Rapid development of broadly influenza neutralizing antibodies through redundant mutations

Abstract

The neutralizing antibody response to influenza virus is dominated by antibodies that bind to the globular head of haemagglutinin, which undergoes a continuous antigenic drift, necessitating the re-formulation of influenza vaccines on an annual basis. Recently, several laboratories have described a new class of rare influenza-neutralizing antibodies that target a conserved site in the haemagglutinin stem. Most of these antibodies use the heavy-chain variable region VH1-69 gene, and structural data demonstrate that they bind to the haemagglutinin stem through conserved heavy-chain complementarity determining region (HCDR) residues. However, the VH1-69 antibodies are highly mutated and are produced by some but not all individuals, suggesting that several somatic mutations may be required for their development. To address this, here we characterize 197 anti-stem antibodies from a single donor, reconstruct the developmental pathways of several VH1-69 clones and identify two key elements that are required for the initial development of most VH1-69 antibodies: a polymorphic germline-encoded phenylalanine at position 54 and a conserved tyrosine at position 98 in HCDR3. Strikingly, in most cases a single proline to alanine mutation at position 52a in HCDR2 is sufficient to confer high affinity binding to the selecting H1 antigen, consistent with rapid affinity maturation. Surprisingly, additional favourable mutations continue to accumulate, increasing the breadth of reactivity and making both the initial mutations and phenylalanine at position 54 functionally redundant. These results define VH1-69 allele polymorphism, rearrangement of the VDJ gene segments and single somatic mutations as the three requirements for generating broadly neutralizing VH1-69 antibodies and reveal an unexpected redundancy in the affinity maturation process.

Overlapping hotspots in CDRs are critical sites for V region diversification

“…For example, there are many mutations in A:T around residue 280, where the A:Ts are between two simple AID hotspots that mutate at an intermediate frequency, and as in the CDR2, there are no AID cold spots. Mutations in FW3 have been observed previously (62), and recent studies (1,30) have revealed the importance of mutations, and even deletions and insertions, in the FWs in the broadly neutralizing capacity of antibodies to HIV and influenza. In addition, it has been suggested that mutations in the FW are necessary to stabilize the structure of the CDRs (31).…”

“…The black vertical lines are mutations in dC on either strand and are presumed to have been initiated by the direct action of AID, whereas the gray vertical lines are mutations in A:T residues and are presumed be a result of error-prone MMR (47). After recent precedents (1,30,48), the CDRs indicated by the horizontal gray bars at the top of the figure are based on the Kabat criteria (49) combined with analysis of the crystal structure of the IGHV3-23*01 V region, rather than the IMGT definition (48). It is obvious that the frequency of these independent and unique somatic mutations in Fig.…”

“…In addition, we are still learning about how the V regions can undergo such high rates of mutation and still assemble their heavy and light chain V regions to produce a stable antigen-binding site that can undergo affinity maturation and changes in fine specificity. Although it is clear that mutations in the complementary determining regions (CDRs) play a critical role in antigen binding, recent studies reveal that some of these highly mutated sites in the CDRs do not directly interact with antigen but have an important role in generating protective broadly neutralizing antibodies to influenza and HIV (1,30,31).Taken together, these findings suggest that the DNA sequence of Ig V regions has evolved so that AID will mutate certain subregions while protecting those parts of the V region that are required to …”

Antibody‐virus co‐evolution in HIV infection: paths for HIV vaccine development

“…The V3-glycan PCDN, BF520-derived and DH270 bnAb lineages share some common traits: 1) differently from previously described V3-gylcan bnAbs (23, 25), their evolution did not involve insertion/deletion (indel) events, demonstrating that indels are not a universal requirement for the V3-glycan bnAb class to acquire neutralization breadth; 2) neutralization breadth was acquired with relatively modest levels of somatic hypermutation that can be achieved through vaccination (57, 60, 61), and 3) the UCA of the V3-glycan lineages did not neutralize or bind autologous TF suggesting the hypothesis that V3-glycan bnAb lineages may arise in response to altered forms of the Env protein.…”

Structure of the apo anti-influenza CH65 Fab

“…These bnAbs bind the influenza hemagglutinin glycoprotein (HA; Wilson et al, 1981) and target functionally conserved epitopes on the HA head and stem regions (Lee & Wilson, 2015). In conjunction with these advances in antibody discovery, the developmental pathways that the HA stem-targeted V H 1-69-encoded antibodies take to convert from germline to affinitymatured forms are an active area of investigation (Lingwood et al, 2012;Avnir et al, 2014;Pappas et al, 2014). These studies show that somatic mutations that arise during the affinitymaturation process can affect the conformations of the antibody complementarity-determining region (CDR) loops.…”