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Formerly a common childhood pathogen, mumps virus (MuV) remains active worldwide, despite relatively high vaccine coverage. MuV is thought to infect the upper respiratory tract before disseminating to other organs; however, the early cellular targets of MuV in vivo are unknown. To address this, we generated a green fluorescent protein (GFP)-tagged vaccine strain (JL5) of MuV to infect leukocytic cell lines and found that replication was greatest in monocytes. Infection of peripheral blood mononuclear cells (PBMCs) also showed that both JL5 and a circulating strain of MuV (Iowa 2006; genotype G), preferentially infected monocytes. Further, monocyte-derived macrophages showed high susceptibility to MuV, with genotype G infecting macrophages to a much greater extent. While mice are generally resistant to MuV infection, we inoculated immunocompetent Rosa26-tdTomato mice intranasally with a GFP and Cre recombinase tagged MuV to determine whether monocytes/macrophages are important targets in vivo. We observed a small population of tdTomato+ cells within the lungs, which included epithelial cells; however, the vast majority were alveolar macrophages (AMs). To validate these findings, we infected murine AMs isolated from Rosa26-tdTomato mice with the GFP and Cre recombinase tagged MuV and found that while MuV could enter AMs, as determined by tdTomato positivity, only a small percentage of these expressed GFP, suggesting that inhibition in murine cells occurs postentry. To translate these findings, we infected cells from human bronchoalveolar lavage fluid with MuV and found that most infected cells were AMs. These findings highlight the high susceptibility of AMs and provide a basis for early MuV pathogenesis and subsequent dissemination. [ABSTRACT FROM AUTHOR]

The therapeutic potential of gene editing technologies hinges on the development of safe and effective delivery methods. In this study, we developed a temperature-sensitive and less immunogenic Sendai virus (ts SeV) as a novel delivery vector for CRISPR-Cas9 and for efficient gene editing in sensitive human cell types with limited induction of an innate immune response. ts SeV demonstrates high transduction efficiency in human CD34+ hematopoietic stem and progenitor cells (HSPCs) including transduction of the CD34+/CD38-/CD45RA-/CD90+(Thy1+)/CD49fhigh stem cell enriched subpopulation. The frequency of CCR5 editing exceeded 90% and bi-allelic CCR5 editing exceeded 70% resulting in significant inhibition of HIV-1 infection in primary human CD14+ monocytes. These results demonstrate the potential of the ts SeV platform as a safe, efficient, and flexible addition to the current gene-editing tool delivery methods, which may help further expand the possibilities in personalized medicine and the treatment of genetic disorders. [ABSTRACT FROM AUTHOR]

We report the emergency development and application of a robust serologic test to evaluate acute and convalescent antibody responses to SARS-CoV-2 in Argentina. The assays, COVIDAR IgG and IgM, which were produced and provided for free to health authorities, private and public health institutions and nursing homes, use a combination of a trimer stabilized spike protein and the receptor binding domain (RBD) in a single enzyme-linked immunosorbent assay (ELISA) plate. Over half million tests have already been distributed to detect and quantify antibodies for multiple purposes, including assessment of immune responses in hospitalized patients and large seroprevalence studies in neighborhoods, slums and health care workers, which resulted in a powerful tool for asymptomatic detection and policy making in the country. Analysis of antibody levels and longitudinal studies of symptomatic and asymptomatic SARS-CoV-2 infections in over one thousand patient samples provided insightful information about IgM and IgG seroconversion time and kinetics, and IgM waning profiles. At least 35% of patients showed seroconversion within 7 days, and 95% within 45 days of symptoms onset, with simultaneous or close sequential IgM and IgG detection. Longitudinal studies of asymptomatic cases showed a wide range of antibody responses with median levels below those observed in symptomatic patients. Regarding convalescent plasma applications, a protocol was standardized for the assessment of end point IgG antibody titers with COVIDAR with more than 500 plasma donors. The protocol showed a positive correlation with neutralizing antibody titers, and was used for clinical trials and therapies across the country. Using this protocol, about 80% of convalescent donor plasmas were potentially suitable for therapies. Here, we demonstrate the importance of providing a robust and specific serologic assay for generating new information about antibody kinetics in infected individuals and mitigation policies to cope with pandemic needs. Author summary: The development of robust and specific serologic assays to detect antibodies to SARS-CoV-2 is essential to understand the pandemic evolution and establish mitigation strategies. Here, we report the emergency development, production and application of a versatile ELISA test for detecting antibodies against the whole spike protein and its receptor binding domain. Over half million tests have been freely distributed in public and private health institutions of Argentina for evaluating immune responses, convalescent plasma programs and for large seroprevalence studies in neighborhoods and health care workers. We are still learning how and when to use serologic testing in different epidemiological settings. This program allowed us to produce large amount of high quality data on antibody levels in symptomatic and asymptomatic SARS-CoV-2 infections and generate relevant information about IgM and IgG seroconversion time and kinetics. We also present standardized protocols for antibody quantification as guidance for convalescent donor plasma selection in hospitals throughout the country for compassionate use and clinical trials. Here, we provide a framework for generating widely available tools, protocols and information of antibody responses for pandemic management. [ABSTRACT FROM AUTHOR]

Viruses have evolved myriad strategies to exploit the translation machinery of host cells to potentiate their replication. However, how paramyxovirus (PMVs) modulate cellular translation for their own benefit has not been systematically examined. Utilizing puromycylation labeling, overexpression of individual viral genes, and infection with wild-type virus versus its gene-deleted counterpart, we found that PMVs significantly inhibit host cells' nascent peptide synthesis during infection, with the viral matrix being the primary contributor to this effect. Using the rNiV-NPL replicon system, we discovered that the viral matrix enhances viral protein translation without affecting viral mRNA transcription and suppresses host protein expression at the translational level. Polysome profile analysis revealed that the HPIV3 matrix promotes the association of viral mRNAs with ribosomes, thereby enhancing their translation efficiency during infection. Intriguingly, our NiV-Matrix interactome identified the core exon-junction complex (cEJC), critical for mRNA biogenesis, as a significant component that interacts with the paramyxoviral matrix predominantly in the cytoplasm. siRNA knockdown of eIF4AIII simulated the restriction of cellular functions by the viral matrix, leading to enhanced viral gene translation and a reduction in host protein synthesis. Moreover, siRNA depletion of cEJC resulted in a 2-3 log enhancement in infectious virus titer for various PMVs but not SARS-CoV-2, enterovirus D68, or influenza virus. Our findings characterize a host translational interference mechanism mediated by viral matrix and host cEJC interactions. We propose that the PMV matrix redirects ribosomes to translate viral mRNAs at the expense of host cell transcripts, enhancing viral replication, and thereby enhancing viral replication. These insights provide a deeper understanding of the molecular interactions between paramyxoviruses and host cells, highlighting potential targets for antiviral strategies., Competing Interests: DECLARATION OF INTERESTS The authors declare no competing interests.

The transformative potential of gene editing technologies hinges on the development of safe and effective delivery methods. In this study, we developed a temperature-sensitive and interferon-silent Sendai virus (ts SeV) as a novel delivery vector for CRISPR-Cas9 and for efficient gene editing in sensitive human cell types without inducing IFN responses. ts SeV demonstrates unprecedented transduction efficiency in human CD34+ hematopoietic stem and progenitor cells (HSPCs) including transduction of the CD34+/CD38-/CD45RA-/CD90+(Thy1+)/CD49f high stem cell enriched subpopulation. The frequency of CCR5 editing exceeded 90% and bi-allelic CCR5 editing exceeded 70% resulting in significant inhibition of HIV-1 infection in primary human CD14+ monocytes. These results demonstrate the potential of the ts SeV platform as a safe, efficient, and flexible addition to the current gene-editing tool delivery methods, which may help to further expand the possibilities in personalized medicine and the treatment of genetic disorders.

Importance: The COVID-19 pandemic remains a significant public health concern for the global population; the development and characterization of therapeutics, especially ones that are broadly effective, will continue to be essential as severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) variants emerge. Neutralizing monoclonal antibodies remain an effective therapeutic strategy to prevent virus infection and spread so long as they recognize and interact with circulating variants. The epitope and binding specificity of a neutralizing anti-SARS-CoV-2 Spike receptor-binding domain antibody clone against many SARS-CoV-2 variants of concern were characterized by generating antibody-resistant virions coupled with cryo-EM structural analysis and VSV-spike neutralization studies. This workflow can serve to predict the efficacy of antibody therapeutics against emerging variants and inform the design of therapeutics and vaccines., Competing Interests: A patent entitled "SARS-COV-2 antibodies and uses thereof" (WO2022 087393A1) has been filed by Icahn School of Medicine at Mount Sinai with D.T. and J.A.D. as inventors.

Rationale: SARS-CoV-2 entry into host cells is facilitated by endogenous and exogenous proteases that proteolytically activate the spike glycoprotein and antiproteases inhibiting this process. Understanding the key actors in viral entry is crucial for advancing knowledge of virus tropism, pathogenesis, and potential therapeutic targets., Objectives: We aimed to investigate the role of naïve serum and alpha-1-antitrypsin (AAT) in inhibiting protease-mediated SARS-CoV-2 entry and explore the implications of AAT deficiency on susceptibility to different SARS-CoV-2 variants., Findings: Our study demonstrates that naïve serum exhibits significant inhibition of SARS-CoV-2 entry, with AAT identified as the major serum protease inhibitor potently restricting entry. Using pseudoparticles, replication-competent pseudoviruses, and authentic SARS-CoV-2, we show that AAT inhibition occurs at low concentrations compared with those in serum and bronchoalveolar tissues, suggesting physiological relevance. Furthermore, sera from subjects with an AAT-deficient genotype show reduced ability to inhibit entry of both Wuhan-Hu-1 (WT) and B.1.617.2 (Delta) but exhibit no difference in inhibiting B.1.1.529 (Omicron) entry., Conclusions: AAT may have a variant-dependent therapeutic potential against SARS-CoV-2. Our findings highlight the importance of further investigating the complex interplay between proteases, antiproteases, and spike glycoprotein activation in SARS-CoV-2 and other respiratory viruses to identify potential therapeutic targets and improve understanding of disease pathogenesis.

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has led to over 760 million cases and >6.8 million deaths worldwide. We developed a panel of human neutralizing monoclonal antibodies (mAbs) targeting the SARS-CoV-2 Spike protein using Harbour H2L2 transgenic mice immunized with Spike receptor binding domain (RBD) (1). Representative antibodies from genetically-distinct families were evaluated for inhibition of replication-competent VSV expressing SARS-CoV-2 Spike (rcVSV-S) in place of VSV-G. One mAb (denoted FG-10A3) inhibited infection of all rcVSV-S variants; its therapeutically-modified version, STI-9167, inhibited infection of all tested SARS-CoV-2 variants, including Omicron BA.1 and BA.2, and limited virus proliferation in vivo (1). To characterize the binding specificity and epitope of FG-10A3, we generated mAb-resistant rcVSV-S virions and performed structural analysis of the antibody/antigen complex using cryo-EM. FG-10A3/STI-9167 is a Class 1 antibody that prevents Spike-ACE2 binding by engaging a region within the Spike receptor binding motif (RBM). Sequencing of mAb-resistant rcVSV-S virions identified F486 as a critical residue for mAb neutralization, with structural analysis revealing that both the variable heavy and light chains of STI-9167 bound the disulfide-stabilized 470-490 loop at the Spike RBD tip. Interestingly, substitutions at position 486 were later observed in emerging variants of concern BA.2.75.2 and XBB. This work provides a predictive modeling strategy to define the neutralizing capacity and limitations of mAb therapeutics against emerging SARS-CoV-2 variants., Importance: The COVID-19 pandemic remains a significant public health concern for the global population; development and characterization of therapeutics, especially ones that are broadly effective, will continue to be essential as SARS-CoV-2 variants emerge. Neutralizing monoclonal antibodies remain an effective therapeutic strategy to prevent virus infection and spread with the caveat that they interact with the circulating variants. The epitope and binding specificity of a broadly neutralizing anti-SARS-CoV-2 Spike RBD antibody clone against many SARS-CoV-2 VOC was characterized by generating antibody-resistant virions coupled with cryo-EM structural analysis. This workflow can serve to predict the efficacy of antibody therapeutics against emerging variants and inform the design of therapeutics and vaccines.

The global COVID-19 pandemic has mobilized efforts to develop vaccines and antibody-based therapeutics, including convalescent plasma therapy, that inhibit viral entry by inducing or transferring neutralizing antibodies (nAbs) against the SARS-CoV-2 spike glycoprotein (CoV2-S). However, rigorous efficacy testing requires extensive screening with live virus under onerous BSL3 conditions which limits high throughput screening of patient and vaccine sera. Myriad BSL-2 compatible surrogate virus neutralization assays (VNAs) have been developed to overcome this barrier. Yet, there is marked variability between VNAs and how their results are presented, making inter-group comparisons difficult. To address these limitations, we developed a standardized VNA using VSVΔG-based CoV-2-S pseudotyped particles (CoV2pp) that can be robustly produced at scale and generate accurate neutralizing titers within 18 hours post-infection. Our standardized CoV2pp VNA showed a strong positive correlation with CoV2-S ELISA and live virus neutralizations in confirmed convalescent patient sera. Three independent groups subsequently validated our standardized CoV2pp VNA (n>120). Our data show that absolute (abs) IC50, IC80, and IC90 values can be legitimately compared across diverse cohorts, highlight the substantial but consistent variability in neutralization potency across these cohorts, and support the use of absIC80 as a more meaningful metric for assessing the neutralization potency of vaccine or convalescent sera. Lastly, we used our CoV2pp in a screen to identify ultra-permissive 293T clones that stably express ACE2 or ACE2+TMPRSS2. When used in combination with our CoV2pp, we can now produce CoV2pp sufficient for 150,000 standardized VNA/week.