Biology subjects

Nussenzweig, M. C.

Publications and source records attributed to Nussenzweig, M. C..

At least 19 recordsLinked to original sources

bioRxiv2026

B cells sustain tumor-specific CD4 T cells to promote response to PD-1 targeted therapy

Immunotherapy transformed cancer treatment, yet precise correlates of response remain to be defined. While intratumoral follicular helper like (Tfhl) CD4 T cells and IgG1+ B cells have been associated with improved outcomes to PD-1 blockade for hepatocellular carcinoma (HCC), their mechanisms contributing to response are unclear. To address this question, we developed a murine model of HCC and examined CD8, CD4 and B cell responses. Increasing CD4-help sensitizes mice to PD-1 blockade, in a CD4- and CD8-dependent manner. Tumor-specific CD4 T cells contained Tfhl and Th1 populations, and both Bcl6 and T-bet were required for efficacy. Antigen-specific B cells were essential for CD4-helper expansion, yet secretion of antibodies was not required for long-term survival. Thus, B cells are critical for effective immunotherapy, but secreted antibodies are not.

immunology↗

bioRxiv2026

Structural and mutational analyses define distinct molecular routes to broad SARS-CoV-2 receptor-binding domain recognition

Broadly reactive antibodies elicited by SARS-CoV-2 infection or vaccination can reveal conserved viral vulnerabilities and inform vaccines with broad coronavirus coverage. Here, we characterize two human-derived monoclonal antibodies, B2014 and C5078, that recognize conserved epitopes on the SARS-CoV-2 RBD and retain activity across antigenically distinct variants. Notably, C5078 also recognizes diverse sarbecoviruses and remains active against currently circulating variants, including XFG and NB.1.8.1. Cryo-EM structures reveal that B2014 recognizes an epitope adjacent to the class 3 antibody site, whereas C5078 targets the highly conserved, cryptic site V epitope. Structural analysis defines how C5078 uses affinity-matured interactions to engage conserved RBD residues, providing a molecular basis for its exceptional breadth. Deep mutational scanning across multiple SARS-CoV-2 variant backgrounds further defines potential pathways of antibody escape, explaining the loss of B2014 activity against antigenically evolved variants while revealing a high barrier to escape from C5078. Together, these findings define distinct structural solutions for broad RBD recognition and highlight conserved, mutationally constrained epitopes that may serve as targets for vaccines designed to elicit antibody responses resilient to ongoing SARS-CoV-2 evolution and future sarbecovirus emergence.

biophysics↗

bioRxiv2026

Biophysical properties of IgD determine thresholds for self-tolerance and selection into germinal centers

Immunoglobulin D (IgD) is among the most conserved antibody isotypes, found in virtually all jawed vertebrates1. Unlike other isotypes, IgD contains an unusually long hinge region of up to 160 amino acids that connects the constant and variable regions. Its expression pattern is also conserved; IgD is co-expressed with IgM on transitional and mature naive B cells. However, the function of IgD has remained enigmatic since its discovery in 19652,3. Here we present and test a biophysical model positing that IgD increases the entropic cost of bivalent antigen binding. Single-molecule measurements revealed that the antigen-binding arms of IgD are substantially more dynamic than those of IgM, suggesting that cell surface IgD would be energetically penalized in bivalent antigen binding. Consistent with the model and biophysical data, we find that the long hinge compromises antigen capture by IgD B cell receptors (BCRs) compared to IgM BCRs. To determine how the difference in antigen binding impacts immunity, we produced mice that express only IgM and IgD, exclusively IgM or IgD, or IgD with a truncated hinge region. The data indicate that the increased entropic cost of antigen binding imposed by the IgD hinge attenuates negative selection by self-antigen while increasing the affinity-based threshold for positive selection into the germinal center (GC). Together the results indicate that IgD functions physiologically to desensitize B cells to antigen, thereby expanding the B cell repertoire while optimizing affinity-based selection into the GC.

immunology↗

bioRxiv2026

A designed overlapping variant immunogen pool elicits broad sarbecovirus neutralization

A central problem in achieving vaccine-based protection against viral infections is eliciting antibodies that are resilient to viral variation. Successive waves of SARS-CoV-2 infection during the COVID19 pandemic were driven by variants that acquired resistance to neutralizing antibodies elicited by prior SARS-CoV-2 variants. To the extent that serum neutralization breadth occurs in individuals with multiple exposures to SARS-CoV-2 antigens, we and others find that it is largely comprised of antibodies that target the variable receptor binding domain (RBD), rather than more conserved spike protein domains. By designing synthetic dimeric RBD immunogens we show that limiting divergence in heterodimeric components favors the generation of cross-reactive B cells and antibodies. We thus devised a vaccine approach based on a two-dose immunization with a pool of five overlapping heterodimeric synthetic RBD variants. Collectively, the RBD heterodimer pool was designed to cover 10% sequence variation and elicited greater antibody cross-reactivity and neutralization breadth than homodimers or heterodimers with highly divergent components. Using an unconventional prospective challenge model in mice, we demonstrate the effectiveness of the RBD heterodimer pool in inducing antibody responses that attenuate infection by future SARS-CoV-2 variants, as well as protection in a challenge model based on a chimeric vesicular stomatitis virus bearing a spike protein from SARS-CoV-1. Significance statementViral antigenic escape undermines both vaccination efforts and the development of herd immunity, resulting in an enormous viral disease burden. A central problem in eliciting vaccine-based protection against some viral infections is achieving antibody neutralization breadth. To elicit collections of antibodies that overcome the problem of limited antibody tolerance of viral variation, we designed a novel strategy based on an overlapping series of immunogens. This immunogen pool conferred at least partial protection against subsequently prevalent SARS-CoV-2 variants as well as a chimeric SARS-CoV-1 based model virus.

microbiology↗

bioRxiv2026

Dynamic Antigen Expression and Intrinsic CTL Resistance in HIV Reservoir Clones

Reservoirs of clonally expanded CD4+ T-cells harboring rebound-competent HIV proviruses persist lifelong during ART. Latency is considered the principal barrier to viral eradication and has resisted pharmacological reversal, yet it appears that sustained immune pressure may still erode reservoirs. Recent advances have yielded glimpses into these exceptionally rare reservoir-harboring cells, implicating intrinsic pro-survival properties in their persistence. Here, we isolate and characterize populations of authentic reservoir clones (ARCs) that robustly proliferate and accumulate while producing infectious virus, without overtly succumbing to viral cytopathic effects. At any given moment, only small fractions of ARCs expressed HIV proteins, a state remarkably unperturbed by potent TCR or mitogenic stimulation. Nevertheless, sustained co-culture with cytotoxic T-lymphocytes (CTL) revealed extensive time-integrated antigenic vulnerability, culling clonal expansion of some ARCs by >90%. Notably, a regulatory T-cell ARC displayed pronounced cell-intrinsic resistance to CTL - a longstanding hypothesis we now directly demonstrate - linked to low oxidative stress and reversed with desferoxamine, a hypoxic stress inducer and FDA-approved therapeutic. Overall, we provide novel insights into the vulnerabilities of reservoir clones to potent, sustained CTL pressure and highlight intrinsic resistance pathways as actionable therapeutic targets, opening opportunities for advancing immune-based HIV cure strategies.

immunology↗

bioRxiv2026

B Lymphocyte Protein Factories produced by Hematopoietic Stem Cell Gene Editing

Long-term in vivo production of therapeutic proteins and development of vaccines that elicit protective levels of broadly neutralizing antibodies (bNAbs) against major pathogens face challenges. Here we report on an alternative gene-editing approach using small numbers of hematopoietic stem and progenitor cells (HSPCs) to direct long-term, high-level expression of antibodies or cargo proteins. Edited B lymphocyte offspring can be activated by cognate antigen to undergo clonal expansion and develop into specific antibody or cargo protein-synthesizing plasma cells. These cells produce long-lasting, therapeutic levels of serum antibody against HIV-1 or malaria and an anti-influenza virus bNAb that mediated universal protection from heterologous lethal challenge. Our data provide a paradigm for cell therapy approaches to prevent or treat disease using self-amplifying B cell protein factories.

immunology↗

bioRxiv2025

Antibody Mediated Diversification of Primary and Secondary 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. SummaryHumoral 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.

immunology↗

bioRxiv2025

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 (TFH) cells. Recent studies have shown that, like GC B cells, TFH cells also undergo antigen-dependent selection, with competition among TFH clones dictated by their ability to recognize and stimulate B cells. This sensitivity-dependent selection process leads to dynamic remodeling of the TFH repertoire over time. Despite the essential role of TFH cells in B cell selection, the functional consequences of the time evolution of the TFH cell population remains poorly understood. To address this gap, we developed a population dynamics model that explicitly incorporates key TFH cell properties and dynamics. Our analysis predicts that dynamic feedback between B and TFH 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 TFH clone sensitivity dictates distinct evolutionary strategies during affinity maturation, with low-sensitivity TFH cells accelerating affinity gain at the expense of B cell diversity, while high-sensitivity TFH cells slow affinity maturation but preserve a broader B cell repertoire. These findings highlight the importance of co-regulation between TFH 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 StatementEffector 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 (TFH) 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 TFH cells provides a robust mechanism for regulating the B:TFH ratio and tuning the tradeoff between the speed of affinity maturation and the diversity of the antibody response.

immunology↗

bioRxiv2025

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 naive 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.

immunology↗

bioRxiv2025

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 StatementNanoparticle 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.

immunology↗

bioRxiv2025

HIV-1 expression is heterogeneous among clones of CD4+ T cells carrying authentic intact latent proviruses

Antiretroviral therapy suppresses HIV-1 infection but is not curative because it fails to eliminate a reservoir of intact latent proviruses that reside primarily in CD4+ T cells. This compartment is composed of rare T cells that predominantly express memory and effector memory markers. The lack of precise understanding of the latent compartment has made it challenging to develop curative strategies for HIV-1 infection. Here we report on the properties of CD4+ T cells clones carrying intact latent proviruses, expanded in vitro from single cells obtained from the reservoir of people living with HIV-1. The latent proviruses in the clones were integrated into ZNF genes, non-genic satellite and centromeric regions, frequently associated with latency. Notably, the transcriptome of the cultured clones resembled their cells of origin. Despite their descent from single cells, only a fraction of the cells ranging from 0.4-14% expressed relatively low levels of HIV-1 that did not measurably alter host gene transcriptome. Latency reversing agents (LRAs) variably increased the number and amount of expression per cell, but the effects were modest and clone and LRA specific. The results suggest that pharmacologic and immunologic approaches to clear the reservoir should be optimized to accommodate intra- and inter-clonal diversity.

immunology↗

bioRxiv2025

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

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

immunology↗

bioRxiv2025

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.

bioengineering↗

bioRxiv2024

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.

immunology↗

bioRxiv2024

Bispecific antibodies with broad neutralization potency against SARS-CoV-2 variants of concern

The ongoing emergence of 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 N-terminal domain (NTD) and receptor binding domain (RBD)-specific monoclonal antibodies previously isolated from 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-EM structural analysis revealing recognition of a distinct NTD epitope outside of the site i antigenic supersite. Given C1596s favorable binding profile, we designed a series of bispecific antibodies (bsAbs) termed CoV2-biRNs, that featured both NTD and RBD specificities. Notably, 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, EG.5.1, and BA.2.86, contrasting the diminished potency of parental antibodies delivered as monotherapies or as a cocktail. Furthermore, prophylactic delivery of CoV2-biRN5 significantly reduced the viral load within the lungs of K18-hACE2 mice following challenge with SARS-CoV-2 XBB.1.5. In conclusion, our NTD-RBD bsAbs offer promising potential for the design of resilient, next-generation antibody therapeutics against SARS-CoV-2 VOCs. One Sentence SummaryBispecific antibodies with a highly cross-reactive NTD antibody demonstrate resilience to SARS-CoV-2 variants of concern.

immunology↗

bioRxiv2024

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

HIV-1 anti-retroviral 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 100-10,000X 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.

immunology↗

bioRxiv2023

SARS-CoV-2 spike glycosylation affects function and neutralization sensitivity

The glycosylation of viral envelope proteins can play important roles in virus biology and immune evasion. The spike (S) glycoprotein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) includes 22 N-linked glycosylation sequons and 17 O-linked glycosites. Here, we investigated the effect of individual glycosylation sites on SARS-CoV-2 S function in pseudotyped virus infection assays and on sensitivity to monoclonal and polyclonal neutralizing antibodies. In most cases, removal of individual glycosylation sites decreased the infectiousness of the pseudotyped virus. For glycosylation mutants in the N-terminal domain (NTD) and the receptor binding domain (RBD), reduction in pseudotype infectivity was predicted by a commensurate reduction in the level of virion-incorporated spike protein. Notably, the presence of a glycan at position N343 within the RBD had diverse effects on neutralization by RBD-specific monoclonal antibodies (mAbs) cloned from convalescent individuals. The N343 glycan reduced overall sensitivity to polyclonal antibodies in plasma from COVID-19 convalescent individuals, suggesting a role for SARS-CoV-2 spike glycosylation in immune evasion. However, vaccination of convalescent individuals produced neutralizing activity that was resilient to the inhibitory effect of the N343 glycan.

microbiology↗

bioRxiv2022

CD4-binding site immunogens elicit heterologous anti-HIV-1 neutralizing antibodies in transgenic and wildtype animals

Passive transfer of broadly neutralizing anti-HIV-1 antibodies (bNAbs) protects against infection, and therefore eliciting bNAbs by vaccination is a major goal of HIV-1 vaccine efforts. bNAbs that target the CD4-binding site (CD4bs) on HIV-1 Env are among the most broadly active, but to date, responses elicited against this epitope in vaccinated animals have lacked potency and breadth. We hypothesized that CD4bs bNAbs resembling the antibody IOMA might be easier to elicit than other CD4bs antibodies that exhibit higher somatic mutation rates, a difficult-to-achieve mechanism to accommodate Envs N276gp120 N-glycan, and rare 5-residue light chain complementarity determining region 3s (CDRL3s). As an initial test of this idea, we developed IOMA germline-targeting Env immunogens and evaluated a sequential immunization regimen in transgenic mice expressing germline-reverted IOMA. These mice developed CD4bs epitope-specific responses with heterologous neutralization, and cloned antibodies overcame neutralization roadblocks including accommodating the N276gp120 glycan, with some neutralizing selected HIV-1 strains more potently than IOMA. The immunization regimen also elicited CD4bs-specific responses in animals containing polyclonal antibody repertoires. Thus, germline-targeting of IOMA-class antibody precursors represents a potential vaccine strategy to induce CD4bs bNAbs.

immunology↗