Michel C. Nussenzweig

Broadly neutralizing antibodies in adult males living with HIV undergoing analytical treatment interruption: secondary and exploratory outcomes of the phase II randomized controlled RIO trial

Mechanisms of antibody-mediated enhancement of immune responses

Experimental study reveals FcγR-dependent enhancement of germinal center responses, indicating distinct mechanisms regulate B cell recruitment thresholds.

Antibody Mediated Diversification of Primary and Secondary Humoral Immune Responses

Humoral immune responses are characterized by increasing antibody affinity and diversity over time. Increased affinity is mediated by a combination of immunoglobulin gene somatic mutation and iterative cycles of selection in germinal centers. Less is understood about how diversity increases. Here we examine the role of antibody feedback in diversifying immune responses in mice that produce B cells that are incapable of secreting antibodies. To this end, we produced two strains of mice, one that expresses only membrane and secreted forms of IgM, and a second that produces only the membrane bound form of IgM. Analysis of primary and secondary immune responses show that antibody feedback significantly diversifies both primary and secondary immune responses even when antibodies are present at levels that are 10–30 fold lower than physiologic. The data have significant implication for sequential vaccination approaches aimed at shepherding immunity to produce broadly neutralizing antibodies to highly diversified pathogens such as HIV-1 and Influenza. Summary Humoral immune responses diversify over time but whether secreted antibodies influence this process is unknown. Using antibody secretion-deficient mice this study shows a profound impact of secreted antibodies on the evolution of B cell diversity after vaccination.

CD8+ T cell stemness precedes post-intervention control of HIV viraemia

Interventions to induce lasting human immunodeficiency virus (HIV) remission are needed to obviate the requirement for lifelong antiretroviral therapy. Durable post-intervention control (PIC) of viraemia has been achieved in a subset of people following administration of broadly neutralizing anti-HIV-1 antibodies (bNAb) and analytical interruption of treatment 1–4 . Previous studies support a role for CD8 + T cells in PIC 5–9 , but the precise features of CD8 + T cells involved remain unclear. Here we mapped and functionally profiled CD8 + T cell responses to autologous HIV epitopes using longitudinal samples from four analytical treatment interruption trials in bNAb recipients. PIC was associated with superior pre-intervention HIV-specific CD8 + T cell proliferative capacity, stem-cell-like memory phenotype and recall cytotoxicity against autologous HIV peptide-pulsed CD4 + T cells. CD8 + T cell stemness was increased further following bNAb administration without emergence of new clonotypes targeting defined HLA-optimal epitopes. Multi-modal single-cell analyses revealed molecular features associated with PIC and HIV-specific CD8 + T cell stemness, including signatures of metabolic fitness and reduced T cell exhaustion. These results identify immune features that precede subsequent PIC to inform the development of combination immunotherapies that will elicit durable HIV remission.

Correlates of HIV-1 control after combination immunotherapy

The identification of therapeutic strategies to induce sustained antiretroviral therapy (ART)-free control of HIV infection is a major priority 1 . Combination immunotherapy including HIV vaccination, immune stimulation, latency reversal and passive transfer of broadly neutralizing antibodies (bNAbs) has shown promise in non-human primate models 2–6 , but few studies have translated such approaches into people. Here we performed a single-arm, proof-of-concept study in ten people living with HIV on ART, combining the following three approaches: (1) therapeutic vaccination with an HIV Gag conserved element-targeted DNA + IL-12 prime/modified vaccinia Ankara (MVA) boost regimen followed by (2) administration of two bNAbs (10-1074, VRC07-523LS) and a toll-like receptor 9 agonist (lefitolimod) during ART suppression, followed by (3) repeat bNAb administration at the time of ART interruption (Clinicaltrials.gov: NCT04357821 ). Seven out of the ten participants exhibited post-intervention control after pausing ART, independent of residual bNAb plasma levels. Robust expansion of activated CD8 + T cells early in response to rebounding virus correlated with a lower median viral load after peak viraemia off ART. These data suggest that combination immunotherapy approaches might prove effective in inducing sustained control of HIV by slowing rebound and improving CD8 + T cell responses, and that these approaches should continue to be optimized.

Modeling the Synergetic Dynamics of B cells and TFH cells in Germinal Center Reactions

B cells producing high-affinity antibodies arise through affinity maturation within germinal centers (GCs), where selection is driven by T follicular helper (T FH ) cells. Recent studies have shown that, like GC B cells, T FH cells also undergo antigen-dependent selection, with competition among T FH clones dictated by their ability to recognize and stimulate B cells. This sensitivity-dependent selection process leads to dynamic remodeling of the T FH repertoire over time. Despite the essential role of T FH cells in B cell selection, the functional consequences of the time evolution of the T FH cell population remains poorly understood. To address this gap, we developed a population dynamics model that explicitly incorporates key T FH cell properties and dynamics. Our analysis predicts that dynamic feedback between B and T FH cell populations provides robust homeostatic regulation of their numbers in the GC, yielding a stable lymphocyte ratio that we verify experimentally. Moreover, our model predicts that T FH clone sensitivity dictates distinct evolutionary strategies during affinity maturation, with low-sensitivity T FH cells accelerating affinity gain at the expense of B cell diversity, while high-sensitivity T FH cells slow affinity maturation but preserve a broader B cell repertoire. These findings highlight the importance of co-regulation between T FH and B cells and suggest that reciprocal stimulation allows the immune system to tune the tradeoff between the speed of affinity gain and the breadth of B cell diversity—a principle that may extend to other adaptive systems. Significance Statement Effector B cells that secrete high-affinity antibodies and form immunological memory are essential for humoral immunity and arise from germinal center (GC) reactions. Within GCs, B cells undergo an accelerated version of Darwinian evolution to enhance antibody affinity. This process is orchestrated by T follicular helper (T FH ) cells which provide stimulatory signals to selected B cells and undergo their own antigen-driven selection. To investigate this co-evolutionary process, we developed a tractable population-level model of the GC reaction. Our analysis reveals that the reciprocal stimulation of B and T FH cells provides a robust mechanism for regulating the B:T FH ratio and tuning the tradeoff between the speed of affinity maturation and the diversity of the antibody response.

Enhanced HIV-1 Control After Antibody Therapy is Associated with Autologous Antibodies and Reservoir Clearance in the RIO trial

RIO is an ongoing double blind randomized placebo controlled human trial in which participants that started antiretroviral therapy (ART) during primary or early-stage infection underwent treatment interruption and were randomly assigned to a group receiving one or two doses of two long acting broadly neutralizing antibodies 3BNC117-LS and 10-1074-LS (Arm A, n = 34), or saline (Arm B, n = 34). The primary clinical study met its primary endpoint and was published elsewhere, demonstrating significantly delayed viral rebound and prolonged ART-free control in participants receiving bNAbs compared to placebo. The infusions were generally safe and well tolerated. Here we report on pre-specified secondary analyses examining viral rebound, antibody sensitivity, and reservoir dynamics, as well as exploratory analyses of rebound virus evolution and autologous antibody activity. Pre-infusion reservoir measurements showed low levels of intact proviral HIV-1 DNA in circulating CD4+ T cells in both arms. The rebounding viruses in participants that received the antibodies, showed significant selection for resistance to 10-1074-LS but not to 3BNC117-LS. Notably, there was a significant correlation between greater initial reservoir sensitivity to autologous antibodies and to 10-1074 and time to rebound. Finally, comparison of pre-infusion and pre-rebound HIV-1 proviral reservoir showed a decrease in intact but not defective proviruses in Arm A but not in Arm B. Together, these findings suggest that baseline humoral immunity and prolonged exposure to LS-bNAbs may shape post-treatment viral rebound dynamics in individuals that initiate ART during primary infection. ClinicalTrials.gov identifier: NCT04319367

B lymphocytes that enter the germinal center late preferentially differentiate into memory cells that recognize subdominant epitopes

Immune responses to pathogens and effective vaccines elicit germinal center (GC) responses wherein B cells undergo affinity maturation and develop into plasma cells (PCs) and memory B cells (MBCs). The GC reaction is initially seeded by a limited group of founder B cells, and subsequently further diversified by continual entry of naïve B cells that compete with GC founder cells for antigen and T cell help. Whether these later-arriving invaders contribute to the development of PCs or MBCs is not known. To investigate the fate of GC invaders we developed a dual-recombinase reporter approach that enables pre- and post-GC B cell lineage tracing and used it to examine immune responses to vaccination and influenza infection. Notably, fate-mapped invaders preferentially give rise to MBCs as opposed to PCs. Moreover, antibodies expressed by invader-derived MBCs harbor fewer somatic mutations, exhibit lower affinity, and their antibodies bind to subdominant antigenic epitopes relative to founder MBCs. Our findings indicate that invader GC B cells are an important source of humoral immune memory diversification after infection or vaccination.

Consistent Induction of Broadly Neutralizing HIV Antibodies by a Novel Two-Step Mechanism Informs Immunogen Design

This research demonstrates a two-step mechanism for eliciting broadly neutralizing antibodies in macaques, suggesting new immunogen designs.

Memory B cell Development in Response to mRNA SARS-CoV-2 and Nanoparticle Immunization in Mice

Nanoparticle immunogens excel at rapidly inducing high levels of circulating antibodies and are being deployed as part of several novel vaccines. However, their ability to elicit memory B cell responses is less well understood. Here we compared serologic and memory B cell responses to prime boost vaccination with either SARS-CoV-2 Wuhan-Hu-1 mRNA vaccine, or protein nanoparticles: SARS-CoV-2 B.1.351 homotypic containing a single receptor binding domain (RBD); (homotypic beta) or a combination of different Sarbecovirus RBDs (mosaic 8b), respectively. The memory B cells elicited by the 3 vaccine regimens showed closely related antibody sequences, similar levels of somatic mutation and clonal diversity. The breadth of serologic responses elicited by the mosaic nanoparticles were comparable to the homotypic nanoparticle and superior to the mRNA vaccine for some mismatched strains. However, serum neutralizing titers to SARS-CoV-2 were highest after mRNA vaccination. The three vaccines elicited memory B cells that produced antibodies specific to a broad range of epitopes on the RBD that differed in a way that may reflect epitope masking. Monoclonal antibodies derived from memory B cells elicited by the mosaic 8b nanoparticle showed greater breadth against a panel of SARS-CoV-2 variants and SARS-CoV. Significance Statement: Nanoparticle vaccines are promising next-generation vaccine candidates, yet their capacity to generate durable memory B cell responses remains incompletely understood. We compared immune responses following SARS-CoV-2 mRNA, homotypic beta nanoparticle, or mosaic 8b nanoparticle vaccination in mice. Serum antibody neutralizing responses against a panel of SARS-CoV-2 variants elicited by an mRNA vaccine were equivalent or superior to those elicited by mosaic 8b nanoparticle vaccines. However, the monoclonal antibodies derived from memory B cells elicited by the mosaic 8b nanoparticle showed better neutralizing breadth against heterologous pseudoviruses. These findings highlight individual strengths of mRNA and nanoparticle vaccines and show that mosaic 8b nanoparticle immunogens can enhance the breadth of memory B cell-derived antibodies.

Genomic specificity of anti-mouse TCR mAbs determined by single-cell RNAseq

Single-cell RNAseq revealed specific TCR gene patterns in T cells, indicating diversity among anti-V mAbs.

Clonal expansion and diversification of germinal center and memory B cell responses to booster immunization in primates

Analysis shows memory B cells undergo clonal expansion in germinal centers after booster immunization, suggesting a robust immune response in primates.

Design and characterization of HIV-1 vaccine candidates to elicit antibodies targeting multiple epitopes

Germline-targeting immunogens elicit broadly neutralizing antibodies in rhesus macaques and mice, suggesting new HIV-1 vaccine strategies.

Clonal Expansion and Diversification of Germinal Center and Memory B Cell Responses to Booster Immunization in Primates

Summary Effective vaccines elicit B cell clonal expansion in germinal centers (GCs) that produce memory B cells and antibody secreting plasma cells. Studies in mice indicate that, whereas the plasma cell compartment is enriched for cells producing high affinity antibodies, the memory pool is more diverse and contains only a relatively small proportion of higher affinity cells. Upon boosting, murine memory B cells producing high affinity antibodies tend to develop into plasma cells but few if any re-enter GCs. However, mice live for only a few weeks in nature, and in keeping with the rather limited requirement for immune memory, this compartment comprises only 1–2% of all B cells. In contrast, memory accounts for nearly 50% of all B cells in primates. Here we examine memory and GC B cell responses in rhesus macaques immunized and boosted ipsilaterally or contralaterally with an mRNA vaccine encoding severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike protein. The neutralizing activity of antibodies cloned from the memory compartment, and the size of the compartment, was independent of the site of boosting. Moreover, in primates, memory B cells enter and undergo iterative expansion in newly developing GCs when boosting is at a site distal to the site of priming. Thus, in primates, high affinity memory B cells constitute a reservoir that actively participates in further development of immunity irrespective of the anatomical site of vaccine boosting. Highlights - Clonal overlap between primate memory and germinal center B cell compartments following booster immunization. - Neutralization activity of the memory and germinal center compartments are independent of the boost site. - Relationship between site of booster immunization (ipsilateral versus contralateral) and development of memory and germinal center (GC) responses in primates

Regulated somatic hypermutation enhances antibody affinity maturation

Germinal centres are specialized microenvironments where B cells undergo affinity maturation. B cells expressing antibodies whose affinity is improved by somatic hypermutation are selected for expansion by limiting numbers of T follicular helper cells. Cell division is accompanied by mutation of the immunoglobulin genes, at what is believed to be a fixed rate of around 1 × 10 −3 per base pair per cell division 1 . As mutagenesis is random, the probability of acquiring deleterious mutations outweighs the probability of acquiring affinity-enhancing mutations. This effect might be heightened, and even become counterproductive, in B cells that express high-affinity antibodies and undergo the greatest number of cell divisions 2 . Here we experimentally examine a theoretical model that explains how affinity maturation could be optimized by varying the rate of somatic hypermutation such that cells that express higher-affinity antibodies divide more but mutate less per division. Data obtained from mice immunized with SARS-CoV-2 vaccines or a model antigen align with the theoretical model and show that cells producing high-affinity antibodies shorten the G0/G1 phases of the cell cycle and reduce their mutation rates. We propose that these mechanisms safeguard high-affinity B cell lineages and enhance the outcomes of antibody affinity maturation.

Bispecific antibodies targeting the N-terminal and receptor binding domains potently neutralize SARS-CoV-2 variants of concern

The ongoing emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) that reduce the effectiveness of antibody therapeutics necessitates development of next-generation antibody modalities that are resilient to viral evolution. Here, we characterized amino-terminal domain (NTD)- and receptor binding domain (RBD)-specific monoclonal antibodies previously isolated from coronavirus disease 2019 (COVID-19) convalescent donors for their activity against emergent SARS-CoV-2 VOCs. Among these, the NTD-specific antibody C1596 displayed the greatest breadth of binding to VOCs, with cryo-electron microscopy structural analysis revealing recognition of a distinct NTD epitope outside of the site i antigenic supersite. Given C1596's favorable binding profile, we designed a series of bispecific antibodies (bsAbs), termed CoV2-biRNs, that featured both NTD and RBD specificities. Two of the C1596-inclusive bsAbs, CoV2-biRN5 and CoV2-biRN7, retained potent in vitro neutralization activity against all Omicron variants tested, including XBB.1.5, BA.2.86, and JN.1, contrasting the diminished potency of parental antibodies delivered as monotherapies or as a cocktail. Furthermore, prophylactic delivery of CoV2-biRN5 reduced the viral load within the lungs of K18-hACE2 mice after challenge with SARS-CoV-2 XBB.1.5. In conclusion, NTD-RBD bsAbs offer promising potential for the design of resilient, next-generation antibody therapeutics against SARS-CoV-2 VOCs.

Design and characterization of HIV-1 vaccine candidates to elicit antibodies targeting multiple epitopes

A primary goal in the development of an AIDS vaccine is the elicitation of broadly neutralizing antibodies (bNAbs) that protect against diverse HIV-1 strains. To this aim, germline-targeting immunogens have been developed to activate bNAb precursors and initiate the induction of bNAbs. While most pre-clinical germline-targeting HIV-1 vaccine candidates only include a single bNAb precursor epitope, an effective HIV-1 vaccine will likely require bNAbs that target multiple epitopes on Env. Here, we report a newly designed germline-targeting Env SOSIP trimer, named 3nv.2, that presents three bNAb epitopes on Env: the CD4bs, V3, and V2 epitopes. 3nv.2 forms a stable trimeric Env and binds to bNAb precursors from each of the desired epitopes. Immunization experiments in rhesus macaques and mice demonstrate 3nv.2 elicits the combined effects of its parent immunogens. Our results provide proof-of-concept for using a germline-targeting immunogen presenting three or more bNAb epitopes and a framework to develop improved next-generation HIV-1 vaccine candidates.

Affinity maturation of antibody responses is mediated by differential plasma cell proliferation

Increased antibody affinity over time after vaccination, known as affinity maturation, is a prototypical feature of immune responses. Recent studies have shown that a diverse collection of B cells, producing antibodies with a wide spectrum of different affinities, is selected into the plasma cell (PC) pathway. How affinity-permissive selection enables PC affinity maturation remains unknown. We found that PC precursors (prePCs) expressing high-affinity antibodies received higher levels of T follicular helper cell (TFH cell)-derived help and divided at higher rates compared with their lower-affinity counterparts once they left the germinal center. Our findings indicate that differential cell division by selected prePCs accounts for how diverse precursors develop into a PC compartment that mediates serological affinity maturation.

Affinity maturation of antibody responses is mediated by differential plasma cell proliferation

Increased antibody affinity over time after vaccination, known as affinity maturation, is a prototypical feature of immune responses. Recent studies have shown that a diverse collection of B cells, producing antibodies with a wide spectrum of different affinities, are selected into the plasma cell (PC) pathway. How affinity-permissive selection enables PC affinity maturation remains unknown. Here we report that PC precursors (prePC) expressing high affinity antibodies receive higher levels of T follicular helper (Tfh)-derived help and divide at higher rates than their lower affinity counterparts once they leave the GC. Thus, differential cell division by selected prePCs accounts for how diverse precursors develop into a PC compartment that mediates serological affinity maturation.

Transcription of HIV-1 at sites of intact latent provirus integration

HIV-1 antiretroviral therapy is highly effective but fails to eliminate a reservoir of latent proviruses, leading to a requirement for life-long treatment. How the site of integration of authentic intact latent proviruses might impact their own or neighboring gene expression or reservoir dynamics is poorly understood. Here, we report on proviral and neighboring gene transcription at sites of intact latent HIV-1 integration in cultured T cells obtained directly from people living with HIV, as well as engineered primary T cells and cell lines. Proviral gene expression was correlated to the level of endogenous gene expression under resting but not activated conditions. Notably, latent proviral promoters were 100–10,000× less active than in productively infected cells and had little or no measurable impact on neighboring gene expression under resting or activated conditions. Thus, the site of integration has a dominant effect on the transcriptional activity of intact HIV-1 proviruses in the latent reservoir, thereby influencing cytopathic effects and proviral immune evasion.