The assembly of neutrophil inflammasomes during COVID-19 is mediated by type I interferons.

The severity of COVID-19 is linked to excessive inflammation. Neutrophils represent a critical arm of the innate immune response and are major mediators of inflammation, but their role in COVID-19 pathophysiology remains poorly understood. We conducted transcriptomic profiling of neutrophils obtained from patients with mild and severe COVID-19, as well as from SARS-CoV-2 infected mice, in comparison to non-infected healthy controls. In addition, we investigated the inflammasome formation potential in neutrophils from patients and mice upon SARS-CoV-2 infection. Transcriptomic analysis of polymorphonuclear cells (PMNs), consisting mainly of mature neutrophils, revealed a striking type I interferon (IFN-I) gene signature in severe COVID-19 patients, contrasting with mild COVID-19 and healthy controls. Notably, low-density granulocytes (LDGs) from severe COVID-19 patients exhibited an immature neutrophil phenotype and lacked this IFN-I signature. Moreover, PMNs from severe COVID-19 patients showed heightened nigericin-induced caspase1 activation, but reduced responsiveness to exogenous inflammasome priming. Furthermore, IFN-I emerged as a priming stimulus for neutrophil inflammasomes. These findings suggest a potential role for neutrophil inflammasomes in driving inflammation during severe COVID-19. Altogether, these findings open promising avenues for targeted therapeutic interventions to mitigate the pathological processes associated with the disease.

Fibrinolysis associated proteins and lipopolysaccharide bioactivity in plasma and cerebrospinal fluid in multiple sclerosis.

The coagulation cascade and fibrinolysis have links with neuroinflammation and increased activation of the coagulation system has been reported in MS patients. We quantified levels of D-dimer, tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) and the bioactivity of bacterial lipopolysaccharide (LPS) in cerebrospinal fluid (CSF) and plasma from newly diagnosed untreated MS patients and controls. These molecules showed multiple correlations with each other as well as with age, HLA-DRB1*15:01, body-mass-index and CSF IgG. Our results confirm previous findings of increased plasma PAI-1 and LPS in MS patients compared to controls indicating changes in platelet function and gut permeability in MS.

Maturing neutrophils of lower density associate with thrombocytopenia in Puumala orthohantavirus-caused hemorrhagic fever with renal syndrome.

Puumala orthohantavirus-caused hemorrhagic fever with renal syndrome (PUUV-HFRS) is characterized by strong neutrophil activation. Neutrophils are the most abundant immune cell type in the circulation and are specially equipped to rapidly respond to infections. They are more heterogenous than previously appreciated, with specific neutrophil subsets recently implicated in inflammation and immunosuppression. Furthermore, neutrophils can be divided based on their density to either low-density granulocytes (LDGs) or "normal density" polymorphonuclear cell (PMN) fractions. In the current study we aimed to identify and characterize the different neutrophil subsets in the circulation of PUUV-HFRS patients. PMNs exhibited an activation of antiviral pathways, while circulating LDGs were increased in frequency following acute PUUV-HFRS. Furthermore, cell surface marker expression analysis revealed that PUUV-associated LDGs are primarily immature and most likely reflect an increased neutrophil production from the bone marrow. Interestingly, both the frequency of LDGs and the presence of a "left shift" in blood associated with the extent of thrombocytopenia, one of the hallmarks of severe HFRS, suggesting that maturing neutrophils could play a role in disease pathogenesis. These results imply that elevated circulating LDGs might be a general finding in acute viral infections. However, in contrast to the COVID-19 associated LDGs described previously, the secretome of PUUV LDGs did not show significant immunosuppressive ability, which suggests inherent biological differences in the LDG responses that can be dependent on the causative virus or differing infection kinetics.

Innate lymphoid cells are activated in HFRS, and their function can be modulated by hantavirus-induced type I interferons.

Hantaviruses cause the acute zoonotic diseases hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Infected patients show strong systemic inflammation and immune cell activation. NK cells are highly activated in HFRS, suggesting that also other innate lymphoid cells (ILCs) might be responding to infection. Here, we characterized peripheral ILC responses, and measured plasma levels of soluble factors and plasma viral load, in 17 Puumala virus (PUUV)-infected HFRS patients. This revealed an increased frequency of ILC2 in patients, in particular the ILC2 lineage-committed c-Kitlo ILC2 subset. Patients' ILCs showed an activated profile with increased proliferation and displayed altered expression of several homing markers. How ILCs are activated during viral infection is largely unknown. When analyzing PUUV-mediated activation of ILCs in vitro we observed that this was dependent on type I interferons, suggesting a role for type I interferons-produced in response to virus infection-in the activation of ILCs. Further, stimulation of naïve ILC2s with IFN-ß affected ILC2 cytokine responses in vitro, causing decreased IL-5 and IL-13, and increased IL-10, CXCL10, and GM-CSF secretion. These results show that ILCs are activated in HFRS patients and suggest that the classical antiviral type I IFNs are involved in shaping ILC functions.

Nanobody engineering for SARS-CoV-2 neutralization and detection.

In response to the ongoing COVID-19 pandemic, the quest for coronavirus inhibitors has inspired research on a variety of small proteins beyond conventional antibodies, including robust single-domain antibody fragments, i.e., "nanobodies." Here, we explore the potential of nanobody engineering in the development of antivirals and diagnostic tools. Through fusion of nanobody domains that target distinct binding sites, we engineered multimodular nanobody constructs that neutralize wild-type SARS-CoV-2 and the Alpha and Delta variants at high potency, with IC50 values as low as 50 pM. Despite simultaneous binding to distinct epitopes, Beta and Omicron variants were more resistant to neutralization by the multimodular nanobodies, which highlights the importance of accounting for antigenic drift in the design of biologics. To further explore the applications of nanobody engineering in outbreak management, we present an assay based on fusions of nanobodies with fragments of NanoLuc luciferase that can detect sub-nanomolar quantities of the SARS-CoV-2 spike protein in a single step. Our work showcases the potential of nanobody engineering to combat emerging infectious diseases. IMPORTANCE: Nanobodies, small protein binders derived from the camelid antibody, are highly potent inhibitors of respiratory viruses that offer several advantages over conventional antibodies as candidates for specific therapies, including high stability and low production costs. In this work, we leverage the unique properties of nanobodies and apply them as building blocks for new therapeutic and diagnostic tools. We report ultra-potent SARS-CoV-2 inhibition by engineered nanobodies comprising multiple modules in structure-guided combinations and develop nanobodies that carry signal molecules, allowing rapid detection of the SARS-CoV-2 spike protein. Our results highlight the potential of engineered nanobodies in the development of effective countermeasures, both therapeutic and diagnostic, to manage outbreaks of emerging viruses.

Mucosal-Associated Invariant T Cells are not susceptible in vitro to SARS-CoV-2 infection but accumulate into the lungs of COVID-19 patients.

Prolonged T cell lymphopenia is common in COVID-19, caused by SARS-CoV-2. While the mechanisms of lymphopenia during COVID-19 remain elusive, it is especially pronounced in a specialized innate-like T cell population called Mucosal Associated Invariant T cells (MAITs). MAITs has been suggested to express Angiotensin-Converting Enzyme 2 (ACE2), which is the well-known cellular receptor for SARS-CoV-2. However, it is still unclear if SARS-CoV-2 can infect or affect MAIT cells directly. In this study, we performed multicolor flow cytometry on peripheral blood mononuclear cells obtained from COVID-19 patients to assess the frequencies of CD8+Vα7.2+CD161+ MAIT subsets at acute and convalescent disease phases. The susceptibility of MAITs and T cells to direct exposure by SARS-CoV-2 was analysed using cells isolated from healthy donor buffy coats by viability assays, virus-specific RT-PCR, and flow cytometry. In situ lung immunofluorescence was used to evaluate retention of T cells, especially MAIT cells, in lung tissues during acute COVID-19. Our study confirms previous reports indicating that circulating MAITs are activated, and their frequency is declined in patients with acute SARS-CoV-2 infection, whereas an accumulation of MAITs and T cells was seen in the lung tissue of individuals with fatal COVID-19. However, despite a fraction of MAITs found to express ACE2, no evidence for the susceptibility of MAITs for direct infection or activation by SARS-CoV-2 particles was observed. Thus, their activation and decline in the circulation is most likely explained by indirect mechanisms involving other immune cells and cytokine-induced pro-inflammatory environment but not by direct exposure to viral particles at the infection site.

Image-based and machine learning-guided multiplexed serology test for SARS-CoV-2.

We present a miniaturized immunofluorescence assay (mini-IFA) for measuring antibody response in patient blood samples. The method utilizes machine learning-guided image analysis and enables simultaneous measurement of immunoglobulin M (IgM), IgA, and IgG responses against different viral antigens in an automated and high-throughput manner. The assay relies on antigens expressed through transfection, enabling use at a low biosafety level and fast adaptation to emerging pathogens. Using severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as the model pathogen, we demonstrate that this method allows differentiation between vaccine-induced and infection-induced antibody responses. Additionally, we established a dedicated web page for quantitative visualization of sample-specific results and their distribution, comparing them with controls and other samples. Our results provide a proof of concept for the approach, demonstrating fast and accurate measurement of antibody responses in a research setup with prospects for clinical diagnostics.

Complete Protection from SARS-CoV-2 Lung Infection in Mice Through Combined Intranasal Delivery of PIKfyve Kinase and TMPRSS2 Protease Inhibitors.

Emerging variants of concern of SARS-CoV-2 can significantly reduce the prophylactic and therapeutic efficacy of vaccines and neutralizing antibodies due to mutations in the viral genome. Targeting cell host factors required for infection provides a complementary strategy to overcome this problem since the host genome is less susceptible to variation during the life span of infection. The enzymatic activities of the endosomal PIKfyve phosphoinositide kinase and the serine protease TMPRSS2 are essential to meditate infection in two complementary viral entry pathways. Simultaneous inhibition in cultured cells of their enzymatic activities with the small molecule inhibitors apilimod dimesylate and nafamostat mesylate synergistically prevent viral entry and infection of native SARS-CoV-2 and vesicular stomatitis virus (VSV)-SARS-CoV-2 chimeras expressing the SARS-CoV-2 surface spike (S) protein and of variants of concern. We now report prophylactic prevention of lung infection in mice intranasally infected with SARS-CoV-2 beta by combined intranasal delivery of very low doses of apilimod dimesylate and nafamostat mesylate, in a formulation that is stable for over 3 months at room temperature. Administration of these drugs up to 6 hours post infection did not inhibit infection of the lungs but substantially reduced death of infected airway epithelial cells. The efficiency and simplicity of formulation of the drug combination suggests its suitability as prophylactic or therapeutic treatment against SARS-CoV-2 infection in households, point of care facilities, and under conditions where refrigeration would not be readily available.

Structural and mechanistic basis of neutralization by a pan-hantavirus protective antibody.

Emerging rodent-borne hantaviruses cause severe diseases in humans with no approved vaccines or therapeutics. We recently isolated a monoclonal broadly neutralizing antibody (nAb) from a Puumala virus-experienced human donor. Here, we report its structure bound to its target, the Gn/Gc glycoprotein heterodimer comprising the viral fusion complex. The structure explains the broad activity of the nAb: It recognizes conserved Gc fusion loop sequences and the main chain of variable Gn sequences, thereby straddling the Gn/Gc heterodimer and locking it in its prefusion conformation. We show that the nAb's accelerated dissociation from the divergent Andes virus Gn/Gc at endosomal acidic pH limits its potency against this highly lethal virus and correct this liability by engineering an optimized variant that sets a benchmark as a candidate pan-hantavirus therapeutic.

Signal peptide mimicry primes Sec61 for client-selective inhibition.

Preventing the biogenesis of disease-relevant proteins is an attractive therapeutic strategy, but attempts to target essential protein biogenesis factors have been hampered by excessive toxicity. Here we describe KZR-8445, a cyclic depsipeptide that targets the Sec61 translocon and selectively disrupts secretory and membrane protein biogenesis in a signal peptide-dependent manner. KZR-8445 potently inhibits the secretion of pro-inflammatory cytokines in primary immune cells and is highly efficacious in a mouse model of rheumatoid arthritis. A cryogenic electron microscopy structure reveals that KZR-8445 occupies the fully opened Se61 lateral gate and blocks access to the lumenal plug domain. KZR-8445 binding stabilizes the lateral gate helices in a manner that traps select signal peptides in the Sec61 channel and prevents their movement into the lipid bilayer. Our results establish a framework for the structure-guided discovery of novel therapeutics that selectively modulate Sec61-mediated protein biogenesis.

Intranasal administration of adenoviral vaccines expressing SARS-CoV-2 spike protein improves vaccine immunity in mouse models.

The ongoing SARS-CoV-2 pandemic is controlled but not halted by public health measures and mass vaccination strategies which have exclusively relied on intramuscular vaccines. Intranasal vaccines can prime or recruit to the respiratory epithelium mucosal immune cells capable of preventing infection. Here we report a comprehensive series of studies on this concept using various mouse models, including HLA class II-humanized transgenic strains. We found that a single intranasal (i.n.) dose of serotype-5 adenoviral vectors expressing either the receptor binding domain (Ad5-RBD) or the complete ectodomain (Ad5-S) of the SARS-CoV-2 spike protein was effective in inducing i) serum and bronchoalveolar lavage (BAL) anti-spike IgA and IgG, ii) robust SARS-CoV-2-neutralizing activity in the serum and BAL, iii) rigorous spike-directed T helper 1 cell/cytotoxic T cell immunity, and iv) protection of mice from a challenge with the SARS-CoV-2 beta variant. Intramuscular (i.m.) Ad5-RBD or Ad5-S administration did not induce serum or BAL IgA, and resulted in lower neutralizing titers in the serum. Moreover, prior immunity induced by an intramuscular mRNA vaccine could be potently enhanced and modulated towards a mucosal IgA response by an i.n. Ad5-S booster. Notably, Ad5 DNA was found in the liver or spleen after i.m. but not i.n. administration, indicating a lack of systemic spread of the vaccine vector, which has been associated with a risk of thrombotic thrombocytopenia. Unlike in otherwise genetically identical HLA-DQ6 mice, in HLA-DQ8 mice Ad5-RBD vaccine was inferior to Ad5-S, suggesting that the RBD fragment does not contain a sufficient collection of helper-T cell epitopes to constitute an optimal vaccine antigen. Our data add to previous promising preclinical results on intranasal SARS-CoV-2 vaccination and support the potential of this approach to elicit mucosal immunity for preventing transmission of SARS-CoV-2.

Risk of lymphoid malignancies increased after Puumala virus infection in Finland, 2009-2019: A retrospective register-based cohort study.

OBJECTIVES: The Puumala virus (PUUV) is a hantavirus that causes hemorrhagic fever with renal syndrome. Studies showing an increased risk of lymphoid malignancies after hantavirus infection, together with the observation that PUUV infects B cells, motivated us to study the risk of lymphoid malignancies after PUUV infection. METHODS: We linked data from the Finnish Cancer Registry and National Infectious Diseases Register for 2009-2019. We used a time-dependent Cox regression model to evaluate the hazard of the lymphoid malignancies grouped according to the HAEMACARE classification. RESULTS: We identified 68 cases of lymphoid malignancies after PUUV infection among 16,075 PUUV-infected individuals during 61,114,826 person-years of observation. A total of 10 cases occurred within 3-<12 months and 38 within 1-<5 years after PUUV infection, and the risk of lymphoid malignancies increased with hazard ratios (HRs) of 2.0 (95% confidence interval [CI], 1.1-3.7) and 1.6 (95% CI, 1.2-2.3), respectively. The group of mature B cell neoplasms showed an increased risk 3-<12 months and 1-<5 years after PUUV infection, HR 2.2 (95% CI, 1.2-4.3) and HR 1.8 (95% CI, 1.3-2.5), respectively. CONCLUSION: PUUV infection is associated with lymphoid malignancies in the Finnish population, supporting the earlier studies. Further research is required to understand the pathophysiological mechanisms behind this association.

Intranasal trimeric sherpabody inhibits SARS-CoV-2 including recent immunoevasive Omicron subvariants.

The emergence of increasingly immunoevasive SARS-CoV-2 variants emphasizes the need for prophylactic strategies to complement vaccination in fighting the COVID-19 pandemic. Intranasal administration of neutralizing antibodies has shown encouraging protective potential but there remains a need for SARS-CoV-2 blocking agents that are less vulnerable to mutational viral variation and more economical to produce in large scale. Here we describe TriSb92, a highly manufacturable and stable trimeric antibody-mimetic sherpabody targeted against a conserved region of the viral spike glycoprotein. TriSb92 potently neutralizes SARS-CoV-2, including the latest Omicron variants like BF.7, XBB, and BQ.1.1. In female Balb/c mice intranasal administration of just 5 or 50 micrograms of TriSb92 as early as 8 h before but also 4 h after SARS-CoV-2 challenge can protect from infection. Cryo-EM and biochemical studies reveal triggering of a conformational shift in the spike trimer as the inhibitory mechanism of TriSb92. The potency and robust biochemical properties of TriSb92 together with its resistance against viral sequence evolution suggest that TriSb92 could be useful as a nasal spray for protecting susceptible individuals from SARS-CoV-2 infection.

Puumala Hantavirus Infections Show Extensive Variation in Clinical Outcome.

The clinical outcome of Puumala hantavirus (PUUV) infection shows extensive variation, ranging from inapparent subclinical infection (70-80%) to severe hemorrhagic fever with renal syndrome (HFRS), with about 0.1% of cases being fatal. Most hospitalized patients experience acute kidney injury (AKI), histologically known as acute hemorrhagic tubulointerstitial nephritis. Why this variation? There is no evidence that there would be more virulent and less virulent variants infecting humans, although this has not been extensively studied. Individuals with the human leukocyte antigen (HLA) alleles B*08 and DRB1*0301 are likely to have a severe form of the PUUV infection, and those with B*27 are likely to have a benign clinical course. Other genetic factors, related to the tumor necrosis factor (TNF) gene and the C4A component of the complement system, may be involved. Various autoimmune phenomena and Epstein-Barr virus infection are associated with PUUV infection, but hantavirus-neutralizing antibodies are not associated with lower disease severity in PUUV HFRS. Wide individual differences occur in ocular and central nervous system (CNS) manifestations and in the long-term consequences of nephropathia epidemica (NE). Numerous biomarkers have been detected, and some are clinically used to assess and predict the severity of PUUV infection. A new addition is the plasma glucose concentration associated with the severity of both capillary leakage, thrombocytopenia, inflammation, and AKI in PUUV infection. Our question, "Why this variation?" remains largely unanswered.

A comprehensive assessment of four whole blood stabilizers for flow-cytometric analysis of leukocyte populations.

Though cryopreservation of cell fractions is widely used in flow cytometry studies, whole blood cryopreservation is more challenging due to the presence of erythrocytes and effects of fixatives commonly used for preservation. Here, we evaluated and compared head-to-head the performance of four commercial whole blood cryopreservation kits; (1) Cytodelics, (2) Stable-Lyse V2 and Stable-Store V2 (SLSS-V2), (3) Proteomic stabilizer (PROT-1), and (4) Transfix. We found that PROT-1, Transfix, and Cytodelics maintained the distribution of major leukocyte subsets-granulocytes, T cells, natural killer cells, and B cells, on a comparable level to unpreserved samples, despite the attenuation of fluorescence intensities in flow cytometric assays. Moreover, these three stabilizers also maintained the activated phenotypes of neutrophils upon stimulation with N-formylmethionyl-leucyl-phenylalanine and lipopolysaccharides. The upregulation of adhesion molecules (CD11b), Fc receptors (CD16), and granule proteins (CD66b), as well as the shedding of surface L-selectin (CD62L), was conserved most efficiently in PROT-1 and Cytodelics when compared to samples only treated with erythrocyte lysing. However, none of the stabilizers provided a reliable detection of CCR7 for accurate quantification of T cell maturation stages. We also evaluated the performance of Cytodelics in longitudinal clinical samples obtained from acute COVID-19 patients, where it allowed reliable detection of lymphopenia and granulocyte expansion. These results support the feasibility of whole blood cryopreservation for immunophenotyping by flow cytometry, particularly in longitudinal studies. In conclusion, the performance of different stabilizers is variable and therefore the choice of stabilizers should depend on cell type of interest, as well as antibody clones and experimental design of each study.

Wild Red Deer (Cervus elaphus) Do Not Play a Role as Vectors or Reservoirs of SARS-CoV-2 in North-Eastern Poland.

Several studies reported a high prevalence of SARS-CoV-2 among white-tailed deer in North America. Monitoring cervids in all regions to better understand SARS-CoV-2 infection and circulation in other deer populations has been urged. To evaluate deer exposure and/or infection to/by SARS-CoV-2 in Poland, we sampled 90 red deer shot by hunters in five hunting districts in north-eastern Poland. Serum and nasopharyngeal swabs were collected, and then an immunofluorescent assay (IFA) to detect anti-SARS-CoV-2 antibodies was performed as well as real-time PCR with reverse transcription for direct virus detection. No positive samples were detected. There is no evidence of spillover of SARS-CoV-2 from the human to deer population in Poland.

Urinary extracellular vesicles carry multiple activators and regulators of coagulation.

Cells shape their extracellular milieu by secreting intracellular products into the environment including extracellular vesicles which are lipid-bilayer limited membrane particles. These vesicles carry out a range of functions, including regulation of coagulation, via multiple contributor mechanisms. Urinary extracellular vesicles are secreted by various cells, lining the urinary space, including the nephron and bladder. They are known to have procoagulant properties, however, the details of this function, beyond tissue factor are not well known. The aim of the study was to access the role of urinary extracellular vesicles in impacting coagulation upon supplementation to plasma. This could indicate their physiological function upon kidney injury or pathology. Supplementation to standard human plasma and plasmas deficient in various coagulation factors was used for this purpose, and calibrated automated thrombogram (CAT®) was the major technique applied. We found that these vesicles contain multiple coagulation-related factors, and their lipid composition affects coagulation activities of plasma upon supplementation. Remarkably, these vesicles can restore thrombin generation in FVII, FVIII, FIX and FXI -deficient plasmas. This study explores the multiple roles of urinary extracellular vesicles in coagulation in in vitro blood coagulation and implies their importance in its regulation by several mechanisms.

Neutralizing Antibody Titers in Hospitalized Patients with Acute Puumala Orthohantavirus Infection Do Not Associate with Disease Severity.

Nephropathia epidemica (NE), a mild form of haemorrhagic fever with renal syndrome (HFRS), is an acute febrile illness caused by Puumala orthohantavirus (PUUV). NE manifests typically with acute kidney injury (AKI), with a case fatality rate of about 0.1%. The treatment and management of hantavirus infections are mainly supportive, although neutralizing monoclonal antibodies and immune sera therapeutics are under investigation. In order to assess the potential use of antibody therapeutics in NE, we sought to determine the relationship between circulating PUUV neutralizing antibodies, PUUV nucleocapsid protein (N) IgG antibodies, and viral loads with markers of disease severity. The study included serum samples of extensively characterized patient cohorts (n = 116) from Tampere University Hospital, Finland. The results showed that upon hospitalization, most patients already had considerable neutralizing and anti-PUUV-N IgG antibody levels. However, contrary to expectations, neutralizing antibody titers from the first day of hospitalization did not appear to protect from AKI or correlate with more favorable disease outcomes. This indicates that further studies are needed to investigate the applicability of neutralizing antibodies as a therapy for hospitalized NE patients.

Experimental Infection of Mink with SARS-COV-2 Omicron Variant and Subsequent Clinical Disease.

We report an experimental infection of American mink with SARS-CoV-2 Omicron variant and show that mink remain positive for viral RNA for days, experience clinical signs and histopathologic changes, and transmit the virus to uninfected recipients. Preparedness is crucial to avoid spread among mink and spillover to human populations.

Human antibody recognizing a quaternary epitope in the Puumala virus glycoprotein provides broad protection against orthohantaviruses.

The rodent-borne hantavirus Puumala virus (PUUV) and related agents cause hemorrhagic fever with renal syndrome (HFRS) in humans. Other hantaviruses, including Andes virus (ANDV) and Sin Nombre virus, cause a distinct zoonotic disease, hantavirus cardiopulmonary syndrome (HCPS). Although these infections are severe and have substantial case fatality rates, no FDA-approved hantavirus countermeasures are available. Recent work suggests that monoclonal antibodies may have therapeutic utility. We describe here the isolation of human neutralizing antibodies (nAbs) against tetrameric Gn/Gc glycoprotein spikes from PUUV-experienced donors. We define a dominant class of nAbs recognizing the "capping loop" of Gn that masks the hydrophobic fusion loops in Gc. A subset of nAbs in this class, including ADI-42898, bound Gn/Gc complexes but not Gn alone, strongly suggesting that they recognize a quaternary epitope encompassing both Gn and Gc. ADI-42898 blocked the cell entry of seven HCPS- and HFRS-associated hantaviruses, and single doses of this nAb could protect Syrian hamsters and bank voles challenged with the highly virulent HCPS-causing ANDV and HFRS-causing PUUV, respectively. ADI-42898 is a promising candidate for clinical development as a countermeasure for both HCPS and HFRS, and its mode of Gn/Gc recognition informs the development of broadly protective hantavirus vaccines.