BACKGROUND Functional B cell responses for both prevention and control of hepatitis B virus (HBV) infection remain poorly understood, including in the context of HBV/HIV coinfection. METHODS Here, we employed high-dimensional single-cell analysis to assess global and hepatitis B surface antigen–specific (HBsAg-specific) B cells in a longitudinal cohort of incident HBV from the Multicenter AIDS Cohort Study, with a subset of the cohort living with HIV-1. RESULTS We observed that prior HIV infection has negative consequences for B cell function in early post-acute HBV infection, including increased frequencies of atypical memory B cells and regulatory B cells, expression of the activation marker CD86 on multiple B cell subsets in chronic HBV (CHB), and restricted expansion of HBsAg-specific B cells. In contrast, in HBV monoinfection, we observed no changes in the global B cell population from prior to infection and robust expansion of HBsAg-specific B cells. These expanded antigen-specific B cells resembled class-switched intermediate and resting memory B cells, with activation phenotypes that may contribute to ongoing HBV control. CONCLUSION HIV infection has a significant impact on B cell responses to subsequent HBV infection that may promote development of CHB in HBV/HIV coinfection. FUNDING Vaccine Research Center, NIAID, Bill & Melinda Gates Foundation, and NIH.

Many HIV-1 broadly neutralizing antibodies (bnAbs) account for envelope (Env) glycan shielding by supplementing antibody-protein interactions with antibody-glycan interactions. Further, bnAbs that interact predominantly via glycans can augment their binding through antigen-binding fragment (Fab) dimerization, generally utilizing non-variable region interactions. Here, we examined a donor whose serum identified glycan-reactivity (antibody 2G12-like) and CD4 binding-site (CD4bs) reactivity and isolated both glycan-reactive and CD4bs-reactive antibodies. The CD4bs antibodies were members of the VRC01-antibody class and neutralized nearly 70% of HIV-1 (208-strain panel). The glycan-reactive antibody had ∼30% breadth, and cryo-EM analysis revealed it to be a Fab-dimerized glycan (FDG)-reactive antibody with a distinct architecture wherein the Fab arms dimerized through a disulfide bond at the tips of the complementarity-determining loops that wedged between two glycans. Overall, we identified an FDG antibody that recognized through a “dual-glycan clamp” epitope and is the second FDG antibody to be isolated from an HIV-infected donor since antibody 2G12.

Microglia are central nervous system (CNS)-resident macrophages, with key roles in immune surveillance, phagocytosis, and synaptic pruning. Yolk sac-derived microglia show minimal turnover from hematopoietic stem/progenitor cells (HSPCs) under steady-state conditions in mice. However, clinical benefits observed in patients receiving HSPC gene therapies for CNS disorders suggest functional integration of HSPC-derived cells. To investigate microglia replacement and the impact of clonal hematopoiesis (CH) on microglia, we analyzed microglia in rhesus macaques receiving barcoded or CRISPR-edited (TET2-mutant) HSPC transplants. We found that <2% microglia were derived from HSPCs many years following transplant, with no evidence of enhanced replacement in CH. The rare HSPC-derived tissue-resident cells exhibited a macrophage-like gene expression profile. Our results demonstrate limited long-term microglia replacement from adult HSPCs, even with CH, contrasting prior human studies. This work provides insights into microglia ontogeny and informs strategies for CNS-targeted HSPC gene therapies and interpretation of CH-related neuroprotection.

Exposure to Mycobacterium tuberculosis (Mtb) leads to a spectrum of outcomes, from latent infection to active disease, but limited insight into protective immune correlates has hampered vaccine development. Using a proteome-wide Mtb microarray, we mapped antibody specificities across individuals with varying Mtb exposure, including humans with controlled latent tuberculosis infection, uncontrolled active TB, or that resist IGRA conversion as well as non-human primates with near sterilized Mtb infection following intravenous BCG. While current TB vaccine antigens were poorly immunogenic across most populations, striking overlap in antibody binding was observed to surface and secreted proteins, all associated with enhanced antibody functions and some also robustly targeted by T cells. Collectively, these data provide an atlas of antibody protein binding across the spectrum of Mtb infection, and further point to a handful of promising Mtb protein candidates to guide the design of antibody-based TB interventions aimed at mitigating disease globally.

Nipah virus (NiV) is a highly pathogenic, zoonotic paramyxovirus with pandemic potential. No licensed vaccines or treatments are available. Therefore, we conducted a phase 1, first-in-human, open-label, dose-escalation trial of a lipid nanoparticle mRNA vaccine, mRNA-1215, encoding a chimeric pre-fusion F (Pre-F) protein linked to glycoprotein G of a NiV Malaysian strain. Forty healthy adults, who met eligibility criteria, were enrolled into the 10-, 25-, 50- or 100-μg dose groups with ten participants per group. Each participant received two doses of mRNA-1215, intramuscularly, at a 4-week interval except for one participant in the 10-μg dose group who received only the first dose. All participants remained in the study until their final study visit. The primary outcome of vaccine safety and tolerability was determined by prespecified end points: the frequency and severity of solicited local and systemic adverse events (AEs), unsolicited AEs, safety laboratory measures, medically attended AEs, AEs of special interest, new chronic medical conditions and serious AEs. The most frequently reported local and systemic AEs were mild pain and tenderness at the injection site (n  = 33; 82%) and mild malaise (n  = 16; 40%), respectively. Overall, the vaccine was well tolerated. No serious AEs occurred during the study. mRNA-1215 elicited robust Pre-F and G binding antibody titers, the prespecified secondary end points. An exploratory analysis found that mRNA-1215 elicited neutralizing titers by 2 weeks after the prime in all dose groups. Responses increased after the boost and remained elevated for at least 1 year after vaccination. These findings demonstrate an initial overall favorable safety profile and immunogenicity results for a first-in-human, structure-based, chimeric mRNA Nipah virus vaccine. mRNA-1215 is a promising vaccine candidate for continued clinical development for populations at risk for regional and potentially larger outbreaks caused by Nipah virus. ClinicalTrials.gov identifier: NCT05398796 .

Reports of HIV-1-specific broadly neutralizing monoclonal antibodies (bNAbs) mediating a potential ‘vaccinal effect’ implicate passively transferred bNAbs in promoting endogenous anti-HIV-1 immune responses. To date, three clinical trials have reported either increased anti-HIV-1 neutralizing antibodies or T cell responses following bNAb administration to people living with HIV. Despite strong enthusiasm for this hypothesis, motivated in large part by its potential application to HIV-1 therapeutic strategies, the mechanism(s) underlying a vaccinal effect remain unclear. Moreover, vaccinal effects on antibody and T cell responses are not consistently replicated. Partly, this inconsistency may be due to numerous difficulties in sensitively measuring a vaccinal effect in the context of human clinical trials. The magnitude of immune response increase following bNAb administration is generally modest, even when it is observed; a far greater enhancement of neutralization or T cell responses is likely required for a biologically meaningful impact. We review clinical and pre-clinical nonhuman primate studies that evaluated HIV-1/SIV monoclonal antibodies for vaccinal effects, with an emphasis on the strengths and limitations of these studies. Considerations for future studies investigating vaccinal effects are discussed, including appropriate comparators and specificity controls. Lastly, immune response characteristics of elite controller cohorts are outlined as potential vaccinal effect endpoints more likely to mediate HIV-1 suppression. As bNAb therapeutic interventions increasingly turn to combination approaches, including incorporation of immunomodulatory agents, attention to study design incorporating appropriate control groups, and relevant immunogenicity assays will enable more conclusive interpretation of vaccinal effects likely to mediate durable control of HIV. In any case, to date, the elicitation of vaccinal effects has been disappointing.

Conventional vaccines have so far failed to elicit the types of antibodies needed for protection against HIV. As an alternative, we evaluated adeno-associated virus (AAV) delivery of rhesus macaque antibodies to the SIV envelope glycoprotein for protection against SIV challenge. AAV vectors encoding a broadly neutralizing antibody (bnAb) and an antibody that only mediates antibody-dependent cellular cytotoxicity (ADCC) were administered individually or together to separate groups of rhesus macaques. Antibody expression was sustained for more than a year with minimal anti-drug antibody responses. All animals that received a control antibody or the ADCC-only antibody became infected after five low-dose, intrarectal challenges with SIVmac239. In contrast, 14 of 16 animals that received the bnAb resisted two rounds of twelve SIVmac239 challenges more than a year apart. Thus, AAV delivery of a single bnAb can afford durable protection against a pathogenic SIV strain that is notoriously difficult to protect against by vaccination.

Broadly neutralizing antibodies (bNAbs) targeting the apex of the HIV-1-envelope (Env) trimer comprise the most potent category of HIV-1 bNAbs and have emerged as promising therapeutics. Here, we investigate the development of the HIV-1 apex-directed PGT145-PGDM1400 antibody lineage and report cryo-EM structures at 3.4 Å resolution of PGDM1400 and of an improved PGT145 variant (PGT145-R100aS), each bound to the BG505 Env trimer. Cross-species-based engineering improves PGT145 IC80 breadth to near that of PGDM1400. Despite similar breadth and potency, the two antibodies differ in their residue-level interactions with important apex features, including N160 glycans and apex cavity, with residue 100i of PGT145 (sulfated tyrosine) penetrating ∼7 Å farther than residue 100i of PGDM1400 (aspartic acid). While apex-directed bNAbs from other donors use maturation pathways that often converge on analogous residue-level recognition, our results demonstrate that divergent residue-level recognition can occur within the same lineage, thereby enabling improved coverage of escape variants.

Toxoplasma gondii is a highly versatile parasite that infects most warm-blooded animals and is a major cause of retinochoroiditis and uveitis in humans. The pathophysiology of these conditions remains poorly understood. Both parasite virulence and host inflammatory response contribute to the development of ocular disease. While CD4+ T cells play a critical role in host resistance to Toxoplasma infection, their kinetics and effector functions, as well as their contribution to the clinical outcome of the infection, including ocular involvement, remain poorly understood. To address this question, we investigated the immune response during acute and convalescent toxoplasmosis and stratified patients further based on the presence or absence of ocular disease. We found that T. gondii infection leads to decreased and increased proportions of central and effector memory CD4+ T cells, respectively. Applying unsupervised analysis, distinct CD4+ T-cell subsets were determined. Among 50 clusters, 10 produced cytotoxic proteins (granzyme B and perforin) and one produced cytokines upon antigen-specific stimulation. We observed that proportions of five CD4+ T-cell clusters out of 50 were different during acute disease between T. gondii-infected patients with and without ocular lesions. Interestingly, three of the five displayed a cytotoxic signature indicating their possible involvement in ocular immunopathology. Taken together, our results reveal that during T. gondii infection, CD4+ T cells not only develop a Th1 cytokine profile, but also acquire previously unappreciated cytotoxic capacity/function. These results, while underscoring the complexity of the CD4+ T-cell response to T. gondii, suggest that specific subsets may be involved in the development of pathology and provide possible targets for therapeutic intervention.

The latent viral reservoir remains the major barrier to HIV cure, placing the burden of strict adherence to antiretroviral therapy (ART) on people living with HIV to prevent recrudescence of viremia. For infants with perinatally acquired HIV, adherence is anticipated to be a lifelong need. In this study, we tested the hypothesis that administration of ART and viral Envelope-specific rhesus-derived IgG1 monoclonal antibodies (RhmAbs) with or without the IL-15 superagonist N-803 early in infection would limit viral reservoir establishment in SIV-infected infant rhesus macaques. Following initiation of ART at 1-2 weeks after oral SIVmac251 infection, we observed biphasic decay of viremia, with first phase decay significantly faster in the ART + SIV RhmAbs-treated group compared to controls that received only ART. In contrast, the addition of N-803 to ART + SIV RhmAbs significantly slowed both the first and second phase viral decay compared to the ART only group. Treatment with a single dose of N-803 resulted in increased frequency of Ki67 expressing NK, CD8+, and CD4+ T cells. Levels of intact SIV proviruses in CD4+ T cells from blood, lymph nodes, and rectum at week 48 of ART did not differ across groups. Similarly, the time to viral rebound following ART interruption was not impacted by the experimental treatments. These results support the concept that the rebound-competent viral reservoir is formed within days after infection and that targeting only productively infected cells for clearance near the time of ART initiation, even during acute infection, may be insufficient to limit reservoir establishment.

Broadly neutralizing antibodies targeting the V2 apex of HIV-1 envelope are desired as vaccine design templates, but few have been described. Here, we report 11 lineages of V2 apex-neutralizing antibodies from simian-human immunodeficiency virus (SHIV)-infected rhesus macaques and determine cryo-EM structures for 9. A single V2 apex-neutralizing lineage accounted for cross-clade breadth in most macaques, and somatic hypermutation relative to breadth was generally low, exemplified by antibody V033-a.01 with <5% nucleotide mutation and 37% breadth (208-strain panel). Envelope complex structures revealed eight different antibody classes (one multi-donor) and the complete repertoire of all five possible recognition topologies, recapitulating canonical human modes of apex insertion and C-strand hydrogen bonding. Despite this diversity in recognition, all rhesus-V2 apex antibodies were derived from reading frame two of the DH3-15*01 gene. Collectively, these results define-in rhesus-the structural and genetic basis of HIV-1 V2 apex recognition and demonstrate unprecedented structural plasticity of a highly selected immunogenetic element.

The membrane-proximal external region (MPER) of the HIV-1 envelope is a target for broadly neutralizing antibodies (bnAbs), and vaccine-elicited MPER-directed antibodies have recently been reported from a human clinical trial. In this study, we sought to identify MPER-directed nAbs in simian immunodeficiency virus (SIV)-infected rhesus macaques. We isolated four lineages of SIV MPER-directed nAbs from two SIV-infected macaques. The nAbs displayed low potency but up to 90% breadth on a 20-strain SIV panel. Crystal structures of representative nAbs in complex with SIV MPER peptides revealed the SIV antibodies to bind a helical epitope at the N-terminal (proximal) region of the MPER, defining a reproducible multi-donor class encompassing all four lineages. HIV-1 comparison showed that this class of SIV MPER-directed antibodies targets a helical region overlapping that targeted by human vaccine-elicited ones. Thus, a prevalent and reproducible class of SIV bnAbs recognizes an epitope similar to that recently observed in an HIV-1-vaccine trial.

Neutrophils are a key component of the innate immune system. While historically considered a circulating cell, recent mouse models indicate that a small proportion of neutrophils could be tissue-localized in homeostasis. Important questions remain: How are neutrophils distributed in the body? What are their circulating and tissue half-lives? What percentage are extravascular? Where in tissues are they localized? To address these, we combined PET/CT, in vivo flow cytometry (serial intravascular staining, SIVS), mathematical modeling, and tissue imaging, in a rhesus macaque model. Live-tracking of ex vivo Zr-89 labeled cells by PET/CT revealed that neutrophils quickly pass through the lungs before migrating to the spleen, liver, and bone marrow, where they remain in a steady state. Using SIVS for direct labeling and tracking of circulating cells, we established that the circulating half-life of rhesus neutrophils is significantly shorter (0.4–8.8 h) than previous human studies have estimated (17 h). Previously circulating neutrophils are detectable in tissues for at least 24 hours. Tissue flow cytometry of in vivo labeled cells revealed that approximately 1% of neutrophils are extravascular in homeostasis. Multiplexed confocal imaging of tissue-localized neutrophils indicates their localization in the spleen is not random but parafollicular, in proximity to migrating B and T cells. Ongoing work is aimed at establishing the spatial localization of neutrophils in other tissues. Cellular Adhesion, Migration, and Inflammation (CAM)

Advances in T cell biology have revealed heterogeneity among T cell populations that is not captured by existing general nomenclature. This issue has caused an ad hoc broadening of core T cell subset definitions and the invention of new subset designations that have not been uniformly delineated. To address this issue, in this Consensus Statement, we propose guidelines that serve three goals. First, they advocate that primary research reports define the experimental basis by which relevant subsets are designated in the methods section of each study. Second, they provide standardized definitions for existing subset designations in popular use, and common experimental criteria for defining each subset are noted. Last, they present an alternative ‘modular nomenclature’ paradigm. The newly proposed modular nomenclature eschews conceptualization of antigen-experienced T cells as belonging to a few idealized subsets, and the nomenclature instead simply indicates individual biological properties present in a T cell population with brief descriptors. Collectively, these guidelines intend to enhance transparency in the literature while facilitating clearer communication of findings and concepts to researchers, students and clinicians. This Consensus Statement clarifies the existing subset-based nomenclature for T cells. Furthermore, it proposes an alternative modular nomenclature that is designed to be brief and flexible and to avoid ambiguity and unwanted implications. The authors also provide guidance on how T cell nomenclature should be described in research papers.

HIV-1 envelope broadly neutralizing antibodies represent a promising component of HIV-1 cure strategies. To evaluate the therapeutic efficacy of combination monoclonal antibodies (mAbs) in a rigorous nonhuman primate model, we tested different combinations of simian immunodeficiency virus (SIV) neutralizing mAbs in SIVmac251-infected rhesus macaques. Antiretroviral therapy-suppressed animals received anti-SIV mAbs targeting multiple Env epitopes spanning analytical treatment interruption (ATI) in 3 groups (n = 7 each): i) no mAb; ii) 4-mAb combination; and iii) 2-mAb combination. Each mAb was administered at 15 mg/kg, and both mAb-treated groups received ITS103.01, a highly potent CD4-binding site targeting antibody. mAb treatment delayed viral rebound, lowered rebound viremia setpoint and viral diversity, and extended animal lifespan. Compared to controls, for which viremia rebounded 2 wk following ATI, mAb infusion delayed rebound for both groups ( P = 0.0003). Animals that received the 4-mAb regimen rebounded 3 to 6 wk post-ATI while the 2-mAb regimen rebounded 5 to 22 wk post-ATI. Envelope escape mutations emerged in rebound virus of mAb-treated animals that abrogated neutralization by ITS103.01, the most potent in the cocktail. These data demonstrate in vivo antiviral activity of SIV mAbs in the context of ATI via immune pressure dominated by the most potent mAb and highlight their potential in adjunctive therapeutic studies.

Noroviruses infect millions each year, and while effective countermeasures are eagerly sought, none have been reported for the GI genogroup, first described more than 50 years ago. Here, to provide insight into GI norovirus neutralization, we isolated a broad GI antibody, 16E10, from a human blood donor and showed it neutralizes noroviruses in human enteroid cultures and abrogates or reduces infection in rhesus macaques. The cryogenic electron microscopy reconstruction of 16E10 with a norovirus protruding-domain dimer at 2.56-Å resolution reveals an exceptionally large binding surface, overlapping an antibody supersite, distal from host receptor-binding or cofactor-binding sites. Cryogenic electron microscopy reconstructions with virus-like particles (VLPs) showed that 16E10 disrupts protruding domains on the VLP surface and disassembles VLPs, altering viral organization required for avidity. While its epitope was generally conserved, 16E10 recognized multiple sequence-divergent residues, binding to which was enabled by corresponding cavities in the 16E10-norovirus interface. Broad recognition of noroviruses can thus incorporate sequence-divergent residues, through a cavity-based mechanism of diversity tolerance.

An alternative to lifelong antiretroviral therapy (ART) is needed to achieve durable control of HIV-1. Here, we show that adeno-associated virus (AAV) delivery of two rhesus macaque antibodies to the simian immunodeficiency virus (SIV) envelope glycoprotein (Env) with potent neutralization and antibody-dependent cellular cytotoxicity can prevent viral rebound in macaques infected with barcoded SIVmac239M after discontinuing suppressive ART. After AAV administration, sustained antibody expression with minimal antidrug antibody responses was achieved in all but one animal. After ART withdrawal, SIV replication rebounded within 2 weeks in all control animals but remained <15 copies per milliliter in plasma for more than a year in four of the eight animals that received AAV vectors encoding Env-specific antibodies. Viral sequences from animals that rebounded with delayed kinetics exhibited restricted clonal diversity and antibody escape mutations in Env. Thus, sustained expression of antibodies with potent antiviral activity can afford durable, ART-free containment of pathogenic SIV infection.

Venezuelan equine encephalitis virus (VEEV) is an arbovirus that causes a disease in which 4%-14% of individuals can develop neurological symptoms. Prior to 1970, VEEV was developed as a biological threat agent due to its stability and high morbidity when administered by aerosol. Currently, no FDA-licensed vaccines nor therapeutics for VEEV exist. Single-domain antibodies (sdAbs) may provide a therapeutic option due to their small size and ability to bind recessed epitopes not recognized by conventional antibodies. This study identified two bivalent sdAbs that were able to protect mice from a lethal challenge against both epizootic and enzootic subtypes of VEEV. Cryo-EM structures of sdAb-VEEV complexes revealed the sdAbs that comprised the bivalent sdAbs to recognize a mixture of conserved and non-conserved regions of the VEEV envelope proteins. While all three of the cryo-EM-characterized epitopes were unique in terms of their recognized VEEV residues, two sdAbs, V2B3 and V2C3, overlapped sterically, explaining why only their combinations with the non-sterically overlapping sdAb V3A8f, which composed the bivalent sdAbs described here, were so particularly effective. Binding and neutralization studies found that the bivalent sdAbs have the potential to be broad-spectrum anti-alphavirus therapeutics as they cross-neutralize multiple alphaviruses.IMPORTANCEAlphaviruses are no longer geographically constrained to one region of the world but are expanding to be of global concern. In many regions of the world, multiple alphaviruses co-circulate; therefore, having a therapeutic that is pan-alphavirus is important. A cocktail of multiple pan-alphavirus binding/neutralizing antibodies (Abs) may provide optimal coverage against alphaviruses while decreasing the prevalence of viral escape mutants, which could cause the therapeutic to no longer be efficacious. Structures of these Abs, defining their recognition, could assist in identifying optimal combinations. A bivalent pan-alphavirus single-domain antibody could be used in a cocktail with already identified alphavirus IgG antibodies.