP). To enable rational antigen design, we solved a cryo-EM structure of the highly potent anti-PfCSP antibody L9 in complex with recombinant PfCSP. We found that L9 Fab binds multivalently to the minor (NPNV) repeat domain, which is stabilized by a unique set of affinity-matured homotypic, antibody-antibody contacts. Molecular dynamics simulations revealed a critical role of the L9 light chain in integrity of the homotypic interface, which likely impacts PfCSP affinity and protective efficacy. These findings reveal the molecular mechanism of the unique NPNV selectivity of L9 and emphasize the importance of anti-homotypic affinity maturation in protective immunity against P. falciparum.

The most advanced P. falciparum circumsporozoite protein (PfCSP)-based malaria vaccine confers partial protection. Here, Pholcharee et al. present crystal structures, binding affinities/kinetics, and in vivo protection of 8 anti-NANP antibodies to understand in vivo protection of PfCSP-targeting antibodies.

Potent and durable vaccine responses will be required for control of malaria caused by Plasmodium falciparum (Pf). RTS,S/AS01 is the first, and to date, the only vaccine that has demonstrated significant reduction of clinical and severe malaria in endemic cohorts in Phase 3 trials. Although the vaccine is protective, efficacy declines over time with kinetics paralleling the decline in antibody responses to the Pf circumsporozoite protein (PfCSP). Although most attention has focused on antibodies to repeat motifs on PfCSP, antibodies to other regions may play a role in protection. Here, we expressed and characterized seven monoclonal antibodies to the C-terminal domain of CSP (ctCSP) from volunteers immunized with RTS,S/AS01. Competition and crystal structure studies indicated that the antibodies target two different sites on opposite faces of ctCSP. One site contains a polymorphic region (denoted α-ctCSP) and has been previously characterized, whereas the second is a previously undescribed site on the conserved β-sheet face of the ctCSP (denoted β-ctCSP). Antibodies to the β-ctCSP site exhibited broad reactivity with a diverse panel of ctCSP peptides whose sequences were derived from field isolates of P. falciparum whereas antibodies to the α-ctCSP site showed very limited cross reactivity. Importantly, an antibody to the β-site demonstrated inhibition activity against malaria infection in a murine model. This study identifies a previously unidentified conserved epitope on CSP that could be targeted by prophylactic antibodies and exploited in structure-based vaccine design.

Incessant antigenic evolution enables the persistence and spread of influenza virus in the human population. As the principal target of the immune response, the hemagglutinin (HA) surface antigen on influenza viruses continuously acquires and replaces N-linked glycosylation sites to shield immunogenic protein epitopes using host-derived glycans. Anti-glycan antibodies, such as 2G12, target the HIV-1 envelope protein (Env), which is even more extensively glycosylated and contains under-processed oligomannose-type clusters on its dense glycan shield. Here, we illustrate that 2G12 can also neutralize human seasonal influenza A H3N2 viruses that have evolved to present similar oligomannose-type clusters on their HAs from around 20 years after the 1968 pandemic. Using structural biology and mass spectrometric approaches, we find that two N-glycosylation sites close to the receptor binding site (RBS) on influenza hemagglutinin represent the oligomannose cluster recognized by 2G12. One of these glycan sites is highly conserved in all human H3N2 strains and the other emerged during virus evolution. These two N-glycosylation sites have also become crucial for fitness of recent H3N2 strains. These findings shed light on the evolution of the glycan shield on influenza virus and suggest 2G12-like antibodies can potentially act as broad neutralizers to target human enveloped viruses.

Lasting protection has long been a goal for malaria vaccines. The major surface antigen on Plasmodium falciparum sporozoites, the circumsporozoite protein (PfCSP), has been an attractive target for vaccine development and most protective antibodies studied to date interact with the central NANP repeat region of PfCSP. However, it remains unclear what structural and functional characteristics correlate with better protection by one antibody over another. Binding to the junctional region between the N-terminal domain and central NANP repeats has been proposed to result in superior protection: this region initiates with the only NPDP sequence followed immediately by NANP. Here, we isolated antibodies in Kymab mice immunized with full-length recombinant PfCSP and two protective antibodies were selected for further study with reactivity against the junctional region. X-ray and EM structures of two monoclonal antibodies, mAb667 and mAb668, shed light on their differential affinity and specificity for the junctional region. Importantly, these antibodies also bind to the NANP repeat region with equal or better affinity. A comparison with an NANP-only binding antibody (mAb317) revealed roughly similar but statistically distinct levels of protection against sporozoite challenge in mouse liver burden models, suggesting that junctional antibody protection might relate to the ability to also cross-react with the NANP repeat region. Our findings indicate that additional efforts are necessary to isolate a true junctional antibody with no or much reduced affinity to the NANP region to elucidate the role of the junctional epitope in protection.