Functional Relevance of Improbable Antibody Mutations for HIV Broadly Neutralizing Antibody Development

摘要

class="head no_bottom_margin" id="sec1title">Main TextThe goal of HIV-1 vaccine development is the reproducible elicitation of potent, broadly neutralizing antibodies (bnAbs) (). BnAbs isolated from infected individuals have one or more unusual traits, including long third complementarity-determining regions (CDR3s) (), autoreactivity (), large insertions and deletions (), and high somatic mutation frequencies (). Somatic hypermutation (SHM) of the B cell receptor is the diversification method within the evolutionary process of affinity maturation that leads B cells to acquire high-specificity antigen recognition (). Not all mutations acquired during antibody maturation are necessary for bnAb development; rather, high mutational levels may reflect the length of time required to elicit bnAbs (, ). Consequently, shorter maturation pathways to neutralization breadth involving a critical subset of mutations are desirable, because antibody mutation levels induced by vaccines seldom reach the mutation frequencies observed in bnAbs (, ). Importantly, within this subset of critical mutations, some mutations may be probable and easy to elicit, whereas other mutations may be improbable and very challenging to elicit due to biases in how mutations arise during affinity maturation.Somatic hypermutation occurs prior to antigen affinity-based selection during affinity maturation (, ). Somatic hypermutation is mediated by activation-induced cytidine deaminase (AID) (), and AID preferentially targets specific nucleotide sequence motifs (“hot spots”), whereas targeting of other nucleotide motifs (“cold spots”) is disfavored (, , href="#bib64" rid="bib64" class=" bibr popnode">Yaari et al., 2013). AID initiates DNA lesions, and their subsequent repair results in a bias for certain bases to be substituted at the targeted position (href="#bib11" rid="bib11" class=" bibr popnode">Cowell and Kepler, 2000). The consequence of this non-uniformly random mutation process is that specific amino acid substitutions occur with varying frequencies prior to antigenic selection. Mutations at AID hotspots can occur frequently in the absence of antigen selection due to immune-activation-associated AID activity (href="#bib6" rid="bib6" class=" bibr popnode">Bonsignori et al., 2016, href="#bib65" rid="bib65" class=" bibr popnode">Yeap et al., 2015). Amino acid substitutions that occur infrequently generally require strong antigenic selection in order to arise during maturation (href="#bib9" rid="bib9" class=" bibr popnode">Brown et al., 1992, href="#bib32" rid="bib32" class=" bibr popnode">Kocks and Rajewsky, 1988). Such rare amino acid substitutions are improbable prior to selection for two reasons: 1) base mutations must occur at AID cold spots, and 2) due to codon mapping, multiple base substitutions must occur for a specific amino acid change. Within the critical subset of mutations that grant broad neutralization capacity to a bnAb lineage, those key mutations that are also improbable prior to selection may represent important events in bnAb maturation and are thus compelling targets for selection in a vaccine setting. We recently described a rare mutation, G57R, in DH270, a V3-glycan bnAb lineage, that conferred broad neutralization, thus demonstrating in one bnAb lineage that functionally important, improbable mutations can be roadblocks in HIV-1 bnAb development (href="#bib7" rid="bib7" class=" bibr popnode">Bonsignori et al., 2017). However, what has remained unclear is whether and to what extent such roadblocks are a general problem for bnAb elicitation. Here we describe the identification of improbable mutations in three bnAb B cell lineages and determine the functional relevance of these mutations for development of bnAb neutralization potency.To determine the role of rare mutational events in bnAb development, we developed a computational program, “antigen receptor mutation analyzer for detection of low-likelihood occurrences” (ARMADiLLO), to identify improbable antibody mutations (see href="#sec2" rid="sec2" class=" sec">STAR Methods). We first applied ARMADiLLO retrospectively to the analysis of the G57R mutation in the DH270 bnAb lineage. ARMADiLLO estimated the G57R mutation to occur with < 1% frequency prior to selection. This mutation was functionally critical because reversion back to G57 in the lineage resulted in loss of heterologous neutralization (href="#bib7" rid="bib7" class=" bibr popnode">Bonsignori et al., 2017). Thus, the ARMADiLLO program can identify a known, key improbable mutation.All BCR mutations arise prior to antigenic selection (href="#bib22" rid="bib22" class=" bibr popnode">Hwang et al., 2015). In HIV-1 infection, antibody heterologous breadth is not directly selected for during bnAb development because BCRs only interact with autologous virus Envs. Since improbable bnAb mutations can confer heterologous breadth, they represent critical events in bnAb development and make compelling targets for focusing selection with immunogens. To test this hypothesis, we analyzed three additional bnAb lineages with ARMADiLLO to identify improbable mutations (defined as < 2% estimated probability of occurring prior to selection; see href="#sec2" rid="sec2" class=" sec">STAR Methods) and then tested for their effect on neutralization during bnAb development. We chose three lineages that allowed for study of different levels of maturation in bnAb development: CH235, mid-stage bnAb development (href="#bib7" rid="bib7" class=" bibr popnode">Bonsignori et al., 2017); VRC01, late-stage bnAb development (href="#bib63" rid="bib63" class=" bibr popnode">Wu et al., 2015); and BF520.1, early-stage bnAb development (href="#bib53" rid="bib53" class=" bibr popnode">Simonich et al., 2016).CH235 is a CD4-binding-site (href="#bib17" rid="bib17" class=" bibr popnode">Gao et al., 2014) bnAb lineage that evolved to 90% neutralization breadth over 5 years of infection and acquired 45 VH amino acid mutations (href="#bib6" rid="bib6" class=" bibr popnode">Bonsignori et al., 2016). We identified improbable mutations in the heavy chain of an early intermediate member of the lineage (also termed CH235), reverted each to their respective germline-encoded amino acids, and then tested antibody mutants for neutralization against heterologous, difficult-to-neutralize (tier 2) (href="#bib51" rid="bib51" class=" bibr popnode">Seaman et al., 2010), CH235-sensitive viruses (href="/pmc/articles/PMC6002614/figure/fig1/" target="figure" class="fig-table-link figpopup" rid-figpopup="fig1" rid-ob="ob-fig1" co-legend-rid="lgnd_fig1">Figures 1A and href="#mmc1" rid="mmc1" class=" supplementary-material">S1A). Single amino acid reversion mutations resulted in either a reduction or an abrogation of neutralization for each of three improbable mutations—K19T, W47L, and G55W—demonstrating that improbable mutations in the CH235 lineage were indeed critical and could confer heterologous neutralization.href="/pmc/articles/PMC6002614/figure/fig1/" target="figure" rid-figpopup="fig1" rid-ob="ob-fig1">class="inline_block ts_canvas" href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&p=PMC3&id=6002614_gr1.jpg" target="tileshopwindow">target="object" href="/pmc/articles/PMC6002614/figure/fig1/?report=objectonly">Open in a separate windowclass="figpopup" href="/pmc/articles/PMC6002614/figure/fig1/" target="figure" rid-figpopup="fig1" rid-ob="ob-fig1">Figure 1Improbable Mutations Confer Heterologous Neutralization in bnAb Development(A–C) BnAbs (A) CH235, (B) VRC01, and (C) BF520.1 and their corresponding mutants with reverted improbable mutations were tested for neutralization against heterologous viruses. The reversion of improbable mutations in all three bnAbs diminished neutralization potency. The chain in which the mutation was made is listed in parentheses. The estimated probabilities of the mutations, as well as the neutralization capacities of the antibody mutants against additional viruses, are included in href="#mmc1" rid="mmc1" class=" supplementary-material">Table S1.