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.
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.
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.
INTRODUCTION: Intracranial atherosclerotic stenosis (ICAS) is a common cause of ischemic stroke. Stroke patients manifesting symptomatic ICAS often have poor prognoses, including worsened functional ...outcome, increased morbidity, and mortality. Despite risk factor management, ICAS exhibits a nonlinear rise in incidence with age, with an increase in unstable lipid-rich plaques. Understanding mechanisms of ICAS is crucial to improve clinical outcomes and reduce stroke incidence. Previous studies have demonstrated that intracranial arteries demonstrate different histopathologic profiles compared to extracranial arteries, likely lowering their risk of atherosclerosis. However, these studies are limited to healthy intracranial arteries. Comprehensive multiomic data examining diseased intracranial arteries are limited. METHODS: We performed bulk transcriptomic analysis in 4 tissues in a post-mortem brain dataset (N=304), with 34 patients with cerebral atherosclerosis confirmed via autopsy, 90 patients with peripheral atherosclerosis, and 180 controls. Cell type deconvolution was conducted to identify differential expression signatures in 7 neuronal and glial subtypes. To examine interferon response further in glial cells, we generated human induced pluripotent stem cell (hiPSC)-derived astrocytes treated with 3 doses of interferon and performed transcriptomic analysis. RESULTS: Cerebral atherosclerosis was significantly (pathway enrichment p<10e-9) associated with interferon signaling compared to peripheral atherosclerosis and controls. Glial cells particularly expressed patterns associated with cerebral atherosclerosis. Astrocytes, oligodendrocytes, excitatory neurons, and endothelial cells were most impacted. In hiPSC-derived astrocytes treated with interferon, we identified upregulation of interferon-inducible genes matching those found in intracranial atherosclerosis in the post-mortem brain, implicating glial reactivity to interferon-related stress. CONCLUSIONS: Our study implicates interferon signaling as critical within the development of cerebral atherosclerosis. Protection of the intracranial arteries from atherosclerosis may occur due to reduced expression of interferon-inducible genes. Targeted treatment of cerebral atherosclerosis should consider interferon-signaling modulators.
In this study we profiled vaccine-induced polyclonal antibodies as well as plasmablast-derived mAbs from individuals who received SARS-CoV-2 spike mRNA vaccine. Polyclonal antibody responses in ...vaccinees were robust and comparable to or exceeded those seen after natural infection. However, the ratio of binding to neutralizing antibodies after vaccination was greater than that after natural infection and, at the monoclonal level, we found that the majority of vaccine-induced antibodies did not have neutralizing activity. We also found a co-dominance of mAbs targeting the NTD and RBD of SARS-CoV-2 spike and an original antigenic-sin like backboost to spikes of seasonal human coronaviruses OC43 and HKU1. Neutralizing activity of NTD mAbs but not RBD mAbs against a clinical viral isolate carrying E484K as well as extensive changes in the NTD was abolished, suggesting that a proportion of vaccine-induced RBD binding antibodies may provide substantial protection against viral variants carrying single E484K RBD mutations.
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•Antibody responses after SARS-CoV-2 mRNA vaccination target RBD, NTD, and S2•SARS-CoV-2 mRNA vaccination induces a high rate of non-neutralizing antibodies•Crossreactive antibodies to seasonal β-coronaviruses are induced by vaccination•Variant mutation N501Y enhances affinity to human ACE2 while E484K reduces it
An analysis of mRNA vaccine-induced polyclonal antibodies and plasmablast-derived monoclonal antibodies from individuals vaccinated against SARS-CoV-2 identifies a high proportion of non-neutralizing antibodies and the induction of cross-reactive antibodies to seasonal coronaviruses and also maps the regions in the spike protein that are targeted, even among viral variants.
HIV is a devastating virus that causes a life-long infection and can lead to acquired immunodeficiency syndrome (AIDS). RNA modifications play important roles in regulating a wide range of biological ...processes linked to human health and disease. Humans also possess multileveled defenses against invading pathogens, such as Type I interferon (IFN), which is a potent antiviral cytokine that induces expression of IFN-stimulated genes (ISGs) to induce an antiviral state. Both RNA modifications and IFN signaling are proposed to play a major role in the biology of HIV infection.Several distinct RNA modifications have been identified in human cellular mRNA such as N6-methyladenosine (m6A) and pseudouridine (Ѱ). HIV RNA contains numerous m6A modifications, which are read by the YTHDF1-3 reader proteins. There remains some controversy about its impact on viral replication. It is unknown whether HIV has Ѱ modifications. We hypothesize that HIV engages several distinct parts of RNA modification machinery to optimize its replication without changing its genome. Our work demonstrates that YTHDF1-3 incorporate into HIV particles in a nucleocapsid-dependent manner, YTHDF3 incorporation into the viral particle limits the next round of infection by blocking reverse transcription, and YTHDF3 is cleaved by HIV protease in the viral particle, a process which is blocked by HIV protease inhibitors used to treat HIV-infected patients. We also identified seven Ѱ sites in HIV RNA, found that single Ѱ mutant viruses are less infectious than wildtype virus, and identified PUS1 and PUS3 to be potential writers of Ѱ on HIV RNA.ISGs act as antiviral effectors, but also as negative regulators of IFN signaling to prevent over-inflammation. ISG15 is one such negative regulator of IFN signaling. Individuals lacking ISG15 have elevated levels of ISGs and chronic mild auto-inflammation, which is sufficient to provide increased cellular resistance to viral infections. We hypothesize that mild auto-inflammation and deregulation of IFN responses may contribute to HIV control. IFN-primed cells from ISG15-deficient patients are less susceptible to HIV infection compared to wildtype cells, complementation with ISG15 rescues this phenotype, and transcriptome analysis of the primary CD4+ T cells with and without ISG15 and with and without IFN stimulation reveal a dedicated set of ISGs is sufficient to reduce susceptibility of primary CD4+ T cells to HIV infection.Overall, these studies demonstrate that RNA modifications and IFN signaling pathways play major roles in regulating HIV infection biology.
Abstract
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).
Immunoglobulin (Ig) M, IgG1, and IgA1 antibodies against severe acute respiratory syndrome coronavirus 2 spike glycoprotein and its receptor-binding domain are present in plasma from patients with convalescent coronavirus disease 2019 (COVID-19). IgG, IgM, and IgA contribute to virus neutralization, providing the basis for optimal selection of convalescent plasma for COVID-19 treatment.
Abstract
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.
Rapid serologic assays are needed to detect antibodies in individuals infected with or recovering from severe acute respiratory syndrome coronavirus 2. The assay described is advantageous in that it is highly accurate, requires only 2.5 hours, uses little antigen/test, and constitutes a high-throughput platform.
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