Mxra8 is a recently described receptor for multiple alphaviruses, including Chikungunya (CHIKV), Mayaro (MAYV), Ross River (RRV), and O'nyong nyong (ONNV) viruses. To determine its role in pathogenesis, we generated mice with mutant Mxra8 alleles: an 8-nucleotide deletion that produces a truncated, soluble form (Mxra8Δ8/Δ8) and a 97-nucleotide deletion that abolishes Mxra8 expression (Mxra8Δ97/Δ97). Mxra8Δ8/Δ8 and Mxra8Δ97/Δ97 fibroblasts show reduced CHIKV infection in culture, and Mxra8Δ8/Δ8 and Mxra8Δ97/Δ97 mice have decreased infection of musculoskeletal tissues with CHIKV, MAYV, RRV, or ONNV. Less foot swelling is observed in CHIKV-infected Mxra8 mutant mice, which correlated with fewer infiltrating neutrophils and cytokines. A recombinant E2-D71A CHIKV with diminished binding to Mxra8 is attenuated in vivo in wild-type mice. Ectopic Mxra8 expression is sufficient to enhance CHIKV infection and lethality in transgenic flies. These studies establish a role for Mxra8 in the pathogenesis of multiple alphaviruses and suggest that targeting this protein may mitigate disease in humans.

The mammalian SWitch/Sucrose Non-Fermentable (SWI/SNF) chromatin-remodeling BAF (BRG1/BRM-associated factor) complex plays an essential role in developmental and pathological processes. We show that the deletion of Baf155, which encodes a subunit of the BAF complex, in the Tie2(+) lineage (Baf155 (CKO) leads to defects in yolk sac myeloid and definitive erythroid (EryD) lineage differentiation from erythromyeloid progenitors (EMPs). The chromatin of myeloid gene loci in Baf155 CKO EMPs is mostly inaccessible and enriched mainly by the ETS binding motif. BAF155 interacts with PU.1 and is recruited to PU.1 target gene loci together with p300 and KDM6a. Treatment of Baf155 CKO embryos with GSK126, an H3K27me2/3 methyltransferase EZH2 inhibitor, rescues myeloid lineage gene expression. This study uncovers indispensable BAF-mediated chromatin remodeling of myeloid gene loci at the EMP stage. Future studies exploiting epigenetics in the generation and application of EMP derivatives for tissue repair, regeneration, and disease are warranted.

SARS-CoV-2 variants that attenuate antibody neutralization could jeopardize vaccine efficacy. We recently reported the protective activity of an intranasally administered spike protein-based chimpanzee adenovirus-vectored vaccine (ChAd-SARS-CoV-2-S) in animals, which has advanced to human trials. Here, we assessed its durability, dose response, and cross-protective activity in mice. A single intranasal dose of ChAd-SARS-CoV-2-S induced durably high neutralizing and Fc effector antibody responses in serum and S-specific IgG and IgA secreting long-lived plasma cells in the bone marrow. Protection against a historical SARS-CoV-2 strain was observed across a 100-fold vaccine dose range and over a 200-day period. At 6 weeks or 9 months after vaccination, serum antibodies neutralized SARS-CoV-2 strains with B.1.351, B.1.1.28, and B.1.617.1 spike proteins and conferred almost complete protection in the upper and lower respiratory tracts after challenge with variant viruses. Thus, in mice, intranasal immunization with ChAd-SARS-CoV-2-S provides durable protection against historical and emerging SARS-CoV-2 strains.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has necessitated the rapid development of antibody-based therapies and vaccines as countermeasures. Here, we use cryoelectron microscopy (cryo-EM) to characterize two protective anti-SARS-CoV-2 murine monoclonal antibodies (mAbs) in complex with the spike protein, revealing similarities between epitopes targeted by human and murine B cells. The more neutralizing mAb, 2B04, binds the receptor-binding motif (RBM) of the receptor-binding domain (RBD) and competes with angiotensin-converting enzyme 2 (ACE2). By contrast, 2H04 binds adjacent to the RBM and does not compete for ACE2 binding. Naturally occurring sequence variants of SARS-CoV-2 and corresponding neutralization escape variants selected in vitro map to our structurally defined epitopes, suggesting that SARS-CoV-2 might evade therapeutic antibodies with a limited set of mutations, underscoring the importance of combination mAb therapeutics. Finally, we show that 2B04 neutralizes SARS-CoV-2 infection by preventing ACE2 engagement, whereas 2H04 reduces host cell attachment without directly disrupting ACE2-RBM interactions, providing distinct inhibitory mechanisms used by RBD-specific mAbs.

Although neutralizing monoclonal antibodies (mAbs) against epitopes within the alphavirus E2 protein can protect against infection, the functional significance of non-neutralizing mAbs is poorly understood. Here, we evaluate the activity of 13 non-neutralizing mAbs against Mayaro virus (MAYV), an emerging arthritogenic alphavirus. These mAbs bind to the MAYV virion and surface of infected cells but fail to neutralize infection in cell culture. Mapping studies identify six mAb binding groups that localize to discrete epitopes within or adjacent to the A domain of the E2 glycoprotein. Remarkably, passive transfer of non-neutralizing mAbs protects against MAYV infection and disease in mice, and their efficacy requires Fc effector functions. Monocytes mediate the protection of non-neutralizing mAbs in vivo, as Fcγ-receptor-expressing myeloid cells facilitate the binding, uptake, and clearance of MAYV without antibody-dependent enhancement of infection. Humoral protection against alphaviruses likely reflects contributions from non-neutralizing antibodies through Fc-dependent mechanisms that accelerate viral clearance.

The teratogenic potential of Zika virus (ZIKV) has made the development of an effective vaccine a global health priority. Here, we generate two gorilla adenovirus-based ZIKV vaccines that encode for pre-membrane (prM) and envelope (E) proteins (GAd-Zvp) or prM and the ectodomain of E protein (GAd-Eecto). Both vaccines induce humoral and cell-mediated immune responses and prevent lethality after ZIKV challenge in mice. Protection is antibody dependent, CD8+ T cell independent, and for GAd-Eecto requires the complement component C1q. Immunization of GAd-Zvp induces antibodies against a key neutralizing epitope on domain III of E protein and confers durable protection as evidenced by memory B and long-lived plasma cell responses and challenge studies 9 months later. In two models of ZIKV infection during pregnancy, GAd-Zvp prevents maternal-to-fetal transmission. The gorilla adenovirus-based vaccine platform encoding full-length prM and E genes is a promising candidate for preventing congenital ZIKV syndrome and possibly infection by other flaviviruses.

The memory B cell response consists of phenotypically distinct subsets that differ in their ability to respond upon antigen re-encounter. However, the pathways regulating the development and function of memory B cell subsets are poorly understood. Here, we show that CD62L and CD44 are progressively expressed on mouse memory B cells and identify transcriptionally and functionally distinct memory B cell subsets. Bcl6 is important in regulating memory B cell subset differentiation with overexpression of Bcl6 resulting in impaired CD62L+ memory B cell development. Bcl6 regulates memory B cell subset development through control of a network of genes, including Bcl2 and Zeb2. Overexpression of Zeb2 impairs the development of CD62L+ memory B cells. Importantly, CD62L is also differentially expressed on human memory B cells following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination and identifies phenotypically distinct populations. Together, these data indicate that CD62L expression marks functionally distinct memory B cell subsets.

Novel influenza A virus (IAV) strains elicit recall immune responses to conserved epitopes, making them favorable antigenic choices for universal influenza virus vaccines. Evaluating these immunogens requires a thorough understanding of the antigenic sites targeted by the polyclonal antibody (pAb) response, which single-particle electron microscopy (EM) can sensitively detect. In this study, we employ EM polyclonal epitope mapping (EMPEM) to extensively characterize the pAb response to hemagglutinin (HA) after H5N1 immunization in humans. Cross-reactive pAbs originating from memory B cells immediately bound the stem of HA and persisted for more than a year after vaccination. In contrast, de novo pAb responses to multiple sites on the head of HA, targeting previously determined key neutralizing sites on H5 HA, expanded after the second immunization and waned quickly. Thus, EMPEM provides a robust tool for comprehensively tracking the specificity and durability of immune responses elicited by novel universal influenza vaccine candidates.

Spondweni virus (SPOV) is the flavivirus that is most closely related to Zika virus (ZIKV). Although SPOV causes sporadic human infections in Africa, recently it was found in Culex mosquitoes in Haiti. To investigate the pathogenic spectrum of SPOV, we developed infection models in mice. Although two SPOV strains failed to cause disease in immunocompetent mice, each accumulated in the brain, spleen, eye, testis, and kidney when type I interferon signaling was blocked and unexpectedly caused infection, immune cell infiltration, and swelling in the ankle. In pregnant mice, SPOV replicated in the placenta and fetus but did not cause placental insufficiency or microcephaly. We identified human antibodies from ZIKV or DENV immune subjects that neutralized SPOV infection and protected against lethal challenge. Our experiments describe similarities and differences in clinical syndromes between SPOV and ZIKV and suggest that their serological relatedness has implications for antibody therapeutics and flavivirus vaccine development.

The development of an effective vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiologic agent of coronavirus disease 2019 (COVID-19), is a global priority. Here, we compare the protective capacity of intranasal and intramuscular delivery of a chimpanzee adenovirus-vectored vaccine encoding a prefusion stabilized spike protein (chimpanzee adenovirus [ChAd]-SARS-CoV-2-S) in Golden Syrian hamsters. Although immunization with ChAd-SARS-CoV-2-S induces robust spike-protein-specific antibodies capable of neutralizing the virus, antibody levels in serum are higher in hamsters vaccinated by an intranasal compared to intramuscular route. Accordingly, against challenge with SARS-CoV-2, ChAd-SARS-CoV-2-S-immunized hamsters are protected against less weight loss and have reduced viral infection in nasal swabs and lungs, and reduced pathology and inflammatory gene expression in the lungs, compared to ChAd-control immunized hamsters. Intranasal immunization with ChAd-SARS-CoV-2-S provides superior protection against SARS-CoV-2 infection and inflammation in the upper respiratory tract. These findings support intranasal administration of the ChAd-SARS-CoV-2-S candidate vaccine to prevent SARS-CoV-2 infection, disease, and possibly transmission.