As the SARS-CoV-2 pandemic progressed, many monoclonal antibodies (mAbs) that neutralized infection against initial strains lost potency against later variants due to the accumulation of mutations in the spike protein. Nonetheless, some mAbs, including the parent of the therapeutically used sotrovimab, S309, remained protective in animals against Omicron variants despite reduced neutralizing potential, with inhibitory activity likely sustained by Fc-mediated effector functions. Here, we identify Fc variants of S309 that confer enhanced protection against SARS-CoV-2 infection in a humanized Fcγ receptor transgenic (Hu-FcγR Tg) mouse model of infection. Versions of S309 that are afucosylated (AFUC) and contain a G236A (GA) mutation in the Fc region showed increased binding to FcγRs IIA, IIIA, and IIIB and enhanced phagocytic activity in cell culture-based assays. Treatment with S309-GA-AFUC resulted in less viral burden, inflammation, and pulmonary ventilatory dysfunction in the lungs of Hu-FcγR Tg mice challenged with SARS-CoV-2 strains compared to the parental S309 mAb or a variant lacking Fc effector functions (S309-GRLR). The enhanced protection in the lung conferred by S309-GA-AFUC required trafficking of CCR2-expressing monocytes to reduce SARS-CoV-2 viral burden and lung injury. Flow cytometry and RNA sequencing analyses showed that compared to the parental S309 mAb, S309-GA-AFUC treatment reduced the inflammatory state and induced a reparative transcriptional signature in monocytes and interstitial macrophages. Overall, our findings demonstrate that Fc engineering to increase antibody binding to activating FcγRs can strengthen effector functions, shape myeloid transcriptional profiles, and enhance protection against SARS-CoV-2 infection in vivo. IMPORTANCE:Although therapeutic antibodies had success in protecting vulnerable individuals from severe COVID-19 during the early stages of the pandemic, many lost effectiveness as SARS-CoV-2 accumulated mutations that compromised neutralizing activity. Our experiments show that antibody protection against SARS-CoV-2 strains can be enhanced by genetically engineering the Fc region or altering its N-linked glycosylation to improve interactions with FcγRs on host immune cells. Modified versions of S309, the parent of the clinically used sotrovimab antibody, more effectively reduce viral burden and inflammation in the lung and shape protective transcriptional responses, which, together, result in improved lung ventilatory function and outcome after SARS-CoV-2 infection. Thus, antibody engineering can serve as a strategy to enhance therapeutic activity against rapidly evolving viruses with the potential to escape neutralization.

The current outbreak of the emerging arthropod-transmitted Oropouche virus (OROV) in South America has been epidemiologically linked to vertical transmissions, microcephaly, and stillbirths. Nevertheless, the impact of OROV infection during pregnancy has not been experimentally evaluated. To address how OROV infection might impact pregnancy outcome, we performed experiments in human cells and mice. Studies in cell cultures showed that the human trophoblast cell lines BeWo and JEG-3 are permissive to OROV infection (strain BeAn19991) and develop type I interferon (IFN)-dependent antiviral response. In our model, loss of type I IFN signaling in the dam resulted in the spread of virus to the placenta and fetus, whereas loss in the fetus alone was not sufficient to cause fetal infection. Collectively, our study shows that placental cells are susceptible to OROV infection and that the outcome for fetus depends on the integrity of the type I IFN immune response in the dam.

The emergence of SARS-CoV-2 Omicron variants has led to viral escape from many clinically approved monoclonal antibodies (mAbs) due to rapid evolution of the receptor-binding domain (RBD). Co-circulation of SARS-CoV-2 variants with unique sets of antigenic substitutions has further complicated therapeutic mAb discovery. New approaches are needed to rapidly discover and characterize mAbs with preferred specificity and functional characteristics. Here we describe and perform epitope-focused mAb discovery using glycan-masked antigens. We isolated and expressed a panel of 303 mAbs, some of which potently neutralize divergent Omicron subvariants by targeting the class 3 antigenic site on SARS-CoV-2 RBD. Epitope mapping of these antibodies revealed a spectrum of cross-reactivity and differential recognition of the class 3 site, validating the utility of this enrichment approach for targeted mAb discovery. Together, this work rationally designs glycan-masked engineered RBDs and uses them to isolate mAbs that potently neutralize antigenically divergent SARS-CoV-2 variants.

Venezuelan (VEEV), eastern (EEEV), and western (WEEV) equine encephalitis viruses are alphaviruses from different serocomplexes that cause neurological disease in humans. Given their antigenic distance, it has been challenging to isolate cross-reactive antibodies that neutralize infection by multiple medically relevant encephalitic alphaviruses. Recently, distinct entry receptors were identified for these encephalitic alphaviruses: LDLRAD3 for VEEV, VLDLR for EEEV and some strains of WEEV, and PCDH10 for WEEV. Here, using structure-guided mutagenesis, we generated a soluble chimeric protein derived from the LA1 domain of LDLRAD3 and the LA2 domain of VLDLR, termed LDLRAD3-vLA1-VLDLR-LA2, which neutralized infection by VEEV, EEEV, and some WEEV strains in cell culture and protected mice from infection. Structural analysis of this engineered decoy revealed binding to distinct sites on each virus, which corresponded to those engaged by their endogenous entry receptors. We extended the neutralizing and protective capacity to contemporary WEEV strains by adding a single extracellular cadherin domain from sparrow PCDH10 through a stabilized symmetric bispecific decoy scaffold. Our designed receptor decoy serves as a possible countermeasure against multiple encephalitic alphaviruses, and this design platform could be harnessed to develop therapeutic agents against viruses from other families.

BACKGROUND:: The glioblastoma (GBM) immunosuppressive tumor microenvironment is a clinical challenge. Oncolytic Zika virus (ZIKV) has emerged as a promising therapy, targeting treatment-resistant glioma stem cells, stimulating CD8+ T-cell-mediated immunity and extends survival in preclinical models but myeloid cell-driven immunosuppression persists. An antagonist of Siglec-15, a myeloid immune checkpoint molecule, is in a phase II trial for non-small cell lung cancer, but its role in CNS malignancies remains unclear. METHODS:: We evaluated Siglec-15 expression in human GBM samples using flow cytometry, mass cytometry, and immunofluorescence, as well as a public database. Using syngeneic glioma models, we tested a blocking antibody against Siglec-15, and Siglec-15 knock out mice, alongside ZIKV and anti-PD-1 therapies. We performed survival studies and analyzed immune responses, T-cell proliferation and phagocytosis, and tumor rechallenge. RESULTS:: Siglec-15 was expressed by human GBM myeloid (16-22%) and tumor (18-19%) cells, and higher expression was associated with shorter survival. In CT2A-bearing mice, ZIKV + anti-Siglec-15 increased long-term survival to 60% (vs. 40% with ZIKV alone), rising to 83% with anti-PD-1 treatment. Triple therapy in SB28 bearing mice yielded 76% long-term survivor rate with 1.7-fold higher CD8+ T-cell activation. Rechallenged mice showed 11-fold expansion of brain resident/effector memory CD8+ T-cells and 80% survival. Siglec-15 loss on myeloid cells enhanced phagocytosis (CT2A: 25%; SB28: 7%) and T-cell responses (activation: 81%; proliferation: 86.8%). CONCLUSION:: Targeting Siglec-15, combined with PD-1 blockade and ZIKV overcomes myeloid immunosuppression and enhances T-cell activation in GBM, promoting durable anti-tumor immunity. These findings support further investigation of this combination therapy.

BACKGROUND:Glioblastoma (GBM) is a lethal brain tumor with limited treatment options, largely due to profound immune suppression within the tumor microenvironment (TME), the failure of current immunotherapies to restore CD8+ T cell function, and persistence of glioma stem cells (GSCs) after treatment. Oncolytic Zika virus (ZIKV) is a promising therapeutic that selectively targets GSCs and remodels the TME to enhance anti-tumor CD8+ T cell responses. In this study we investigated how ZIKV efficacy in GBM is driven through monocytes. METHODS:We performed single-cell RNA sequencing and T cell receptor (TCR) sequencing to evaluate CD8+ T cell responses following ZIKV treatment. We used CellChat to define signaling networks between ZIKV-activated CCR2+ monocytes and CD8+ T cells in the TME. We used syngeneic, immunocompetent murine GBM models to validate mechanisms in vivo, applying genetic and antibody-based approaches to impair CCR2+ monocyte trafficking and function. RESULTS:ZIKV-induced clonal expansion of tumor-infiltrating CD8+ T cells enriched in granzyme B and perforin-1, with reduced expression of exhaustion markers. CCR2+ monocytes were essential for the recruitment, proliferation, and effector functions of anti-tumor CD8+ T cells in the TME. Disruption of monocyte trafficking or function impaired these responses, diminishing cytotoxic activity and T cell recruitment. CONCLUSIONS:ZIKV-driven activation and recruitment of CCR2+ monocytes supports robust anti-tumor CD8+ T cell responses by enhancing cytotoxicity and limiting exhaustion. These findings highlight the previously unappreciated therapeutic potential of modulating monocyte-T cell crosstalk to overcome immune suppression in GBM.

The protease TMPRSS2 facilitates coronavirus infections, yet its mechanism of viral glycoprotein recognition remains unclear. Here we show that, following ACE2 engagement of the SARS-CoV-2 spike (S) inducing the early fusion intermediate conformation (E-FIC), TMPRSS2 cleaves the R815 S2 ' site and promotes fusogenic conformational changes leading to viral entry. We unveil TMPRSS2 recognition of S2 ', identify key residues modulating binding specificity and demonstrate that S2 ' site-directed broadly neutralizing antibodies target E-FIC and inhibit viral entry by blocking TMPRSS2 access. We computationally designed stabilized E-FIC as a vaccine candidate, overcoming the transient nature of this state. We describe a TMPRSS2-directed monoclonal antibody inhibiting several coronaviruses, including SARS-CoV-2 variants and protecting mice against SARS-CoV-2 challenge. These results outline the mechanistic role of TMPRSS2 and S2 ' site-directed antibodies in coronavirus entry.

Functional gastrointestinal (GI) tract disorders affect a substantial proportion of the global population and are often preceded by intestinal infections that cause injury to enteric neurons and glia through unrestrained immune responses. However, the mechanisms that limit infection-induced inflammation and protect the enteric nervous system remain poorly understood. Here, we defined such neuron-glia-macrophage interactions after West Nile virus (WNV) infection; this model neurotropic virus causes GI tract dysmotility in mice via injury of enteric neurons through a T cell-mediated cytolytic mechanism. In response to WNV infection, RNA sequencing analysis showed that resident muscularis macrophages upregulate antiviral, proinflammatory, and immunomodulatory genes. Whereas pharmacological depletion of resident macrophages did not affect the viral burden in the GI tract, it instead reshaped the enteric glial response to WNV, resulting in excessive production of T cell and neutrophil chemoattractants. The amplified recruitment of these immune cell types worsened enteric neuronal injury. Together, our findings identify resident muscularis macrophages as key regulators of glia-driven inflammation during enteric viral infection and reveal their role in protecting enteric neurons from immune-mediated damage.

Encephalitic alphaviruses, including Eastern equine encephalitis virus (EEEV), cause severe neurological disease with high mortality rates, and thus are a public health threat. Although members of the low-density lipoprotein receptor (LDLR) family, including VLDLR, LRP8 (ApoER2), and LDLR recently were identified as receptors for EEEV, residual infection in receptor-deficient cells suggests that additional entry factors exist. Using a CRISPR-based activation screen, we identified LDLR-related protein 4 (LRP4) as a candidate entry factor for EEEV and several related alphaviruses (Western equine encephalitis, Semliki Forest, and Sindbis viruses). LRP4 mediates viral attachment and internalization, and its ligand-binding domain binds directly to virions. Soluble LRP4 decoy proteins potently inhibit EEEV infection in primary mouse neuronal cells, male mice, and human brain organoids, suggesting possible therapeutic applications. Mammalian and avian LRP4 orthologs demonstrate conserved functions in promoting EEEV infection, supporting a possible role in its host range of infection and transmission. Our findings establish LRP4 as a shared entry receptor for multiple alphaviruses and expand our understanding of alphavirus tropism, pathogenesis, and countermeasure development.

The continued evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has compromised neutralizing antibody responses elicited by prior infection or vaccination and abolished the utility of most monoclonal antibody therapeutics. We previously described a computationally-designed, homotrimeric miniprotein inhibitor, designated TRI2-2, that protects mice against pre-Omicron SARS-CoV-2 variants. Here, we show that TRI2-2 exhibits broadly neutralizing activity of SARS-CoV-2 variants and protects mice against BQ.1.1, XBB.1.5 and BA.2.86 challenge when administered intranasally post-exposure. The resistance of TRI2-2 to viral escape by most variants and the ability to deliver it directly to the upper airways highlight the potential of the multivalent miniprotein inhibitor as an alternative therapeutic modality.

Bile acids (BAs) a complex family of immunometabolites can shape myeloid cell function; their role in antiviral immunity and clinical outcomes in human infection remains unclear. We identified an early plasma specific BA signature that distinguished moderate from severe COVID-19 trajectories (M vs. SCHP) in >1,100 hospitalized patients. Single-cell RNA and ATAC sequencing of PBMCs identified a TGR5⁺ monocyte subset in MCHP characterized by increased chromatin accessibility and expression of interferon-responsive antiviral and MHC class II pathways, and reduced IL-10 signaling. External datasets confirmed enrichment of this program in MCHP and healthy donors. In TGR5⁺ monocytes of MCHP, these BAs synergized with IFN-γ to enhance STAT1 activation, ERK/CREB1 signaling, and H3K27ac; independently promoting trained immunity recall responses. We reveal a previously unrecognized antiviral axis whereby these specific BAs activate TGR5 to drive transcriptional and epigenetic programs associated with upregulation of antiviral responses, trained immunity, and favorable clinical outcomes in COVID-19

Oral antibiotics can predispose to joint inflammation, but this phenomenon remains poorly understood. Here, we leverage mouse models of alphavirus-induced arthritis to investigate the roles of gut commensals, metabolites, and host immune mechanisms in promoting musculoskeletal inflammation. Mice treated with a short course of oral antibiotics exhibited worsened arthritis after chikungunya (CHIKV) or Mayaro virus infections. This phenotype was associated with loss of short-chain fatty acids (SCFAs), greater intestinal permeability, and activation of gut-associated immune cells and required TLR4 signaling, MyD88 expression, monocytes, antigen-specific and bystander CD4 + T cells, and proinflammatory cytokines. Administration of exogenous SCFAs or colonization of mice with bacterial species that generate SCFAs mitigated CHIKV-induced joint inflammation. scRNA-seq revealed that gut-derived SCFAs ameliorate the inflammatory phenotype of synovial CD4 + T cells, infiltrating monocytes, and resident osteoclast-like cells. Thus, antibiotic-triggered gut dysbiosis exacerbates alphavirus arthritis by shaping the inflammatory profile of both infiltrating and resident immune cells in joint tissues.

Despite advances in immunotherapy, the prognosis for patients with glioblastoma (GBM) remains poor. The efficacy of GBM-targeted immunotherapies is limited by the paucity of functional T cells in the tumor microenvironment, a consequence of the local and systemic immunosuppression prevalent in patients with GBM. To overcome these challenges, here we develop a treatment strategy we term "expand and pull," which uses systemic administration of rhIL-7-hyFc, a long-acting recombinant human interleukin-7, to increase peripheral T cell abundance ("expand"), followed by intratumoral oncolytic virus treatment to recruit these cells to the tumor microenvironment ("pull"). We show that rhIL-7-hyFc improves the efficacy of multiple oncolytic viral therapies in syngeneic immuno-resistant mouse models of glioma. Combining rhIL-7-hyFc and Zika virus (ZIKV) increases systemic and intratumoral T cell abundance, improves cytotoxic T cell function, and delays expression of inhibitory checkpoint receptors, resulting in long-term tumor-free survival. We observe similar survival efficacy in experiments using a safer, genetically modified Δ10 3'-UTR ZIKV, as well as the clinically tested oncolytic adenovirus, Delta24-RGD. Collectively, our findings demonstrate that augmentation of both the systemic and local immune responses improves the utility of GBM-targeted immunotherapies.

Arenaviruses are divided into Old World (OW) and New World (NW) groups. OW arenaviruses enter cells through a pH-dependent receptor switch from a plasma-membrane factor to an endolysosomal receptor for subsequent membrane fusion, whereas clade B NW arenaviruses use transferrin receptor 1 without a secondary receptor. Using a vesicular stomatitis virus (VSV) chimera expressing the glycoprotein complex (GPC) of the clade A NW arenavirus Pichindé virus, we performed a genome-wide CRISPR loss-of-function screen and identified the endolysosomal sialomucin CD164 as an essential host factor. CD164 knockout cells were resistant to VSV chimeras bearing the GPCs of Pichindé, Paraná, and Flexal viruses, and to authentic Pichindé and Paraná virus, with susceptibility restored by complementation. The requirement mapped to the cysteine-rich domain of CD164, which bound GP1 in a pH-dependent manner through main-chain interactions. These findings define CD164 as an endolysosomal receptor for clade A NW arenaviruses expanding the receptor switching paradigm.

Chikungunya virus, a mosquito-borne alphavirus, causes outbreaks of both acute and chronic musculoskeletal diseases. Despite the recent approval of a live-attenuated and virus-like particle-based vaccine, a stable, safe and efficacious vaccine that can be manufactured at low cost is lacking. To address this need, we engineered Escherichia coli to produce robust biopolymer particles (BPs) densely coated with CHIKV envelope glycoproteins E2 and E1, forming a natively folded heterodimer mimicking the virus surface (E2-BP-E1). Native E2-E1 heterodimer formation was confirmed by monoclonal antibodies binding to five neutralizing epitopes and by binding of the receptor Mxra8. The structural model of BP-tethered E2-E1 aligned with the crystal structure of mature E2-E1 complex. In vitro, E2-BP-E1 activated dendritic cells (DCs) to produce Th1 cytokines, present MHC class I/II T cell epitopes, and stimulate CD4+ and CD8+ T cell proliferation. In vivo, vaccination without adjuvant induced potent neutralizing antibodies and protective immunity, with a ∼5 log10 reduction in viremia. Histological analysis of muscle and joints confirmed reduced inflammation and pathology in vaccinated mice. E2-BP-E1 was produced using standard E. coli fermentation suggesting safe, cost-effective and scalable manufacturability offering advantages over current vaccines. Overall, we developed a stable particulate CHIKV vaccine that is safe and efficiently protects against infection without the need of an adjuvant.

T cell immunity has a crucial role in vaccine-induced protection against respiratory viruses, yet a detailed characterization of T cell responses and epitopes in Syrian hamsters, a highly utilized preclinical, small animal model for SARS-CoV-2 research, is lacking. In this study, using an intranasal Chimpanzee adenoviral vectored vaccine (ChAd-SARS-CoV-2-S), we characterized the T cell response to the spike protein of SARS-CoV-2 in Syrian hamsters and identified immunogenic CD4+ and CD8+ T cell epitopes using IFN-γ ELISpot assays and cell depletions. The mucosal ChAd-SARS-CoV-2-S vaccine elicited strong T cell responses, with evidence of CD4+ and CD8+ T cell activation in both lymphoid and mucosal tissues. Responses were directed toward the non-receptor-binding domain regions of the spike protein, indicating that dominant T cell epitopes for hamsters reside elsewhere in this structural protein. Six different T cell epitopes (4 for CD4 and 2 for CD8) were identified in the spike protein, and epitope-specific responses were detected in hamsters from 2 vendors, suggesting genetic similarity in terms of major histocompatibility complex allele expression. Identifying T cell epitopes and characterizing T cell responses in lymphoid and mucosal compartments enhances the utility of Syrian hamsters as a preclinical model for SARS-CoV-2 vaccine studies.

The emergence of highly pathogenic avian H5N1 influenza viruses in dairy cows and humans has increased the potential for another pandemic. To address this risk, we developed chimpanzee adenoviral (ChAd)-vectored H5 hemagglutinin-targeted vaccines and tested their immunogenicity and efficacy in rodents. Immunization with ChAd-Texas (clade 2.3.4.4b) vaccine in mice elicits neutralizing antibody responses and confers protection against viral infection and mortality upon challenge with a human H5N1 isolate (A/Michigan/90/2024, clade 2.3.4.4b). Intranasal delivery of the ChAd-Texas vaccine elicits mucosal antibody and T cell responses and confers greater protection than intramuscular immunization. In Syrian hamsters, a single intranasal dose of ChAd-Texas vaccine prevents weight loss and reduces airway infection after H5N1 A/Michigan/90/2024 or A/Texas/37/2024 challenge. Importantly, prior seasonal influenza vaccination does not impair antibody responses or protection after intranasal delivery of the ChAd-Texas vaccine. These results support the development of mucosally administered ChAd-Texas HA vaccines as an effective platform for HPAI H5N1 preparedness.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to cause significant respiratory disease, particularly in vulnerable populations. Although ACE2 is the primary receptor for viral entry, previous studies have identified a naturally occurring, ACE2-independent entry pathway in certain airway-derived cell lines. Utilization of this pathway depends on surface heparan sulfates and requires the E484D substitution within the receptor-binding domain of the viral Spike (S) protein. In this study, we expand the panel of airway-derived cell lines that support ACE2-independent, S-E484D-dependent replication and identify the host lysosomal transmembrane protein TMEM106B as a critical host factor for infection. Knockout of TMEM106B completely abolishes infection by SARS-CoV-2 S E484D in NCI-H522 and NCI-H661 cells. Moreover, ectopic expression of the luminal C-terminal domain (CTD) of TMEM106B-either alone or redirected to the plasma membrane - is sufficient to enable viral entry and infection in otherwise non-permissive cells. We further show that Fc-TMEM106B-CTD decoy protein blocks infection by SARS-CoV-2 S E484D , supporting a direct interaction between the S-E484D protein and TMEM106B-CTD. Finally, passaging experiments with a chimeric VSV-SARS-CoV-2 S E484D identify additional mutations within the heptad repeat 1 that enhance TMEM106B utilization and viral spread in the ACE2-independent cell models. Together, these findings demonstrate that TMEM106B is a key mediator of a naturally occurring ACE2-independent pathway in multiple airway-derived cells lines and suggest that variation in the Spike protein can expand receptor usage by SARS-CoV-2.

The continued evolution of SARS-CoV-2 variants that evade immunity highlights a need to develop vaccines that elicit variant-specific antibodies and neutralize emerging strains. However, immune imprinting from antecedent SARS-CoV-2 exposure can limit the generation of such antibodies. Here, we evaluate strategies to enhance variant-specific antibody responses in female mice primed with Wuhan-1 spike-based mRNA or chimpanzee adenoviral-vectored (ChAd) vaccines and boosted with Omicron variant-matched vaccines. Altering the intramuscular injection site did not substantially affect variant-specific serum antibody responses. However, increasing booster antigen doses, performing repeated boosters, and administering booster vaccines intranasally enhanced variant-specific responses against the vaccine-matched Omicron strain. Boosting intranasally with a ChAd vaccine encoding the spike protein of Omicron XBB.1.5 elicited stronger XBB.1.5-specific responses in serum, bronchoalveolar lavage fluid, and draining lymph nodes than intramuscular boosting with the same vaccine. Regardless of booster regimen, neutralizing activity against XBB.1.5 was predominantly mediated by antibodies that were non-reactive to Wuhan-1 spike. These findings establish that in mice, intranasal or repeated variant-matched boosting can overcome the effects of imprinting and enhance immunity against SARS-CoV-2 strains.

Powassan virus (POWV) is a tick-borne orthoflavivirus that can cause severe neuroinvasive disease. There are no approved vaccines or therapeutics, and the incidence of POWV infection in humans is rising. Here, we isolated and characterized a panel of human monoclonal antibodies (mAbs) from a convalescent donor using single B-cell sorting with POWV EDIII and full-length E ectodomain as antigen bait. Binding and neutralization assays with both POWV reporter virus particles and an authentic strain identified multiple neutralizing mAbs that target distinct epitopes across the E glycoprotein, including EDIII lateral ridge/C-C' loop and non-EDIII regions. Four neutralizing mAbs were evaluated in a lethal mouse challenge, two of which conferred substantial protection. These findings define key targets of the human antibody response to POWV and highlight candidates for the development of human monoclonal antibody therapy for this emerging virus. IMPORTANCE:Powassan virus is an emerging tick-borne orthoflavivirus with steadily increasing case numbers. With an approximately 10% case-fatality rate and no approved therapeutics or vaccines available, POWV represents a potential public health threat. Our results showing that human monoclonal antibodies can protect mice against POWV in a lethal challenge model provide a foundation for developing future effective immunotherapies against this virus.