Mutational fitness landscapes reveal genetic and structural improvement pathways for a vaccine-elicited HIV-1 broadly neutralizing antibody

Vaccine-based elicitation of broadly neutralizing antibodies holds great promise for preventing HIV-1 transmission. However, the key biophysical markers of improved antibody recognition remain uncertain in the diverse landscape of potential antibody mutation pathways, and a more complete understanding of anti–HIV-1 fusion peptide (FP) antibody development will accelerate rational vaccine designs. Here we survey the mutational landscape of the vaccine-elicited anti-FP antibody, vFP16.02, to determine the genetic, structural, and functional features associated with antibody improvement or fitness. Using site-saturation mutagenesis and yeast display functional screening, we found that 1% of possible single mutations improved HIV-1 envelope trimer (Env) affinity, but generally comprised rare somatic hypermutations that may not arise frequently in vivo. We observed that many single mutations in the vFP16.02 Fab could enhance affinity >1,000-fold against soluble FP, although affinity improvements against the HIV-1 trimer were more measured and rare. The most potent variants enhanced affinity to both soluble FP and Env, had mutations concentrated in antibody framework regions, and achieved up to 37% neutralization breadth compared to 28% neutralization of the template antibody. Altered heavy- and light-chain interface angles and conformational dynamics, as well as reduced Fab thermal stability, were associated with improved HIV-1 neutralization breadth and potency. We also observed parallel sets of mutations that enhanced viral neutralization through similar structural mechanisms. These data provide a quantitative understanding of the mutational landscape for vaccine-elicited FP-directed broadly neutralizing antibody and demonstrate that numerous antigen-distal framework mutations can improve antibody function by enhancing affinity simultaneously toward HIV-1 Env and FP.

The tremendous circulating sequence diversity of HIV-1 and its capacity to evade host immunity pose unique challenges for vaccine design (reviewed in refs. 1, 2). Broadly neutralizing antibodies (bNAbs) identified from HIV-1 patients target conserved vulnerable epitopes on the HIV-1 envelope protein (Env) to prevent HIV-1 infection, and HIV-1 bNAb elicitation has become a major goal for HIV-1 vaccine design (3, 4). Several HIV-1 vulnerable epitopes have been described (5) including the following: the V1V2 apex (6, 7), the CD4-binding site (8–10), the membrane-proximal external region (11, 12), the glycan-V3 region (also known as N332 glycan supersite) (10, 13), the highly glycosylated region at the center of the silent face on the gp120 subunit (14), and the fusion peptide (FP), which is required for viral entry (15–17). Several complementary approaches seek to develop immunogens that elicit broadly neutralizing HIV-1 antibodies, with promising results (4, 18, 19). However, vaccine-elicited HIV-1 antibodies are often either less potent or less broad than many of the bNAbs identified from human patients. There is a pressing need to better understand bNAb developmental pathways and outline the genetic and structural antibody features that can provide enhanced neutralization breadth and potency for HIV-1 vaccines.Clinical data show that broadly neutralizing serum antibodies develop naturally in around 20% of individuals with chronic HIV-1 infection (20, 21) and that a smaller number of individuals show highly potent neutralization (22, 23). bNAbs develop via the accumulation of somatic hypermutations (SHM) and affinity maturation following their initial B-cell selection and expansion. While HIV-1–infected individuals may have high titers against HIV-1 antigens, the rarity of bNAbs suggests that the mutations acquired during bNAb development are correspondingly rare and/or that the mutational pathways to effective bNAb development are explored only in the context of chronic HIV-1 antigen exposure over years of viral infection (24). Evidence that high viral load is correlated with higher HIV-1 serum breadth and potency also suggests that larger numbers of sampled antibody maturation pathways are correlated with broad HIV-1 neutralization (20). A major question for HIV-1 vaccine design is thus how to quickly and effectively induce B-cell maturation to bNAbs from a small number of controlled immunizations.HIV-1 bNAbs are often highly somatically mutated (25–28), and reverting these mutations to antibody germline sequences results in drastic reductions of neutralization breadth and potency (9, 29–31). Complementarity determining regions (CDRs) are known to be mutational hotspots and are often in direct contact with antigen to enable recognition. Antibody framework region (FR) mutations can also modulate neutralization breadth and potency by altering the structural orientation, particularly at heavy:light interface alignments, and by altering the intrinsic flexibility of the paratope (16, 32–35). Not all observed bNAb somatic mutations are required for high neutralization breadth and potency (36), and a better understanding of the critical mutations and antibody structural features (both naturally elicited and vaccine-induced) to improve neutralization breadth and potency would enhance our understanding of bNAb development and guide efficient HIV-1 vaccine strategies (16, 17, 37–41).Among various immunization approaches reported for eliciting broadly neutralizing antibodies (16–19, 38–42), one strategy targets the HIV-1 FP epitope and has elicited 59 and 31% HIV-1 neutralization breadth in rhesus macaques and mice, respectively, against a broad panel of 208 HIV-1 strains (16, 17). Priming the immune response with soluble FP followed by three Env trimer boosts elicited antibodies with FP-targeted trimer recognition and broad HIV-1 neutralization; however, the structural mechanisms that enable effective anti-FP antibody maturation and pathway selection are not fully understood. Several key unanswered questions include the following: 1) What are the structural features of FP recognition vs. trimer recognition that may appropriately guide antibody development?; 2) What critical parameters control virus neutralization of FP-targeting bNAbs?; and 3) How do beneficial mutations fit into the entire landscape of possible mutational pathways, and what fraction of possible mutations provide increased affinity, neutralization breadth, and potency?To address these questions, we characterized the genetic and functional fitness landscape of an anti-FP bNAb and used biophysical and structural techniques to follow the mechanisms of antibody improvement. We implemented these screening strategies using the anti-FP bNAb vFP16.02, a vaccine-elicited antibody that neutralizes ∼30% of HIV-1 viral isolates (16). We applied yeast display and fluorescence-activated cell sorting (FACS) coupled with next-generation sequencing (NGS) to identify single mutations that enhanced binding or fitness to multiple HIV-1 BG505 Env trimer variants (Fig. 1). We mapped possible single mutations by their functional impacts and identified a panel of mutations with enhanced binding affinity and neutralization. Structural analyses of a subset of these mutations provided insights into the mechanisms of enhanced neutralization. These data confirm that several parallel mutational pathways exist for HIV-1 bNAb improvement and underscore the importance of improved Env trimer affinity for enhancing neutralization potency and breadth in HIV-1 vaccine designs.Open in a separate windowFig. 1.Experimental workflow for comprehensive functional analysis of all possible single mutations in an HIV-1 bNAb. SSM libraries were designed for VH and VL regions of the anti–HIV-1 FP bNAb vFP16.02. SSM libraries were cloned into yeast Fab surface display libraries and screened by FACS to determine the functional impact of each possible single mutation. Single-mutation display libraries were sorted for their affinity against a BG505 DS-SOSIP HIV-1 Env trimer with two different FP sequences. Sorted yeast libraries were prepped for NGS to enable quantitative variant tracking across three screening rounds; bioinformatic analyses of NGS data were used to interpret the functional impact of each possible amino acid mutation. Selected variants were characterized for neutralization activity and affinity against soluble FP and against HIV-1 Env. Structural analyses were performed to understand the mechanistic basis of anti–HIV-1 FP bNAb improvement.