Antihomotypic affinity maturation improves human B cell responses against a repetitive epitope

Affinity maturation selects B cells expressing somatically mutated antibody variants with improved antigen-binding properties to protect from invading pathogens.We determined the molecular mechanism underlying the clonal selection and affinity maturation of human B cells expressing protective antibodies against the circumsporozoite protein of the malaria parasite Plasmodium falciparum (PfCSP).We show in molecular detail that the repetitive nature of PfCSP facilitates direct homotypic interactions between two PfCSP repeat-bound monoclonal antibodies, thereby improving antigen affinity and B cell activation. These data provide a mechanistic explanation for the strong selection of somatic mutations that mediate homotypic antibody interactions after repeated parasite exposure in humans. Our findings demonstrate a different mode of antigen-mediated affinity maturation to improve antibody responses to PfCSP and presumably other repetitive antigens.

for a NANP 5-mer peptide (NANP 5 ) between 10 −6 and 10 −9 M ( Fig. 1A and table S1) (9). Antigen binding was abrogated when the original Ig Vk1-5 light chain was replaced by Vk2-28 or when the native Ig heavy chains were paired with a Vk1-5 light chain with 9-amino acid-long KCDR3 (Fig.  1B), demonstrating the importance of these specific Ig features in antigen recognition.
The majority of NANP-reactive V H 3-33-Vk1-5-KCDR3:8 B cells belonged to clonally expanded and somatic hypermutation (SHM)-diversified cell clusters with strong selection for replacement mutations in HCDR1 (H.S31) and HCDR2 (H.V50 and H.N56), as well as KCDR3 [KCDR3 S 93 (K.S93)], likely as a result of affinity maturation (Fig. 1, E and F) (9). The introduction of missing somatic mutations (mut) or reversions (rev) at H.V50 and, to a lesser extent, H.S31 revealed a role in binding to a minimal NANP 3 peptide (10,11), as demonstrated for the germline antibody 2163 and the low-mutated antibody 1210 (Fig. 1, G and H, and table S3). In contrast, exchanges at H.N56 and K.S93, either alone (in antibodies 1210_H.K56_N rev , 1210_K. N93_S rev , and 2163_H.N56_K mut ) or in combination (in 1210_NS and 2163_KN), showed no significant effect (Fig. 1, G and H, and table S3). Thus, affinity maturation to the repeat explained the strong selection for only two of the four characteristic replacement mutations in V H 3-33-Vk1-5-KCDR3:8 anti-NANP antibodies.
We next determined the cocrystal structure of the 1210 antigen-binding fragment (Fab) with NANP 5 (Fig. 2, fig. S1A, and tables S4 to S6). The NANP core epitope contained a type I b turn and an elongated conformation (Fig. 2, A and C, and fig. S1B), similar to NANP bound to a chimeric 2140 Ig heavy chain-1210 Ig k antibody and in line with previous observations (fig. S1C and tables S4 and S7) (10)(11)(12)(13)(14). Main-chain atoms in KCDR3 were optimally positioned to mediate H bonds with the repeat, likely contributing to the strong selection of KCDR3:8 (Fig. 2, B and C, and tables S2, S5, and S10). V H 3-33 germline residues, notably H.V50 and H.W52 (the residue encoded only by IGHV3-33 alleles), as well as H.Y52A and H.Y58 in HCDR2, mediated the majority of antigen contacts (table S5 and fig. S2) (15). Affinity maturation at H.V50 and H.S31 may be explained by strengthened van der Waals interactions with the repeat (Fig. 2C).
Notably, our crystal structure also revealed that two 1210 Fabs (designated 1210 Fab-A and Fab-B) bound to one NANP 5 peptide in a headto-head configuration at a 133°angle ( Fig. 2D  and fig. S3). This binding mode led to six homotypic antibody-antibody H bonds providing 263 Å 2 of buried surface area (BSA) between the two Fabs and an additional~120 Å 2 of BSA between the Fabs and the repeat (Fig. 2, E and F, and tables S5, S6, and S10). Two highly selected mutations, H.N56_K and K.S93_N (Fig. 1, E and F), formed H bonds with H.Y52A and H.S99 in the opposing Fab, thereby stabilizing the head-tohead configuration (Fig. 2, G and H). KCDR3:8 optimally contacted HCDR3 of the opposite 1210 molecule, providing another explanation for the length restriction in KCDR3.
To investigate homotypic interactions, we next measured the Fab affinities for NANP 5 and NANP 3 for 1210, 1210_NS (which lacks the selected mutations involved in homotypic binding), a 1210 H.D100_Y mut K.N92_Y mut mutant (1210_YY, designed to disrupt head-to-head binding through steric clashes), and a 1210 germline antibody (1210_GL) (Fig. 2I and fig. S4). Compared with 1210, 1210_YY and 1210_NS showed significantly weakened affinity for NANP 5 but not for NANP 3 , whereas 1210_GL was significantly worse than 1210 at binding both peptides ( Fig. 2I and fig. S4) (16). These data suggest that only 1210 efficiently recognized the repeat in a high-affinity homotypic head-to-head binding configuration. An analysis of full-length PfCSP with 38 NANP repeats confirmed this hypothesis. Approximately twelve 1210 Fabs bound PfCSP and recognized the NANP repeat in a head-to-head binding configuration similar to the 1210 Fab-NANP 5 crystal structure (Fig. 2, J and K, and fig. S3D) (11,17).
Furthermore, 1210_YY IgG, with its restricted ability to engage in homotypic antibody interactions, showed a lower binding avidity to fulllength PfCSP than 1210 ( fig. S5). Thus, affinity maturation selects for mutations that improve homotypic antibody interactions, thereby indirectly increasing PfCSP NANP binding.
To better understand the selection of SHM at the cellular level, we measured the degree of B cell activation in response to NANP 5 of transgenic B cell lines expressing 1210 or variant B cell receptors (BCRs) (Fig. 3, A to D). BCR signaling was delayed in cells expressing 1210_GL compared with that in cells expressing 1210. This effect was even more pronounced in 1210_YY mutant cells. As expected, 1210_H.V50_I mut (1210 with HCDR2 V 50 →I), with high repeat affinity, mediated stronger signals than 1210, especially with low antigen concentrations, whereas 1210_NS showed no significant differences (Fig. 3D). Thus, B cell activation is promoted by both direct NANP binding and homotypic antibody interactions. Despite a 2-log difference in NANP 3 affinities (Fig. 1, G and H) and the varied potential of these antibodies to engage in homotypic interactions, all showed similar capacities to inhibit Pf sporozoites in vitro ( Fig. 3E and fig. S6). Likewise, all antibodies conferred similar levels of dose-dependent protection from the development of blood-stage parasites after passive immunization in mice, presumably because of strong avidity effects (Fig. 3F). These data provide a mechanistic explanation for the strong in vivo selection of antihomotypic antibody mutants by affinity maturation, independently of their protective efficacy as soluble antibodies.