Structural and functional basis of VLDLR usage by Eastern equine encephalitis virus.

The very-low-density lipoprotein receptor (VLDLR) comprises eight LDLR type A (LA) domains and supports entry of distantly related alphaviruses, including Eastern equine encephalitis virus (EEEV) and Semliki Forest virus (SFV). Here, by resolving multiple cryo-electron microscopy structures of EEEV-VLDLR complexes and performing mutagenesis and functional studies, we show that EEEV uses multiple sites (E1/E2 cleft and E2 A domain) to engage more than one LA domain simultaneously. However, no single LA domain is necessary or sufficient to support efficient EEEV infection. Whereas all EEEV strains show conservation of two VLDLR-binding sites, the EEEV PE-6 strain and a few other EEE complex members feature a single amino acid substitution that enables binding of LA domains to an additional site on the E2 B domain. These structural and functional analyses informed the design of a minimal VLDLR decoy receptor that neutralizes EEEV infection and protects mice from lethal challenge.

Vertebrate-class-specific binding modes of the alphavirus receptor MXRA8.

MXRA8 is a receptor for chikungunya (CHIKV) and other arthritogenic alphaviruses with mammalian hosts. However, mammalian MXRA8 does not bind to alphaviruses that infect humans and have avian reservoirs. Here, we show that avian, but not mammalian, MXRA8 can act as a receptor for Sindbis, western equine encephalitis (WEEV), and related alphaviruses with avian reservoirs. Structural analysis of duck MXRA8 complexed with WEEV reveals an inverted binding mode compared with mammalian MXRA8 bound to CHIKV. Whereas both domains of mammalian MXRA8 bind CHIKV E1 and E2, only domain 1 of avian MXRA8 engages WEEV E1, and no appreciable contacts are made with WEEV E2. Using these results, we generated a chimeric avian-mammalian MXRA8 decoy-receptor that neutralizes infection of multiple alphaviruses from distinct antigenic groups in vitro and in vivo. Thus, different alphaviruses can bind MXRA8 encoded by different vertebrate classes with distinct engagement modes, which enables development of broad-spectrum inhibitors.

ZBTB46 coordinates angiogenesis and immunity to control tumor outcome.

Tumor angiogenesis and immunity show an inverse correlation in cancer progression and outcome1. Here, we report that ZBTB46, a repressive transcription factor and a widely accepted marker for classical dendritic cells (DCs)2,3, controls both tumor angiogenesis and immunity. Zbtb46 was downregulated in both DCs and endothelial cells by tumor-derived factors to facilitate robust tumor growth. Zbtb46 downregulation led to a hallmark pro-tumor microenvironment (TME), including dysfunctional vasculature and immunosuppressive conditions. Analysis of human cancer data revealed a similar association of low ZBTB46 expression with an immunosuppressive TME and a worse prognosis. In contrast, enforced Zbtb46 expression led to TME changes to restrict tumor growth. Mechanistically, Zbtb46-deficient endothelial cells were highly angiogenic, and Zbtb46-deficient bone marrow progenitors upregulated Cebpb and diverted the DC program to immunosuppressive myeloid lineage output, potentially explaining the myeloid lineage skewing phenomenon in cancer4. Conversely, enforced Zbtb46 expression normalized tumor vessels and, by suppressing Cebpb, skewed bone marrow precursors toward immunostimulatory myeloid lineage output, leading to an immune-hot TME. Remarkably, Zbtb46 mRNA treatment synergized with anti-PD1 immunotherapy to improve tumor management in preclinical models. These findings identify ZBTB46 as a critical factor for angiogenesis and for myeloid lineage skewing in cancer and suggest that maintaining its expression could have therapeutic benefits.

Pan-protective anti-alphavirus human antibodies target a conserved E1 protein epitope.

Alphaviruses are emerging, mosquito-transmitted pathogens that cause musculoskeletal and neurological disease in humans. Although neutralizing antibodies that inhibit individual alphaviruses have been described, broadly reactive antibodies that protect against both arthritogenic and encephalitic alphaviruses have not been reported. Here, we identify DC2.112 and DC2.315, two pan-protective yet poorly neutralizing human monoclonal antibodies (mAbs) that avidly bind to viral antigen on the surface of cells infected with arthritogenic and encephalitic alphaviruses. These mAbs engage a conserved epitope in domain II of the E1 protein proximal to and within the fusion peptide. Treatment with DC2.112 or DC2.315 protects mice against infection by both arthritogenic (chikungunya and Mayaro) and encephalitic (Venezuelan, Eastern, and Western equine encephalitis) alphaviruses through multiple mechanisms, including inhibition of viral egress and monocyte-dependent Fc effector functions. These findings define a conserved epitope recognized by weakly neutralizing yet protective antibodies that could be targeted for pan-alphavirus immunotherapy and vaccine design.

A SARS-CoV-2 Infection Model in Mice Demonstrates Protection by Neutralizing Antibodies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic with millions of human infections. One limitation to the evaluation of potential therapies and vaccines to inhibit SARS-CoV-2 infection and ameliorate disease is the lack of susceptible small animals in large numbers. Commercially available laboratory strains of mice are not readily infected by SARS-CoV-2 because of species-specific differences in their angiotensin-converting enzyme 2 (ACE2) receptors. Here, we transduced replication-defective adenoviruses encoding human ACE2 via intranasal administration into BALB/c mice and established receptor expression in lung tissues. hACE2-transduced mice were productively infected with SARS-CoV-2, and this resulted in high viral titers in the lung, lung pathology, and weight loss. Passive transfer of a neutralizing monoclonal antibody reduced viral burden in the lung and mitigated inflammation and weight loss. The development of an accessible mouse model of SARS-CoV-2 infection and pathogenesis will expedite the testing and deployment of therapeutics and vaccines.

A Single-Dose Intranasal ChAd Vaccine Protects Upper and Lower Respiratory Tracts against SARS-CoV-2.

The coronavirus disease 2019 pandemic has made deployment of an effective vaccine a global health priority. We evaluated the protective activity of a chimpanzee adenovirus-vectored vaccine encoding a prefusion stabilized spike protein (ChAd-SARS-CoV-2-S) in challenge studies with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and mice expressing the human angiotensin-converting enzyme 2 receptor. Intramuscular dosing of ChAd-SARS-CoV-2-S induces robust systemic humoral and cell-mediated immune responses and protects against lung infection, inflammation, and pathology but does not confer sterilizing immunity, as evidenced by detection of viral RNA and induction of anti-nucleoprotein antibodies after SARS-CoV-2 challenge. In contrast, a single intranasal dose of ChAd-SARS-CoV-2-S induces high levels of neutralizing antibodies, promotes systemic and mucosal immunoglobulin A (IgA) and T cell responses, and almost entirely prevents SARS-CoV-2 infection in both the upper and lower respiratory tracts. Intranasal administration of ChAd-SARS-CoV-2-S is a candidate for preventing SARS-CoV-2 infection and transmission and curtailing pandemic spread.

Cryo-EM Structure of Chikungunya Virus in Complex with the Mxra8 Receptor.

Mxra8 is a receptor for multiple arthritogenic alphaviruses that cause debilitating acute and chronic musculoskeletal disease in humans. Herein, we present a 2.2 Å resolution X-ray crystal structure of Mxra8 and 4 to 5 Å resolution cryo-electron microscopy reconstructions of Mxra8 bound to chikungunya (CHIKV) virus-like particles and infectious virus. The Mxra8 ectodomain contains two strand-swapped Ig-like domains oriented in a unique disulfide-linked head-to-head arrangement. Mxra8 binds by wedging into a cleft created by two adjacent CHIKV E2-E1 heterodimers in one trimeric spike and engaging a neighboring spike. Two binding modes are observed with the fully mature VLP, with one Mxra8 binding with unique contacts. Only the high-affinity binding mode was observed in the complex with infectious CHIKV, as viral maturation and E3 occupancy appear to influence receptor binding-site usage. Our studies provide insight into how Mxra8 binds CHIKV and creates a path for developing alphavirus entry inhibitors.

Natural Killer Cells Control Tumor Growth by Sensing a Growth Factor.

Many tumors produce platelet-derived growth factor (PDGF)-DD, which promotes cellular proliferation, epithelial-mesenchymal transition, stromal reaction, and angiogenesis through autocrine and paracrine PDGFRß signaling. By screening a secretome library, we found that the human immunoreceptor NKp44, encoded by NCR2 and expressed on natural killer (NK) cells and innate lymphoid cells, recognizes PDGF-DD. PDGF-DD engagement of NKp44 triggered NK cell secretion of interferon gamma (IFN)-γ and tumor necrosis factor alpha (TNF-α) that induced tumor cell growth arrest. A distinctive transcriptional signature of PDGF-DD-induced cytokines and the downregulation of tumor cell-cycle genes correlated with NCR2 expression and greater survival in glioblastoma. NKp44 expression in mouse NK cells controlled the dissemination of tumors expressing PDGF-DD more effectively than control mice, an effect enhanced by blockade of the inhibitory receptor CD96 or CpG-oligonucleotide treatment. Thus, while cancer cell production of PDGF-DD supports tumor growth and stromal reaction, it concomitantly activates innate immune responses to tumor expansion.

Structural Basis of Zika Virus-Specific Antibody Protection.

Zika virus (ZIKV) infection during pregnancy has emerged as a global public health problem because of its ability to cause severe congenital disease. Here, we developed six mouse monoclonal antibodies (mAbs) against ZIKV including four (ZV-48, ZV-54, ZV-64, and ZV-67) that were ZIKV specific and neutralized infection of African, Asian, and American strains to varying degrees. X-ray crystallographic and competition binding analyses of Fab fragments and scFvs defined three spatially distinct epitopes in DIII of the envelope protein corresponding to the lateral ridge (ZV-54 and ZV-67), C-C' loop (ZV-48 and ZV-64), and ABDE sheet (ZV-2) regions. In vivo passive transfer studies revealed protective activity of DIII-lateral ridge specific neutralizing mAbs in a mouse model of ZIKV infection. Our results suggest that DIII is targeted by multiple type-specific antibodies with distinct neutralizing activity, which provides a path for developing prophylactic antibodies for use in pregnancy or designing epitope-specific vaccines against ZIKV.

A potently neutralizing SARS-CoV-2 antibody inhibits variants of concern by utilizing unique binding residues in a highly conserved epitope.

With the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with increased transmissibility and potential resistance, antibodies and vaccines with broadly inhibitory activity are needed. Here, we developed a panel of neutralizing anti-SARS-CoV-2 monoclonal antibodies (mAbs) that bound the receptor binding domain of the spike protein at distinct epitopes and blocked virus attachment to its host receptor, human angiotensin converting enzyme-2 (hACE2). Although several potently neutralizing mAbs protected K18-hACE2 transgenic mice against infection caused by ancestral SARS-CoV-2 strains, others induced escape variants in vivo or lost neutralizing activity against emerging strains. One mAb, SARS2-38, potently neutralized all tested SARS-CoV-2 variants of concern and protected mice against challenge by multiple SARS-CoV-2 strains. Structural analysis showed that SARS2-38 engaged a conserved epitope proximal to the receptor binding motif. Thus, treatment with or induction of neutralizing antibodies that bind conserved spike epitopes may limit the loss of potency of therapies or vaccines against emerging SARS-CoV-2 variants.

Profiling B cell immunodominance after SARS-CoV-2 infection reveals antibody evolution to non-neutralizing viral targets.

Dissecting the evolution of memory B cells (MBCs) against SARS-CoV-2 is critical for understanding antibody recall upon secondary exposure. Here, we used single-cell sequencing to profile SARS-CoV-2-reactive B cells in 38 COVID-19 patients. Using oligo-tagged antigen baits, we isolated B cells specific to the SARS-CoV-2 spike, nucleoprotein (NP), open reading frame 8 (ORF8), and endemic human coronavirus (HCoV) spike proteins. SARS-CoV-2 spike-specific cells were enriched in the memory compartment of acutely infected and convalescent patients several months post symptom onset. With severe acute infection, substantial populations of endemic HCoV-reactive antibody-secreting cells were identified and possessed highly mutated variable genes, signifying preexisting immunity. Finally, MBCs exhibited pronounced maturation to NP and ORF8 over time, especially in older patients. Monoclonal antibodies against these targets were non-neutralizing and non-protective in vivo. These findings reveal antibody adaptation to non-neutralizing intracellular antigens during infection, emphasizing the importance of vaccination for inducing neutralizing spike-specific MBCs.

Broadly Neutralizing Alphavirus Antibodies Bind an Epitope on E2 and Inhibit Entry and Egress.

We screened a panel of mouse and human monoclonal antibodies (MAbs) against chikungunya virus and identified several with inhibitory activity against multiple alphaviruses. Passive transfer of broadly neutralizing MAbs protected mice against infection by chikungunya, Mayaro, and O'nyong'nyong alphaviruses. Using alanine-scanning mutagenesis, loss-of-function recombinant proteins and viruses, and multiple functional assays, we determined that broadly neutralizing MAbs block multiple steps in the viral lifecycle, including entry and egress, and bind to a conserved epitope on the B domain of the E2 glycoprotein. A 16 Å resolution cryo-electron microscopy structure of a Fab fragment bound to CHIKV E2 B domain provided an explanation for its neutralizing activity. Binding to the B domain was associated with repositioning of the A domain of E2 that enabled cross-linking of neighboring spikes. Our results suggest that B domain antigenic determinants could be targeted for vaccine or antibody therapeutic development against multiple alphaviruses of global concern.

Affinity-Restricted Memory B Cells Dominate Recall Responses to Heterologous Flaviviruses.

Memory B cells (MBCs) can respond to heterologous antigens either by molding new specificities through secondary germinal centers (GCs) or by selecting preexisting clones without further affinity maturation. To distinguish these mechanisms in flavivirus infections and immunizations, we studied recall responses to envelope protein domain III (DIII). Conditional deletion of activation-induced cytidine deaminase (AID) between heterologous challenges of West Nile, Japanese encephalitis, Zika, and dengue viruses did not affect recall responses. DIII-specific MBCs were contained mostly within the plasma-cell-biased CD80+ subset, and few GCs arose following heterologous boosters, demonstrating that recall responses are confined by preexisting clonal diversity. Measurement of monoclonal antibody (mAb) binding affinity to DIII proteins, timed AID deletion, single-cell RNA sequencing, and lineage tracing experiments point to selection of relatively low-affinity MBCs as a mechanism to promote diversity. Engineering immunogens to avoid this MBC diversity may facilitate flavivirus-type-specific vaccines with minimized potential for infection enhancement.

Human Antibodies Targeting Influenza B Virus Neuraminidase Active Site Are Broadly Protective.

Influenza B virus (IBV) infections can cause severe disease in children and the elderly. Commonly used antivirals have lower clinical effectiveness against IBV compared to influenza A viruses (IAV). Neuraminidase (NA), the second major surface protein on the influenza virus, is emerging as a target of broadly protective antibodies that recognize the NA active site of IAVs. However, similarly broadly protective antibodies against IBV NA have not been identified. Here, we isolated and characterized human monoclonal antibodies (mAbs) that target IBV NA from an IBV-infected patient. Two mAbs displayed broad and potent capacity to inhibit IBV NA enzymatic activity, neutralize the virus in vitro, and protect against lethal IBV infection in mice in prophylactic and therapeutic settings. These mAbs inserted long CDR-H3 loops into the NA active site, engaging residues highly conserved among IBV NAs. These mAbs provide a blueprint for the development of improved vaccines and therapeutics against IBVs.

Structure of Venezuelan equine encephalitis virus in complex with the LDLRAD3 receptor.

LDLRAD3 is a recently defined attachment and entry receptor for Venezuelan equine encephalitis virus (VEEV)1, a New World alphavirus that causes severe neurological disease in humans. Here we present near-atomic-resolution cryo-electron microscopy reconstructions of VEEV virus-like particles alone and in a complex with the ectodomains of LDLRAD3. Domain 1 of LDLRAD3 is a low-density lipoprotein receptor type-A module that binds to VEEV by wedging into a cleft created by two adjacent E2-E1 heterodimers in one trimeric spike, and engages domains A and B of E2 and the fusion loop in E1. Atomic modelling of this interface is supported by mutagenesis and anti-VEEV antibody binding competition assays. Notably, VEEV engages LDLRAD3 in a manner that is similar to the way that arthritogenic alphaviruses bind to the structurally unrelated MXRA8 receptor, but with a much smaller interface. These studies further elucidate the structural basis of alphavirus-receptor interactions, which could inform the development of therapies to mitigate infection and disease against multiple members of this family.

In vivo monoclonal antibody efficacy against SARS-CoV-2 variant strains.

Rapidly emerging SARS-CoV-2 variants jeopardize antibody-based countermeasures. Although cell culture experiments have demonstrated a loss of potency of several anti-spike neutralizing antibodies against variant strains of SARS-CoV-21-3, the in vivo importance of these results remains uncertain. Here we report the in vitro and in vivo activity of a panel of monoclonal antibodies (mAbs), which correspond to many in advanced clinical development by Vir Biotechnology, AbbVie, AstraZeneca, Regeneron and Lilly, against SARS-CoV-2 variant viruses. Although some individual mAbs showed reduced or abrogated neutralizing activity in cell culture against B.1.351, B.1.1.28, B.1.617.1 and B.1.526 viruses with mutations at residue E484 of the spike protein, low prophylactic doses of mAb combinations protected against infection by many variants in K18-hACE2 transgenic mice, 129S2 immunocompetent mice and hamsters, without the emergence of resistance. Exceptions were LY-CoV555 monotherapy and LY-CoV555 and LY-CoV016 combination therapy, both of which lost all protective activity, and the combination of AbbVie 2B04 and 47D11, which showed a partial loss of activity. When administered after infection, higher doses of several mAb cocktails protected in vivo against viruses with a B.1.351 spike gene. Therefore, many-but not all-of the antibody products with Emergency Use Authorization should retain substantial efficacy against the prevailing variant strains of SARS-CoV-2.

LDLRAD3 is a receptor for Venezuelan equine encephalitis virus.

Venezuelan equine encephalitis virus (VEEV) is a neurotropic alphavirus transmitted by mosquitoes that causes encephalitis and death in humans1. VEEV is a biodefence concern because of its potential for aerosol spread and the current lack of sufficient countermeasures. The host factors that are required for VEEV entry and infection remain poorly characterized. Here, using a genome-wide CRISPR-Cas9-based screen, we identify low-density lipoprotein receptor class A domain-containing 3 (LDLRAD3)-a highly conserved yet poorly characterized member of the scavenger receptor superfamily-as a receptor for VEEV. Gene editing of mouse Ldlrad3 or human LDLRAD3 results in markedly reduced viral infection of neuronal cells, which is restored upon complementation with LDLRAD3. LDLRAD3 binds directly to VEEV particles and enhances virus attachment and internalization into host cells. Genetic studies indicate that domain 1 of LDLRAD3 (LDLRAD3(D1)) is necessary and sufficient to support infection by VEEV, and both anti-LDLRAD3 antibodies and an LDLRAD3(D1)-Fc fusion protein block VEEV infection in cell culture. The pathogenesis of VEEV infection is abrogated in mice with deletions in Ldlrad3, and administration of LDLRAD3(D1)-Fc abolishes disease caused by several subtypes of VEEV, including highly virulent strains. The development of a decoy-receptor fusion protein suggests a strategy for the prevention of severe VEEV infection and associated disease in humans.

Characterization of the SARS-CoV-2 BA.5.5 and BQ.1.1 Omicron variants in mice and hamsters.

The continued evolution and emergence of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have resulted in challenges to vaccine and antibody efficacy. The emergence of each new variant necessitates the need to re-evaluate and refine animal models used for countermeasure testing. Here, we tested a recently circulating SARS-CoV-2 Omicron lineage variant, BQ.1.1, in multiple rodent models including K18-human ACE2 (hACE2) transgenic, C57BL/6J, and 129S2 mice, and Syrian golden hamsters. In contrast to a previously dominant BA.5.5 Omicron variant, inoculation of K18-hACE2 mice with BQ.1.1 resulted in substantial weight loss, a characteristic seen in pre-Omicron variants. BQ.1.1 also replicated to higher levels in the lungs of K18-hACE2 mice and caused greater lung pathology than the BA.5.5 variant. However, in C57BL/6J mice, 129S2 mice, and Syrian hamsters, BQ.1.1 did not cause increased respiratory tract infection or disease compared to animals administered BA.5.5. Moreover, the rates of direct contact or airborne transmission in hamsters were not significantly different after BQ.1.1 and BA.5.5 infections. Taken together, these data suggest that the BQ.1.1 Omicron variant has increased virulence in rodent species that express hACE2, possibly due to the acquisition of unique spike mutations relative to earlier Omicron variants. IMPORTANCE As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, there is a need to rapidly assess the efficacy of vaccines and antiviral therapeutics against newly emergent variants. To do so, the commonly used animal models must also be re-evaluated. Here, we determined the pathogenicity of the BQ.1.1 SARS-CoV-2 variant in multiple SARS-CoV-2 animal models including transgenic mice expressing human ACE2 (hACE2), two strains of conventional laboratory mice, and Syrian hamsters. While BQ.1.1 and BA.5.5 infection resulted in similar levels of viral burden and clinical disease in hamsters and the conventional strains of laboratory mice tested, increases in lung infection were detected in hACE2-expressing transgenic mice, which corresponded with greater levels of pro-inflammatory cytokines and lung pathology. Taken together, our data highlight important differences in two closely related Omicron SARS-CoV-2 variant strains and provide a foundation for evaluating countermeasures.

The secreted protein Cowpox Virus 14 contributes to viral virulence and immune evasion by engaging Fc-gamma-receptors.

The genome of cowpoxvirus (CPXV) could be considered prototypical for orthopoxviridae (OXPV) since it contains many open reading frames (ORFs) absent or lost in other OPXV, including vaccinia virus (VACV). These additional ORFs are non-essential for growth in vitro but are expected to contribute to the broad host range, virulence and immune evasion characteristics of CPXV. For instance, unlike VACV, CPXV encodes proteins that interfere with T cell stimulation, either directly or by preventing antigen presentation or co-stimulation. When studying the priming of naïve T cells, we discovered that CPXV, but not VACV, encodes a secreted factor that interferes with activation and proliferation of naïve CD8+ and CD4+ T cells, respectively, in response to anti-CD3 antibodies, but not to other stimuli. Deletion mapping revealed that the inhibitory protein is encoded by CPXV14, a small secreted glycoprotein belonging to the poxvirus immune evasion (PIE) family and containing a smallpoxvirus encoded chemokine receptor (SECRET) domain that mediates binding to chemokines. We demonstrate that CPXV14 inhibition of antibody-mediated T cell activation depends on the presence of Fc-gamma receptors (FcγRs) on bystander cells. In vitro, CPXV14 inhibits FcγR-activation by antigen/antibody complexes by binding to FcγRs with high affinity and immobilized CPXV14 can trigger signaling through FcγRs, particularly the inhibitory FcγRIIB. In vivo, CPXV14-deleted virus showed reduced viremia and virulence resulting in reduced weight loss and death compared to wildtype virus whereas both antibody and CD8+ T cell responses were increased in the absence of CPXV14. Furthermore, no impact of CPXV14-deletion on virulence was observed in mice lacking the inhibitory FcγRIIB. Taken together our results suggest that CPXV14 contributes to virulence and immune evasion by binding to host FcγRs.

Antigen-guided depletion of anti-HLA antibody-producing cells by HLA-Fc fusion proteins.

Platelet transfusion and transplantation of allogeneic stem cells and solid organs are life-saving therapies. Unwanted alloantibodies to nonself human leukocyte antigens (HLAs) on donor cells increase the immunological barrier to these therapies and are important causes of platelet transfusion refractoriness and graft rejection. Although the specificities of anti-HLA antibodies can be determined at the allelic level, traditional treatments for antibody-mediated rejection nonselectively suppress humoral immunity and are not universally successful. We designed HLA-Fc fusion proteins with a bivalent targeting module derived from extracellular domains of HLA and an Fc effector module from mouse IgG2a. We found that HLA-Fc with A2 (A2Fc) and B7 (B7Fc) antigens lowered HLA-A2- and HLA-B7-specific reactivities, respectively, in sera from HLA-sensitized patients. A2Fc and B7Fc bound to B-cell hybridomas bearing surface immunoglobulins with cognate specificities and triggered antigen-specific and Fc-dependent cytotoxicity in vitro. In immunodeficient mice carrying HLA-A2-specific hybridoma cells, A2Fc treatment lowered circulating anti-HLA-A2 levels, abolished the outgrowth of hybridoma cells, and prolonged survival compared with control groups. In an in vivo anti-HLA-A2-mediated platelet transfusion refractoriness model, A2Fc treatment mitigated refractoriness. These results support HLA-Fc being a novel strategy for antigen-specific humoral suppression to improve transfusion and transplantation outcomes. With the long-term goal of targeting HLA-specific memory B cells for desensitization, further studies of HLA-Fc's efficacy in immune-competent animal models are warranted.