A novel CSP C-terminal epitope targeted by an antibody with protective activity against Plasmodium falciparum

Abstract

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.

Fig 1. In vitro binning of ctCSP specific mAbs against recombinant CSP.

(A) Immunogenetics ctCSP-specific mAbs. mAbs were isolated in a previous study (36) from donors undergoing both standard vaccination (months 0, 1, 2; 012M) and delayed fractional dosing (full dose at months 0, 1 + 1/5 dose at month 7; Fx017M). All mAbs were isolated from unique donors, except mAbs 234 + 236, and 1504 + 1550 were each derived from the same individual. The germline genes were identified using IgBLAST (NIH) with the three top hits reported, and the heavy/light chain somatic hypermutations (H/L SHM) of amino acid residues were derived from the comparison with the first top-hit gene. The complementarity-determining regions (CDRs) were defined based on the Kabat system. (B) Binning experiments were performed using bio-layer interferometry on an Octet HTX at 25°C. An in-tandem binning assay format was set up. Biotinylated PfCSP was loaded onto streptavidin sensors. Loaded sensors were dipped into saturating Ab followed by competing Ab. Values are displayed as percent inhibition of binding to PfCSP (see Materials and Methods). Pairs with greater than 50% inhibition were considered competing pairs.

Fig 2. Crystal structures of anti-ctCSP Fabs in complex with the αTSR domain.

(A) Alignment of Fab234, 236, 352, 1488, and 1512 complexes (colored as shown) interacting with the αTSR domain (grey). The complexes are shown in ribbon cartoon representation. (B) Ribbon representation of the αTSR domain with important regions labeled and colored. The epitopes on the αTSR domain can be classified into: 1) the α-epitope region recognized by Fabs 234, 236, 352, and 1488; and 2) the β-epitope region recognized by Fab 1512. The Fab heavy chain and light chain are colored orange and yellow, respectively. (C) The epitopes of the Fabs are represented as surfaces and colored based on the different regions on the αTSR domain as in (B). The paratopes are shown as cartoons with side chains as sticks. The heavy chain and light chain are colored as in (B).

Fig 3. Sequences of αTSR in different haplotypes of P. falciparum and binding of mAbs to peptides corresponding to different haplotypes.

A panel of ctCSP peptides were generated for mAb affinity measurements. (A) All ctCSP haplotypes that represent strains found across East and West Africa (31) were referenced against the C-terminal domain of PfCSP isolate 3D7. The Th2R, RII+, Th3R, and CS.T3 regions are enclosed in green, cyan, magenta, and blue boxes respectively. The combined α-ctCSP epitopes from mAbs 234, 236, 352, and 1488 are highlighted in wheat, whereas the β-ctCSP epitope of mAb 1512 is shown in pink. Residues that are present in both the α-ctCSP and β-ctCSP epitopes are highlighted in orange. The transparent surfaces overlaid with ribbons of ctCSP are also shown and the combined α-ctCSP epitopes, β-ctCSP epitope, and overlapping epitopes are colored in pink, wheat, and orange as above. (B) Dissociation constant (K_D) of all mAbs was measured against all generated ctCSP peptides using surface plasmon resonance (SPR). The strongest to weakest affinities are represented as a gradient from red to yellow and green, respectively. Boxes labeled with NB (no binding) represent haplotypes that had binding responses less than the negative control (humanized mAb 5D5, a PfCSP N-terminus specific antibody).

Fig 4. Confocal microscopy images of P. falciparum sporozoites bound by anti-CSP antibodies.

P. falciparum sporozoites were dissected from infected mosquito salivary glands and purified. The binding of anti-PfCSP mAbs specific to N-terminal region of PfCSP (humanized mAb 5D5), NANP repeats (mAb311), β-ctCSP (mAb1512), and α-ctCSP (mAb236) was observed using the secondary antibody goat-anti-human Alexa 488 (green). A polyclonal anti-HSP70 antibody targeting P. falciparum heat shock protein 70 was used as a positive control and stained with the secondary antibody, goat anti-mouse Rhodamine Red-X (red). The sporozoite nuclei were stained with DAPI (blue). The merged images are also displayed. Images were acquired using a Zeiss LSM880 with Airyscan Confocal Microscope.

Fig 5. Antibody inhibition of malaria infection in mice.

Mice, five per group in three separate experiments, were injected IV with the mAbs and 16h later challenged with chimeric P. berghei sporozoites expressing full-length P. falciparum CSP. Mice were injected with D-Luciferin and imaged with the IVIS spectrum to measure the bioluminescence expressed by the chimeric parasites. Data are presented as % inhibition of parasite burden in the liver (A) or as total flux (B) in each mouse as compared to the mean of the untreated control group (0%). In both data representations, mAb1512 at both 300 μg and 100 μg and mAb236 at both 300 μg and 100 μg exhibited significant inhibition compared to the control group (* = p < 0.035 or **** = p < 0.0001 by Kruskal-Wallis test).