B Cell Function in the Tumor Microenvironment.

The tumor microenvironment (TME) is a heterogeneous, complex organization composed of tumor, stroma, and endothelial cells that is characterized by cross talk between tumor and innate and adaptive immune cells. Over the last decade, it has become increasingly clear that the immune cells in the TME play a critical role in controlling or promoting tumor growth. The function of T lymphocytes in this process has been well characterized. On the other hand, the function of B lymphocytes is less clear, although recent data from our group and others have strongly indicated a critical role for B cells in antitumor immunity. There are, however, a multitude of populations of B cells found within the TME, ranging from naive B cells all the way to terminally differentiated plasma cells and memory B cells. Here, we characterize the role of B cells in the TME in both animal models and patients, with an emphasis on dissecting how B cell heterogeneity contributes to the immune response to cancer.

Germinal Centers.

Germinal centers (GCs) are microanatomical sites of B cell clonal expansion and antibody affinity maturation. Therein, B cells undergo the Darwinian process of somatic diversification and affinity-driven selection of immunoglobulins that produces the high-affinity antibodies essential for effective humoral immunity. Here, we review recent developments in the field of GC biology, primarily as it pertains to GCs induced by infection or immunization. First, we summarize the phenotype and function of the different cell types that compose the GC, focusing on GC B cells. Then, we review the cellular and molecular bases of affinity-dependent selection within the GC and the export of memory and plasma cells. Finally, we present an overview of the emerging field of GC clonal dynamics, focusing on how GC and post-GC selection shapes the diversity of antibodies secreted into serum.

Evolutionary Landscapes of Host-Virus Arms Races.

Vertebrate immune systems suppress viral infection using both innate restriction factors and adaptive immunity. Viruses mutate to escape these defenses, driving hosts to counterevolve to regain fitness. This cycle recurs repeatedly, resulting in an evolutionary arms race whose outcome depends on the pace and likelihood of adaptation by host and viral genes. Although viruses evolve faster than their vertebrate hosts, their proteins are subject to numerous functional constraints that impact the probability of adaptation. These constraints are globally defined by evolutionary landscapes, which describe the fitness and adaptive potential of all possible mutations. We review deep mutational scanning experiments mapping the evolutionary landscapes of both host and viral proteins engaged in arms races. For restriction factors and some broadly neutralizing antibodies, landscapes favor the host, which may help to level the evolutionary playing field against rapidly evolving viruses. We discuss the biophysical underpinnings of these landscapes and their therapeutic implications.

Vaccines and Broadly Neutralizing Antibodies for HIV-1 Prevention.

Development of improved approaches for HIV-1 prevention will likely be required for a durable end to the global AIDS pandemic. Recent advances in preclinical studies and early phase clinical trials offer renewed promise for immunologic strategies for blocking acquisition of HIV-1 infection. Clinical trials are currently underway to evaluate the efficacy of two vaccine candidates and a broadly neutralizing antibody (bNAb) to prevent HIV-1 infection in humans. However, the vast diversity of HIV-1 is a major challenge for both active and passive immunization. Here we review current immunologic strategies for HIV-1 prevention, with a focus on current and next-generation vaccines and bNAbs.

The Antibody Response to Plasmodium falciparum: Cues for Vaccine Design and the Discovery of Receptor-Based Antibodies.

Plasmodium falciparum remains a serious public health problem and a continuous challenge for the immune system due to the complexity and diversity of the pathogen. Recent advances from several laboratories in the characterization of the antibody response to the parasite have led to the identification of critical targets for protection and revealed a new mechanism of diversification based on the insertion of host receptors into immunoglobulin genes, leading to the production of receptor-based antibodies. These advances have opened new possibilities for vaccine design and passive antibody therapies to provide sterilizing immunity and control blood-stage parasites.

Unfinished Business: Evolution of the MHC and the Adaptive Immune System of Jawed Vertebrates.

The major histocompatibility complex (MHC) is a large genetic region with many genes, including the highly polymorphic classical class I and II genes that play crucial roles in adaptive as well as innate immune responses. The organization of the MHC varies enormously among jawed vertebrates, but class I and II genes have not been found in other animals. How did the MHC arise, and are there underlying principles that can help us to understand the evolution of the MHC? This review considers what it means to be an MHC and the potential importance of genome-wide duplication, gene linkage, and gene coevolution for the emergence and evolution of an adaptive immune system. Then it considers what the original antigen-specific receptor and MHC molecule might have looked like, how peptide binding might have evolved, and finally the importance of adaptive immunity in general.

IgA Function in Relation to the Intestinal Microbiota.

IgA is the dominant immunoglobulin isotype produced in mammals, largely secreted across the intestinal mucosal surface. Although induction of IgA has been a hallmark feature of microbiota colonization following colonization in germ-free animals, until recently appreciation of the function of IgA in host-microbial mutualism has depended mainly on indirect evidence of alterations in microbiota composition or penetration of microbes in the absence of somatic mutations in IgA (or compensatory IgM). Highly parallel sequencing techniques that enable high-resolution analysis of either microbial consortia or IgA sequence diversity are now giving us new perspectives on selective targeting of microbial taxa and the trajectory of IgA diversification according to induction mechanisms, between different individuals and over time. The prospects are to link the range of diversified IgA clonotypes to specific antigenic functions in modulating the microbiota composition, position and metabolism to ensure host mutualism.

A potent pan-sarbecovirus neutralizing antibody resilient to epitope diversification.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolution has resulted in viral escape from clinically authorized monoclonal antibodies (mAbs), creating a need for mAbs that are resilient to epitope diversification. Broadly neutralizing coronavirus mAbs that are sufficiently potent for clinical development and retain activity despite viral evolution remain elusive. We identified a human mAb, designated VIR-7229, which targets the viral receptor-binding motif (RBM) with unprecedented cross-reactivity to all sarbecovirus clades, including non-ACE2-utilizing bat sarbecoviruses, while potently neutralizing SARS-CoV-2 variants since 2019, including the recent EG.5, BA.2.86, and JN.1. VIR-7229 tolerates extraordinary epitope variability, partly attributed to its high binding affinity, receptor molecular mimicry, and interactions with RBM backbone atoms. Consequently, VIR-7229 features a high barrier for selection of escape mutants, which are rare and associated with reduced viral fitness, underscoring its potential to be resilient to future viral evolution. VIR-7229 is a strong candidate to become a next-generation medicine.

Analysis of the diverse antigenic landscape of the malaria protein RH5 identifies a potent vaccine-induced human public antibody clonotype.

The highly conserved and essential Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) has emerged as the leading target for vaccines against the disease-causing blood stage of malaria. However, the features of the human vaccine-induced antibody response that confer highly potent inhibition of malaria parasite invasion into red blood cells are not well defined. Here, we characterize 236 human IgG monoclonal antibodies, derived from 15 donors, induced by the most advanced PfRH5 vaccine. We define the antigenic landscape of this molecule and establish that epitope specificity, antibody association rate, and intra-PfRH5 antibody interactions are key determinants of functional anti-parasitic potency. In addition, we identify a germline IgG gene combination that results in an exceptionally potent class of antibody and demonstrate its prophylactic potential to protect against P. falciparum parasite challenge in vivo. This comprehensive dataset provides a framework to guide rational design of next-generation vaccines and prophylactic antibodies to protect against blood-stage malaria.

Adaptive multi-epitope targeting and avidity-enhanced nanobody platform for ultrapotent, durable antiviral therapy.

Pathogens constantly evolve and can develop mutations that evade host immunity and treatment. Addressing these escape mechanisms requires targeting evolutionarily conserved vulnerabilities, as mutations in these regions often impose fitness costs. We introduce adaptive multi-epitope targeting with enhanced avidity (AMETA), a modular and multivalent nanobody platform that conjugates potent bispecific nanobodies to a human immunoglobulin M (IgM) scaffold. AMETA can display 20+ nanobodies, enabling superior avidity binding to multiple conserved and neutralizing epitopes. By leveraging multi-epitope SARS-CoV-2 nanobodies and structure-guided design, AMETA constructs exponentially enhance antiviral potency, surpassing monomeric nanobodies by over a million-fold. These constructs demonstrate ultrapotent, broad, and durable efficacy against pathogenic sarbecoviruses, including Omicron sublineages, with robust preclinical results. Structural analysis through cryoelectron microscopy and modeling has uncovered multiple antiviral mechanisms within a single construct. At picomolar to nanomolar concentrations, AMETA efficiently induces inter-spike and inter-virus cross-linking, promoting spike post-fusion and striking viral disarmament. AMETA's modularity enables rapid, cost-effective production and adaptation to evolving pathogens.

Bispecific dendritic-T cell engager potentiates anti-tumor immunity.

Immune checkpoint inhibition treatment using aPD-1 monoclonal antibodies is a promising cancer immunotherapy approach. However, its effect on tumor immunity is narrow, as most patients do not respond to the treatment or suffer from recurrence. We show that the crosstalk between conventional type I dendritic cells (cDC1) and T cells is essential for an effective aPD-1-mediated anti-tumor response. Accordingly, we developed a bispecific DC-T cell engager (BiCE), a reagent that facilitates physical interactions between PD-1+ T cells and cDC1. BiCE treatment promotes the formation of active dendritic/T cell crosstalk in the tumor and tumor-draining lymph nodes. In vivo, single-cell and physical interacting cell analysis demonstrates the distinct and superior immune reprogramming of the tumors and tumor-draining lymph nodes treated with BiCE as compared to conventional aPD-1 treatment. By bridging immune cells, BiCE potentiates cell circuits and communication pathways needed for effective anti-tumor immunity.

Comparison of protection against mpox following mRNA or modified vaccinia Ankara vaccination in nonhuman primates.

In 2022, mpox virus (MPXV) spread worldwide, causing 99,581 mpox cases in 121 countries. Modified vaccinia Ankara (MVA) vaccine use reduced disease in at-risk populations but failed to deliver complete protection. Lag in manufacturing and distribution of MVA resulted in additional MPXV spread, with 12,000 reported cases in 2023 and an additional outbreak in Central Africa of clade I virus. These outbreaks highlight the threat of zoonotic spillover by Orthopoxviruses. mRNA-1769, an mRNA-lipid nanoparticle (LNP) vaccine expressing MPXV surface proteins, was tested in a lethal MPXV primate model. Similar to MVA, mRNA-1769 conferred protection against challenge and further mitigated symptoms and disease duration. Antibody profiling revealed a collaborative role between neutralizing and Fc-functional extracellular virion (EV)-specific antibodies in viral restriction and ospinophagocytic and cytotoxic antibody functions in protection against lesions. mRNA-1769 enhanced viral control and disease attenuation compared with MVA, highlighting the potential for mRNA vaccines to mitigate future pandemic threats.

Complement in breast milk modifies offspring gut microbiota to promote infant health.

Breastfeeding offers demonstrable benefits to newborns and infants by providing nourishment and immune protection and by shaping the gut commensal microbiota. Although it has been appreciated for decades that breast milk contains complement components, the physiological relevance of complement in breast milk remains undefined. Here, we demonstrate that weanling mice fostered by complement-deficient dams rapidly succumb when exposed to murine pathogen Citrobacter rodentium (CR), whereas pups fostered on complement-containing milk from wild-type dams can tolerate CR challenge. The complement components in breast milk were shown to directly lyse specific members of gram-positive gut commensal microbiota via a C1-dependent, antibody-independent mechanism, resulting in the deposition of the membrane attack complex and subsequent bacterial lysis. By selectively eliminating members of the commensal gut community, complement components from breast milk shape neonate and infant gut microbial composition to be protective against environmental pathogens such as CR.

SARS-CoV-2 BA.2.86 enters lung cells and evades neutralizing antibodies with high efficiency.

BA.2.86, a recently identified descendant of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron BA.2 sublineage, contains ∼35 mutations in the spike (S) protein and spreads in multiple countries. Here, we investigated whether the virus exhibits altered biological traits, focusing on S protein-driven viral entry. Employing pseudotyped particles, we show that BA.2.86, unlike other Omicron sublineages, enters Calu-3 lung cells with high efficiency and in a serine- but not cysteine-protease-dependent manner. Robust lung cell infection was confirmed with authentic BA.2.86, but the virus exhibited low specific infectivity. Further, BA.2.86 was highly resistant against all therapeutic antibodies tested, efficiently evading neutralization by antibodies induced by non-adapted vaccines. In contrast, BA.2.86 and the currently circulating EG.5.1 sublineage were appreciably neutralized by antibodies induced by the XBB.1.5-adapted vaccine. Collectively, BA.2.86 has regained a trait characteristic of early SARS-CoV-2 lineages, robust lung cell entry, and evades neutralizing antibodies. However, BA.2.86 exhibits low specific infectivity, which might limit transmissibility.

Mosaic sarbecovirus nanoparticles elicit cross-reactive responses in pre-vaccinated animals.

Immunization with mosaic-8b (nanoparticles presenting 8 SARS-like betacoronavirus [sarbecovirus] receptor-binding domains [RBDs]) elicits more broadly cross-reactive antibodies than homotypic SARS-CoV-2 RBD-only nanoparticles and protects against sarbecoviruses. To investigate original antigenic sin (OAS) effects on mosaic-8b efficacy, we evaluated the effects of prior COVID-19 vaccinations in non-human primates and mice on anti-sarbecovirus responses elicited by mosaic-8b, admix-8b (8 homotypics), or homotypic SARS-CoV-2 immunizations, finding the greatest cross-reactivity for mosaic-8b. As demonstrated by molecular fate mapping, in which antibodies from specific cohorts of B cells are differentially detected, B cells primed by WA1 spike mRNA-LNP dominated antibody responses after RBD-nanoparticle boosting. While mosaic-8b- and homotypic-nanoparticles boosted cross-reactive antibodies, de novo antibodies were predominantly induced by mosaic-8b, and these were specific for variant RBDs with increased identity to RBDs on mosaic-8b. These results inform OAS mechanisms and support using mosaic-8b to protect COVID-19-vaccinated/infected humans against as-yet-unknown SARS-CoV-2 variants and animal sarbecoviruses with human spillover potential.

Vaccine induction of CD4-mimicking HIV-1 broadly neutralizing antibody precursors in macaques.

The CD4-binding site (CD4bs) is a conserved epitope on HIV-1 envelope (Env) that can be targeted by protective broadly neutralizing antibodies (bnAbs). HIV-1 vaccines have not elicited CD4bs bnAbs for many reasons, including the occlusion of CD4bs by glycans, expansion of appropriate naive B cells with immunogens, and selection of functional antibody mutations. Here, we demonstrate that immunization of macaques with a CD4bs-targeting immunogen elicits neutralizing bnAb precursors with structural and genetic features of CD4-mimicking bnAbs. Structures of the CD4bs nAb bound to HIV-1 Env demonstrated binding angles and heavy-chain interactions characteristic of all known human CD4-mimicking bnAbs. Macaque nAb were derived from variable and joining gene segments orthologous to the genes of human VH1-46-class bnAb. This vaccine study initiated in primates the B cells from which CD4bs bnAbs can derive, accomplishing the key first step in the development of an effective HIV-1 vaccine.

Potent efficacy of an IgG-specific endoglycosidase against IgG-mediated pathologies.

Endo-ß-N-acetylglucosaminidases (ENGases) that specifically hydrolyze the Asn297-linked glycan on immunoglobulin G (IgG) antibodies, the major molecular determinant of fragment crystallizable (Fc) γ receptor (FcγR) binding, are exceedingly rare. All previously characterized IgG-specific ENGases are multi-domain proteins secreted as an immune evasion strategy by Streptococcus pyogenes strains. Here, using in silico analysis and mass spectrometry techniques, we identified a family of single-domain ENGases secreted by pathogenic corynebacterial species that exhibit strict specificity for IgG antibodies. By X-ray crystallographic and surface plasmon resonance analyses, we found that the most catalytically efficient IgG-specific ENGase family member recognizes both protein and glycan components of IgG. Employing in vivo models, we demonstrated the remarkable efficacy of this IgG-specific ENGase in mitigating numerous pathologies that rely on FcγR-mediated effector functions, including T and B lymphocyte depletion, autoimmune hemolytic anemia, and antibody-dependent enhancement of dengue disease, revealing its potential for treating and/or preventing a wide range of IgG-mediated diseases in humans.

Follicular Helper T Cells.

Although T cell help for B cells was described several decades ago, it was the identification of CXCR5 expression by B follicular helper T (Tfh) cells and the subsequent discovery of their dependence on BCL6 that led to the recognition of Tfh cells as an independent helper subset and accelerated the pace of discovery. More than 20 transcription factors, together with RNA-binding proteins and microRNAs, control the expression of chemotactic receptors and molecules important for the function and homeostasis of Tfh cells. Tfh cells prime B cells to initiate extrafollicular and germinal center antibody responses and are crucial for affinity maturation and maintenance of humoral memory. In addition to the roles that Tfh cells have in antimicrobial defense, in cancer, and as HIV reservoirs, regulation of these cells is critical to prevent autoimmunity. The realization that follicular T cells are heterogeneous, comprising helper and regulatory subsets, has raised questions regarding a possible division of labor in germinal center B cell selection and elimination.

Exploiting Mucosal Immunity for Antiviral Vaccines.

Mucosal surfaces provide a remarkably effective barrier against potentially dangerous pathogens. Therefore, enhancing mucosal immunity through vaccines-strengthening that first line of defense-holds significant promise for reducing the burden of viral diseases. The large and varied class of viral pathogens, however, continues to present thorny challenges to vaccine development. Two primary difficulties exist: Viruses exhibit a stunning diversity of strategies for evading the host immune response, and even when we understand the nature of effective immune protection against a given virus, eliciting that protection is technically challenging. Only a few mucosal vaccines have surmounted these obstacles thus far. Recent developments, however, could greatly improve vaccine design. In this review, we first sketch out our understanding of mucosal immunity and then compare the herpes simplex virus, human immunodeficiency virus, and influenza virus to illustrate the distinct challenges of developing successful vaccines and to outline potential solutions.

Continuous germinal center invasion contributes to the diversity of the immune response.

Antibody responses are characterized by increasing affinity and diversity over time. Affinity maturation occurs in germinal centers by a mechanism that involves repeated cycles of somatic mutation and selection. How antibody responses diversify while also undergoing affinity maturation is not as well understood. Here, we examined germinal center (GC) dynamics by tracking B cell entry, division, somatic mutation, and specificity. Our experiments show that naive B cells continuously enter GCs where they compete for T cell help and undergo clonal expansion. Consistent with late entry, invaders carry fewer mutations but can contribute up to 30% or more of the cells in late-stage germinal centers. Notably, cells entering the germinal center at later stages of the reaction diversify the immune response by expressing receptors that show low affinity to the immunogen. Paradoxically, the affinity threshold for late GC entry is lowered in the presence of high-affinity antibodies.