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.

Hantavirus infection-induced B cell activation elevates free light chains levels in circulation.

In humans, orthohantaviruses can cause hemorrhagic fever with renal syndrome (HFRS) or hantavirus pulmonary syndrome (HPS). An earlier study reported that acute Andes virus HPS caused a massive and transient elevation in the number of circulating plasmablasts with specificity towards both viral and host antigens suggestive of polyclonal B cell activation. Immunoglobulins (Igs), produced by different B cell populations, comprise heavy and light chains; however, a certain amount of free light chains (FLCs) is constantly present in serum. Upregulation of FLCs, especially clonal species, associates with renal pathogenesis by fibril or deposit formations affecting the glomeruli, induction of epithelial cell disorders, or cast formation in the tubular network. We report that acute orthohantavirus infection increases the level of Ig FLCs in serum of both HFRS and HPS patients, and that the increase correlates with the severity of acute kidney injury in HFRS. The fact that the kappa to lambda FLC ratio in the sera of HFRS and HPS patients remained within the normal range suggests polyclonal B cell activation rather than proliferation of a single B cell clone. HFRS patients demonstrated increased urinary excretion of FLCs, and we found plasma cell infiltration in archival patient kidney biopsies that we speculate to contribute to the observed FLC excreta. Analysis of hospitalized HFRS patients' peripheral blood mononuclear cells showed elevated plasmablast levels, a fraction of which stained positive for Puumala virus antigen. Furthermore, B cells isolated from healthy donors were susceptible to Puumala virus in vitro, and the virus infection induced increased production of Igs and FLCs. The findings propose that hantaviruses directly activate B cells, and that the ensuing intense production of polyclonal Igs and FLCs may contribute to acute hantavirus infection-associated pathological findings.

Correction: Monocyte subset redistribution from blood to kidneys in patients with Puumala virus caused hemorrhagic fever with renal syndrome.

[This corrects the article DOI: 10.1371/journal.ppat.1009400.].

Monocyte subset redistribution from blood to kidneys in patients with Puumala virus caused hemorrhagic fever with renal syndrome.

Innate immune cells like monocytes patrol the vasculature and mucosal surfaces, recognize pathogens, rapidly redistribute to affected tissues and cause inflammation by secretion of cytokines. We previously showed that monocytes are reduced in blood but accumulate in the airways of patients with Puumala virus (PUUV) caused hemorrhagic fever with renal syndrome (HFRS). However, the dynamics of monocyte infiltration to the kidneys during HFRS, and its impact on disease severity are currently unknown. Here, we examined longitudinal peripheral blood samples and renal biopsies from HFRS patients and performed in vitro experiments to investigate the fate of monocytes during HFRS. During the early stages of HFRS, circulating CD14-CD16+ nonclassical monocytes (NCMs) that patrol the vasculature were reduced in most patients. Instead, CD14+CD16- classical (CMs) and CD14+CD16+ intermediate monocytes (IMs) were increased in blood, in particular in HFRS patients with more severe disease. Blood monocytes from patients with acute HFRS expressed higher levels of HLA-DR, the endothelial adhesion marker CD62L and the chemokine receptors CCR7 and CCR2, as compared to convalescence, suggesting monocyte activation and migration to peripheral tissues during acute HFRS. Supporting this hypothesis, increased numbers of HLA-DR+, CD14+, CD16+ and CD68+ cells were observed in the renal tissues of acute HFRS patients compared to controls. In vitro, blood CD16+ monocytes upregulated CD62L after direct exposure to PUUV whereas CD16- monocytes upregulated CCR7 after contact with PUUV-infected endothelial cells, suggesting differential mechanisms of activation and response between monocyte subsets. Together, our findings suggest that NCMs are reduced in blood, potentially via CD62L-mediated attachment to endothelial cells and monocytes are recruited to the kidneys during HFRS. Monocyte mobilization, activation and functional impairment together may influence the severity of disease in acute PUUV-HFRS.

Characterization of low-density granulocytes in COVID-19.

Severe COVID-19 is characterized by extensive pulmonary complications, to which host immune responses are believed to play a role. As the major arm of innate immunity, neutrophils are one of the first cells recruited to the site of infection where their excessive activation can contribute to lung pathology. Low-density granulocytes (LDGs) are circulating neutrophils, whose numbers increase in some autoimmune diseases and cancer, but are poorly characterized in acute viral infections. Using flow cytometry, we detected a significant increase of LDGs in the blood of acute COVID-19 patients, compared to healthy controls. Based on their surface marker expression, COVID-19-related LDGs exhibit four different populations, which display distinctive stages of granulocytic development and most likely reflect emergency myelopoiesis. Moreover, COVID-19 LDGs show a link with an elevated recruitment and activation of neutrophils. Functional assays demonstrated the immunosuppressive capacities of these cells, which might contribute to impaired lymphocyte responses during acute disease. Taken together, our data confirms a significant granulocyte activation during COVID-19 and suggests that granulocytes of lower density play a role in disease progression.

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.

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.

Urokinase plasminogen activator mediates changes in human astrocytes modeling fragile X syndrome.

The function of astrocytes intertwines with the extracellular matrix, whose neuron and glial cell-derived components shape neuronal plasticity. Astrocyte abnormalities have been reported in the brain of the mouse model for fragile X syndrome (FXS), the most common cause of inherited intellectual disability, and a monogenic cause of autism spectrum disorder. We compared human FXS and control astrocytes generated from human induced pluripotent stem cells and we found increased expression of urokinase plasminogen activator (uPA), which modulates degradation of extracellular matrix. Several pathways associated with uPA and its receptor function were activated in FXS astrocytes. Levels of uPA were also increased in conditioned medium collected from FXS hiPSC-derived astrocyte cultures and correlated inversely with intracellular Ca2+ responses to activation of L-type voltage-gated calcium channels in human astrocytes. Increased uPA augmented neuronal phosphorylation of TrkB within the docking site for the phospholipase-Cγ1 (PLCγ1), indicating effects of uPA on neuronal plasticity. Gene expression changes during neuronal differentiation preceding astrogenesis likely contributed to properties of astrocytes with FXS-specific alterations that showed specificity by not affecting differentiation of adenosine triphosphate (ATP)-responsive astrocyte population. To conclude, our studies identified uPA as an important regulator of astrocyte function and demonstrated that increased uPA in human FXS astrocytes modulated astrocytic responses and neuronal plasticity.

Serological and molecular findings during SARS-CoV-2 infection: the first case study in Finland, January to February 2020.

The first case of coronavirus disease (COVID-19) in Finland was confirmed on 29 January 2020. No secondary cases were detected. We describe the clinical picture and laboratory findings 3-23 days since the first symptoms. The SARS-CoV-2/Finland/1/2020 virus strain was isolated, the genome showing a single nucleotide substitution to the reference strain from Wuhan. Neutralising antibody response appeared within 9 days along with specific IgM and IgG response, targeting particularly nucleocapsid and spike proteins.

Interferons Induce STAT1-Dependent Expression of Tissue Plasminogen Activator, a Pathogenicity Factor in Puumala Hantavirus Disease.

Hantaviruses are zoonotic viruses that show various degrees of vasculopathy in humans. In this study, we analyzed the regulation of 2 fibrinolytic parameters, tissue plasminogen activator (tPA) and its physiological inhibitor, plasminogen activator inhibitor 1 (PAI-1), in Puumala hantavirus (PUUV)-infected patients and in human microvascular endothelial cells. We detected strong upregulation of tPA in the acute phase of illness and in PUUV-infected macaques and found the tPA level to positively correlate with disease severity. The median levels of PAI-1 during the acute stage did not differ from those during the recovery phase. In concordance, hantaviruses induced tPA but not PAI-1 in microvascular endothelial cells, and the induction was demonstrated to be dependent on type I interferon. Importantly, type I and II interferons directly upregulated tPA through signal transducer and activator of transcription 1 (STAT1), which regulated tPA gene expression via a STAT1-responsive enhancer element. These results suggest that tPA may be a general factor in the immunological response to viruses.

Vaccinia virus-free rescue of fluorescent replication-defective vesicular stomatitis virus and pseudotyping with Puumala virus glycoproteins for use in neutralization tests.

Puumala virus (PUUV) grows slowly in cell culture. To study antigenic properties of PUUV, an amenable method for their expression would be beneficial. To achieve this, a replication-defective recombinant vesicular stomatitis virus, rVSVΔG*EGFP, was rescued using BSRT7/5 and encephalomyocarditis virus (EMCV) internal ribosomal entry site (IRES)-enabled rescue plasmids. Using these particles, pseudotypes bearing PUUV Sotkamo strain glycoproteins were produced, with titres in the range 105-108, and were used in pseudotype focus reduction neutralization tests (pFRNTs) with neutralizing monoclonal antibodies and patient sera. The results were compared with those from orthodox focus reduction neutralization tests (oFRNTs) using native PUUV with the same samples and showed a strong positive correlation (rs = 0.82) between the methods. While developing the system we identified three amino acids which were mutated in the Vero E6 cell culture adapted PUUV prototype Sotkamo strain sequence, and changing these residues was critical for expression and neutralizing antibody binding of PUUV glycoproteins.

Food limitation constrains host immune responses to nematode infections.

Trade-offs in the allocation of finite-energy resources among immunological defences and other physiological processes are believed to influence infection risk and disease severity in food-limited wildlife populations. However, this prediction has received little experimental investigation. Here we test the hypothesis that food limitation impairs the ability of wild field voles (Microtus agrestis) to mount an immune response against parasite infections. We conducted a replicated experiment on vole populations maintained in large outdoor enclosures during boreal winter, using food supplementation and anthelmintic treatment of intestinal nematodes. Innate immune responses against intestinal parasite infections were compared between food-supplemented and non-supplemented voles. Voles with high food availability mounted stronger immune responses against intestinal nematode infections than food-limited voles. No food effects were seen in immune responses to intracellular coccidian parasites, possibly owing to their ability to avoid activation of innate immune pathways. Our findings demonstrate that food availability constrains vole immune responses against nematode infections, and support the concept that spatio-temporal heterogeneity in food availability creates variation in infectious disease susceptibility.

Acute hantavirus infection induces galectin-3-binding protein.

Hantaviruses are zoonotic viruses that cause life-threatening diseases when transmitted to humans. Severe hantavirus infection is manifested by impairment of renal function, pulmonary oedema and capillary leakage. Both innate and adaptive immune responses contribute to the pathogenesis, but the underlying mechanisms are not fully understood. Here, we showed that galectin-3-binding protein (Gal-3BP) was upregulated as a result of hantavirus infection both in vitro and in vivo. Gal-3BP is a secreted glycoprotein found in human serum, and increased Gal-3BP levels have been reported in chronic viral infections and in several types of cancer. Our in vitro experiments showed that, whilst Vero E6 cells (an African green monkey kidney cell line) constitutively expressed and secreted Gal-3BP, this protein was detected in primary human cells only as a result of hantavirus infection. Analysis of Gal-3BP levels in serum samples of cynomolgus macaques infected experimentally with hantavirus indicated that hantavirus infection induced Gal-3BP also in vivo. Finally, analysis of plasma samples collected from patients hospitalized because of acute hantavirus infection showed higher Gal-3BP levels during the acute than the convalescent phase. Furthermore, the Gal-3BP levels in patients with haemorrhagic fever with renal syndrome correlated with increased complement activation and with clinical variables reflecting the severity of acute hantavirus infection.

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.

Structural determinants of growth factor binding and specificity by VEGF receptor 2.

Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel formation through activation of three receptor tyrosine kinases, VEGFR-1, -2, and -3. The extracellular domain of VEGF receptors consists of seven immunoglobulin homology domains, which, upon ligand binding, promote receptor dimerization. Dimerization initiates transmembrane signaling, which activates the intracellular tyrosine kinase domain of the receptor. VEGF-C stimulates lymphangiogenesis and contributes to pathological angiogenesis via VEGFR-3. However, proteolytically processed VEGF-C also stimulates VEGFR-2, the predominant transducer of signals required for physiological and pathological angiogenesis. Here we present the crystal structure of VEGF-C bound to the VEGFR-2 high-affinity-binding site, which consists of immunoglobulin homology domains D2 and D3. This structure reveals a symmetrical 22 complex, in which left-handed twisted receptor domains wrap around the 2-fold axis of VEGF-C. In the VEGFs, receptor specificity is determined by an N-terminal alpha helix and three peptide loops. Our structure shows that two of these loops in VEGF-C bind to VEGFR-2 subdomains D2 and D3, while one interacts primarily with D3. Additionally, the N-terminal helix of VEGF-C interacts with D2, and the groove separating the two VEGF-C monomers binds to the D2/D3 linker. VEGF-C, unlike VEGF-A, does not bind VEGFR-1. We therefore created VEGFR-1/VEGFR-2 chimeric proteins to further study receptor specificity. This biochemical analysis, together with our structural data, defined VEGFR-2 residues critical for the binding of VEGF-A and VEGF-C. Our results provide significant insights into the structural features that determine the high affinity and specificity of VEGF/VEGFR interactions.

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.

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.

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.

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.