Somatic Mutations of the Immunoglobulin Framework Are Generally Required for Broad and Potent HIV-1 Neutralization

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Significantly higher levels of plasma CXCL13 [chemokine (C-X-C motif) ligand 13] were associated with the generation of broadly neutralizing antibodies (bnAbs) against HIV in a large longitudinal cohort of HIV-infected individuals. Germinal centers (GCs) perform the remarkable task of optimizing B-cell Ab responses. GCs are required for almost all B-cell receptor affinity maturation and will be a critical parameter to monitor if HIV bnAbs are to be induced by vaccination. However, lymphoid tissue is rarely available from immunized humans, making the monitoring of GC activity by direct assessment of GC B cells and germinal center CD4 + T follicular helper (GC Tfh) cells problematic. The CXCL13-CXCR5 [chemokine (C-X-C motif) receptor 5] chemokine axis plays a central role in organizing both B-cell follicles and GCs. Because GC Tfh cells can produce CXCL13, we explored the potential use of CXCL13 as a blood biomarker to indicate GC activity. In a series of studies, we found that plasma CXCL13 levels correlated with GC activity in draining lymph nodes of immunized mice, immunized macaques, and HIV-infected humans. Furthermore, plasma CXCL13 levels in immunized humans correlated with the magnitude of Ab responses and the frequency of ICOS + (inducible T-cell costimulator) Tfh-like cells in blood. Together, these findings support the potential use of CXCL13 as a plasma biomarker of GC activity in human vaccine trials and other clinical settings.T he germinal center (GC) reaction is a critical immunological process that occurs in draining lymph nodes after immunization (1). The GC response consists of antigen-specific B cells undergoing affinity maturation through a process of somatic hypermutation (SHM) of the B-cell receptor. SHM is necessary for producing high-affinity Ab responses after immunizations and infections. Influenza neutralizing Abs have substantial SHM. Particularly high levels of SHM, 15-30% amino acid mutation (2, 3), are present and necessary for broad Ab neutralization of diverse HIV strains (4, 5). Therefore, as candidate influenza and HIV vaccines are evaluated for the ability to induce broadly neutralizing antibodies (bnAbs), the quantitation and functional characterization of GC responses will be a key parameter for study. Serological analysis of vaccine-specific Ab titers provides important information, but those data are limited. Serological outcomes are measured at time points long after initial immunizations. Neutralizing Ab responses are commonly only measurable after multiple boosts. Those outcomes likely depend on GC activity and affinity maturation at much earlier time points. Several state of the art HIV vaccine strategies rely on long, multistage immunization protocols (6, 7). With bnAb responses as the goal, means of early analysis of the immune response will be essential to understand and improve on vaccination schemes that may end in failure or only partial success. One critical parameter to assess will be the ability of each immunization to generate GC responses.Central to the GC re...

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confidence: 99%

CXCL13 is a plasma biomarker of germinal center activity

Havenar-Daughton

Lindqvist

Heit

et al. 2016

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“…For example, framework mutations can compensate for the destabilizing effect of mutations at CDRs needed for antigen binding (15). In other cases, non-CDR SHMs have been shown to be required for the neutralization activity of broadly neutralizing anti-HIV antibodies (16).…”

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confidence: 99%

“…Thus far, the roles of SHM have been studied primarily by examining the functional consequences of reverting the somatic mutation of selected antibodies back to the germline sequences (15)(16)(17)(18)(19)(20)(21). Although these studies have demonstrated that antibodies depend on somatic mutations to achieve high-affinity antigen binding, the approach is limited to the small set of mutations present in a given antibody.…”

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confidence: 99%

Mutational landscape of antibody variable domains reveals a switch modulating the interdomain conformational dynamics and antigen binding

Koenig¹,

Lee²,

Walters³

et al. 2017

Somatic mutations within the antibody variable domains are critical to the immense capacity of the immune repertoire. Here, via a deep mutational scan, we dissect how mutations at all positions of the variable domains of a high-affinity anti-VEGF antibody G6.31 impact its antigen-binding function. The resulting mutational landscape demonstrates that large portions of antibody variable domain positions are open to mutation, and that beneficial mutations can be found throughout the variable domains. We determine the role of one antigen-distal light chain position 83, demonstrating that mutation at this site optimizes both antigen affinity and thermostability by modulating the interdomain conformational dynamics of the antigen-binding fragment. Furthermore, by analyzing a large number of human antibody sequences and structures, we demonstrate that somatic mutations occur frequently at position 83, with corresponding domain conformations observed for G6.31. Therefore, the modulation of interdomain dynamics represents an important mechanism during antibody maturation in vivo. Deciphering the molecular mechanism of SHMs in improving antibodies is critical for understanding the evolution of the immune repertoire. It is well recognized that mutations at the CDRs or FWR structurally adjacent to the CDRs can modulate and optimize the antigen-binding interface (4-7), and several studies have used saturated mutagenesis of CDR position to explore the effect of various mutations on affinity and specificity (8-11).The role of SHM in antigen-distal FWR is less well understood, however. Previous studies have suggested that the antigen-distal FWR contributes primarily to the variable domain structural integrity; thus, mutations at these positions would be either detrimental to or neutral for antigen-binding function (12)(13)(14). Other authors have characterized the potential of antigen-distal framework mutation as beneficial for antigen-binding function. For example, framework mutations can compensate for the destabilizing effect of mutations at CDRs needed for antigen binding (15). In other cases, non-CDR SHMs have been shown to be required for the neutralization activity of broadly neutralizing anti-HIV antibodies (16).Thus far, the roles of SHM have been studied primarily by examining the functional consequences of reverting the somatic mutation of selected antibodies back to the germline sequences (15-21). Although these studies have demonstrated that antibodies depend on somatic mutations to achieve high-affinity antigen binding, the approach is limited to the small set of mutations present in a given antibody.Here we took a systematic approach to assessing the mutability of the whole variable domain for maintaining or improving folding stability and antigen binding. We used a welloptimized anti-vascular endothelial cell growth factor (VEGF), G6.31, with high affinity (K d = 0.4 nM) and excellent thermostability [fragment antigen-binding (Fab) melting temperature (T m ) = 84°C]. G6.31 derives its HC and LC variable domains ...

“…Tfhs are essential to help germinal center B cells undergo somatic hypermutation and affinity selection (34,35). It is noteworthy that the frequencies of total circulating "Tfh-like" cells correlate with the production of broadly neutralizing antibodies (34), which are typically highly mutated (36).…”

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confidence: 99%

Antibody persistence and T-cell balance: Two key factors confronting HIV vaccine development

Lewis

DeVico

Gallo

2014

The quest for a prophylactic AIDS vaccine is ongoing, but it is now clear that the successful vaccine must elicit protective antibody responses. Accordingly, intense efforts are underway to identify immunogens that elicit these responses. Regardless of the mechanism of antibodymediated protection, be it neutralization, Fc-mediated effector function, or both, antibody persistence and appropriate T-cell help are significant problems confronting the development of a successful AIDS vaccine. Here, we discuss the evidence illustrating the poor persistence of antibody responses to Env, the envelope glycoprotein of HIV-1, and the related problem of CD4 + T-cell responses that compromise vaccine efficacy by creating excess cellular targets of HIV-1 infection. Finally, we propose solutions to both problems that are applicable to all Envbased AIDS vaccines regardless of the mechanism of antibody-mediated protection.