Structural and biophysical correlation of anti-NANP antibodies within vivoprotection againstP. falciparum

Abstract

The most advanced P. falciparum circumsporozoite protein (PfCSP)-based malaria vaccine, RTS,S/AS01 (RTS,S), confers partial protection but with antibody titers that wane relatively rapidly, highlighting the need to elicit more potent and durable antibody responses. Here, we elucidate crystal structures, binding affinities and kinetics, and in vivo protection of eight anti-NANP antibodies (Abs) derived from an RTS,S phase 2a trial and encoded by three different heavy-chain germline genes. The structures reinforce the importance of homotypic Fab-Fab interactions in protective Abs and the overwhelmingly dominant preference for a germline-encoded aromatic residue for recognition of the NANP motif. A number of biophysical properties were analyzed and antibody affinity correlated best with protection in an in vivo mouse model, with the more potent antibodies also recognizing epitopes with repeating secondary structural motifs of type I β- and Asn pseudo 310 turns. Such insights can be incorporated into design of more effective immunogens as well as antibodies for passive immunization.

The light chain of the L9 antibody is critical for binding circumsporozoite protein minor repeats and preventing malaria

“…Lastly, structural studies showed that F10HL9κ and L9HF10κ both bound NPNV in the minimal peptide NANPNVDP in a type-1 β-turn that was near-identical to the NPNA type-1 βturn commonly observed for NANP-preferring mAbs (Imkeller et al, 2018;Oyen et al, 2017;Pholcharee et al, 2021), suggesting that recognition of single NPNV motifs does not differ between these mAbs. The convergent conformations adopted by these subtly different tetrapeptides is in line with a previous study which also observed that cross-reactive PfCSP mAbs bind different repeat epitopes in near-identical conformations (Murugan et al, 2020).…”

“…To gain structural insight into the NPNV binding mechanism of L9, we crystallized Fabs of F10HL9κ and L9HF10κ in complex with the NANPNVDP peptide and solved their structures to 1.89 Å and 2.23 Å, respectively (Figure 2A,C). F10HL9κ Fab binds NANPNVDP with the NPNV motif adopting a type-1 β-turn (Figure S1B) that the NPNA motif was shown to adopt (Ghasparian et al, 2006) and is commonly found in NPNA-preferring repeat mAbs (Figure 2B) (Imkeller et al, 2018;Oyen et al, 2017;Pholcharee et al, 2021). All three HCDRs and the KCDR1 and KCDR3 bind NANPNVDP with a total buried surface area (BSA) of ~373 Å 2 , ~203 Å 2 from the IgH and ~170 Å 2 from Igκ (Figure 2E).…”

Functional human IgA targets a conserved site on malaria sporozoites

“…S8C to E ). Additionally, since the two copies of either MAD2-6 IgA or IgG Fabs come into close contact while simultaneously binding the peptide, they display homotypic Fab-Fab interactions, which were first observed in anti-NANP antibodies ( 27 – 29 ). These interactions are mainly between the heavy chain of one Fab and light chain of the other Fab and include extensive hydrogen bonds and salt bridges between CDRs H2 and L2 ( Fig.…”

“…S8G ). A corresponding interchain disulfide between L Cys 214 and H Cys 215 in IgG is sometimes observed in Fab crystal structures, such as anti-NANP Ab 366 ( 29 ) ( fig. S8G ).…”

“…Thus, MAD2-6 exhibits an inherent propensity for homotypic Fab-Fab interactions that are further strengthened by heavy-chain somatic hypermutation, potentially explaining its low affinity in vitro and increased avidity in vivo. However, this avidity effect can be even more prominent for anti-NANP antibodies that simultaneously bind multiple epitopes formed by tandem NANP repeats ( 27 , 29 ).…”