ISG15 deficiency restricts HIV-1 infection.

Type I interferons (IFN-Is) are a group of potent inflammatory and antiviral cytokines. They induce IFN stimulated genes (ISGs), which act as proinflammatory mediators, antiviral effectors, and negative regulators of the IFN-I signaling cascade itself. One such regulator is interferon stimulated gene 15 (ISG15). Humans with complete ISG15 deficiency express persistently elevated levels of ISGs, and consequently, exhibit broad spectrum resistance to viral infection. Here, we demonstrate that IFN-I primed fibroblasts derived from ISG15-deficient individuals are more resistant to infection with single-cycle HIV-1 compared to healthy control fibroblasts. Complementation with both wild-type (WT) ISG15 and ISG15ΔGG (incapable of ISGylation while retaining negative regulation activity) was sufficient to reverse this phenotype, restoring susceptibility to infection to levels comparable to WT cells. Furthermore, CRISPR-edited ISG15ko primary CD4+ T cells were less susceptible to HIV-1 infection compared to cells treated with non-targeting controls. Transcriptome analysis of these CRISPR-edited ISG15ko primary CD4+ T cells recapitulated the ISG signatures of ISG15 deficient patients. Taken together, we document that the increased broad-spectrum viral resistance in ISG15-deficiency also extends to HIV-1 and is driven by a combination of T-cell-specific ISGs, with both known and unknown functions, predicted to target HIV-1 replication at multiple steps.

Role of Immunoglobulin M and A Antibodies in the Neutralization of Severe Acute Respiratory Syndrome Coronavirus 2.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions of people globally. Virus infection requires the receptor-binding domain (RBD) of the spike protein. Although studies have demonstrated anti-spike and -RBD antibodies to be protective in animal models, and convalescent plasma as a promising therapeutic option, little is known about immunoglobulin isotypes capable of blocking infection. METHODS: We studied spike- and RBD-specific immunoglobulin isotypes in convalescent and acute plasma/serum samples using a multiplex bead assay. We also determined virus neutralization activities in plasma and serum samples, and purified immunoglobulin fractions using a vesicular stomatitis pseudovirus assay. RESULTS: Spike- and RBD-specific immunoglobulin (Ig) M, IgG1, and IgA1 were produced by all or nearly all subjects at variable levels and detected early after infection. All samples displayed neutralizing activity. Regression analyses revealed that IgM and IgG1 contributed most to neutralization, consistent with IgM and IgG fractions' neutralization potency. IgA also exhibited neutralizing activity, but with lower potency. CONCLUSION: IgG, IgM, and IgA are critical components of convalescent plasma used for treatment of coronavirus disease 2019 (COVID-19).

Role of IgM and IgA Antibodies in the Neutralization of SARS-CoV-2.

BACKGROUND: SARS-CoV-2 has infected millions of people globally. Virus infection requires the receptor-binding domain (RBD) of the spike protein. Although studies have demonstrated anti-spike and - RBD antibodies to be protective in animal models, and convalescent plasma as a promising therapeutic option, little is known about immunoglobulin (Ig) isotypes capable of blocking infection. METHODS: We studied spike- and RBD-specific Ig isotypes in convalescent and acute plasma/sera using a multiplex bead assay. We also determined virus neutralization activities in plasma, sera, and purified Ig fractions using a VSV pseudovirus assay. RESULTS: Spike- and RBD-specific IgM, IgG1, and IgA1 were produced by all or nearly all subjects at variable levels and detected early after infection. All samples displayed neutralizing activity. Regression analyses revealed that IgM and IgG1 contributed most to neutralization, consistent with IgM and IgG fractions' neutralization potency. IgA also exhibited neutralizing activity, but with lower potency. CONCLUSION: IgG, IgM and IgA are critical components of convalescent plasma used for COVID-19 treatment.

A High-Throughput Assay for Circulating Antibodies Directed Against the S Protein of Severe Acute Respiratory Syndrome Coronavirus 2.

More than 24 million infections with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were confirmed globally by September 2020. While polymerase chain reaction-based assays are used for diagnosis, there is a need for high-throughput, rapid serologic methods. A Luminex binding assay was developed and used to assess simultaneously the presence of coronavirus disease 2019 (COVID-19)-specific antibodies in human serum and plasma. Clear differentiation was achieved between specimens from infected and uninfected subjects, and a wide range of serum/plasma antibody levels was delineated in infected subjects. All 25 specimens from 18 patients with COVID-19 were positive in the assays with both the trimeric spike and the receptor-binding domain proteins. None of the 13 specimens from uninfected subjects displayed antibodies to either antigen. There was a highly statistically significant difference between the antibody levels of COVID-19-infected and -uninfected specimens (P < .0001). This high-throughput antibody assay is accurate, requires only 2.5 hours, and uses 5 ng of antigen per test.

A serological assay to detect SARS-CoV-2 seroconversion in humans.

SARS-Cov-2 (severe acute respiratory disease coronavirus 2), which causes Coronavirus Disease 2019 (COVID19) was first detected in China in late 2019 and has since then caused a global pandemic. While molecular assays to directly detect the viral genetic material are available for the diagnosis of acute infection, we currently lack serological assays suitable to specifically detect SARS-CoV-2 antibodies. Here we describe serological enzyme-linked immunosorbent assays (ELISA) that we developed using recombinant antigens derived from the spike protein of SARS-CoV-2. Using negative control samples representing pre-COVID 19 background immunity in the general adult population as well as samples from COVID19 patients, we demonstrate that these assays are sensitive and specific, allowing for screening and identification of COVID19 seroconverters using human plasma/serum as early as two days post COVID19 symptoms onset. Importantly, these assays do not require handling of infectious virus, can be adjusted to detect different antibody types and are amendable to scaling. Such serological assays are of critical importance to determine seroprevalence in a given population, define previous exposure and identify highly reactive human donors for the generation of convalescent serum as therapeutic. Sensitive and specific identification of coronavirus SARS-Cov-2 antibody titers may, in the future, also support screening of health care workers to identify those who are already immune and can be deployed to care for infected patients minimizing the risk of viral spread to colleagues and other patients.

A High Through-put Assay for Circulating Antibodies Directed against the S Protein of Severe Acute Respiratory Syndrome Corona virus 2.

BACKGROUND: More than one million infections with the severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) have been confirmed. While PCR-based assays are used for diagnosis, high through-put serologic methods are needed to detect antibodies for seroserveillance and for identification of seroconversion, potential plasma donors, and the nature of the immune response to this pathogen. METHODS: A Luminex binding assay was used to assess the presence of antibodies in human sera from COVID-19-infected and -uninfected individuals specific for two recombinant proteins of SARS-CoV-2. FINDINGS: Fluorochrome-labeled beads were coated with a recombinant soluble stabilized trimeric SARS-CoV-2 S protein ectodomain or its central portion, the receptor binding domain (RBD). Coated beads were incubated with sera, followed by incubation with biotinylated anti-human total Ig antibodies and phycoerythrin (PE)-labeled streptavidin. Readout using a Luminex analyzer clearly differentiated between sera of the infected and uninfected subject, delineating a wide range of serum antibody levels in infected subjects. INTERPRETATION: Antibody assays of sera can identify individuals who are infected with SARS-CoV-2 and have seroconverted, as well as subjects who have been infected and recovered. The use of the Luminex binding Ab assay has the advantage that it can be run in approximately 2.5 hours, uses very little antigen, and permits a high through-put of samples/day. FUNDING: NIAID contracts and grants, Department of Veterans Affairs grants, the Microbiology Laboratory Clinical Services, Translational Science Hub, and Personalized Virology Initiative, and Department of Medicine of Mount Sinai Health System and Icahn School of Medicine at Mount Sinai.

A serological assay to detect SARS-CoV-2 seroconversion in humans.

Here, we describe a serological enzyme-linked immunosorbent assay for the screening and identification of human SARS-CoV-2 seroconverters. This assay does not require the handling of infectious virus, can be adjusted to detect different antibody types in serum and plasma and is amenable to scaling. Serological assays are of critical importance to help define previous exposure to SARS-CoV-2 in populations, identify highly reactive human donors for convalescent plasma therapy and investigate correlates of protection.

HIV protease cleaves the antiviral m6A reader protein YTHDF3 in the viral particle.

N6-methyladenosine (m6A) is the most abundant HIV RNA modification but the interplay between the m6A reader protein YTHDF3 and HIV replication is not well understood. We found that knockout of YTHDF3 in human CD4+ T-cells increases infection supporting the role of YTHDF3 as a restriction factor. Overexpression of the YTHDF3 protein in the producer cells reduces the infectivity of the newly produced viruses. YTHDF3 proteins are incorporated into HIV particles in a nucleocapsid-dependent manner permitting the m6A reader protein to limit infection in the new target cell at the step of reverse transcription. Importantly, HIV protease cleaves the virion-incorporated full-length YTHDF3 protein, a process which is blocked by HIV protease inhibitors used to treat HIV infected patients. Mass-spectrometry confirmed the proteolytic processing of YTHDF3 in the virion. Thus, HIV protease cleaves the virion-encapsidated host m6A effector protein in addition to the viral polyproteins to ensure optimal infectivity of the mature virion.

Influenza A Virus Infection Damages Zebrafish Skeletal Muscle and Exacerbates Disease in Zebrafish Modeling Duchenne Muscular Dystrophy.

INTRODUCTION: Both genetic and infectious diseases can result in skeletal muscle degeneration, inflammation, pain, and/or weakness. Duchenne muscular dystrophy (DMD) is the most common congenital muscle disease. DMD causes progressive muscle wasting due to mutations in Dystrophin. Influenza A and B viruses are frequently associated with muscle complications, especially in children. Infections activate an immune response and immunosuppressant drugs reduce DMD symptoms. These data suggest that the immune system may contribute to muscle pathology. However, roles of the immune response in DMD and Influenza muscle complications are not well understood. Zebrafish with dmd mutations are a well-characterized model in which to study the molecular and cellular mechanisms of DMD pathology. We recently showed that zebrafish can be infected by human Influenza A virus (IAV). Thus, the zebrafish is a powerful system with which to ask questions about the etiology and mechanisms of muscle damage due to genetic and/or infectious diseases. METHODS: We infected zebrafish with IAV and assayed muscle tissue structure, sarcolemma integrity, cell-extracellular matrix (ECM) attachment, and molecular and cellular markers of inflammation in response to IAV infection alone or in the context of DMD. RESULTS: We find that IAV-infected zebrafish display mild muscle degeneration with sarcolemma damage and compromised ECM adhesion. An innate immune response is elicited in muscle in IAV-infected zebrafish: NFkB signaling is activated, pro-inflammatory cytokine expression is upregulated, and neutrophils localize to sites of muscle damage. IAV-infected dmd mutants display more severe muscle damage than would be expected from an additive effect of dmd mutation and IAV infection, suggesting that muscle damage caused by Dystrophin-deficiency and IAV infection is synergistic. DISCUSSION: These data demonstrate the importance of preventing IAV infections in individuals with genetic muscle diseases. Elucidating the mechanisms of immune-mediated muscle damage will not only apply to DMD and IAV, but also to other conditions where the immune system, inflammation, and muscle tissue are known to be affected, such as autoimmune diseases, cancer, and aging.