No abstract

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.

The prevention and treatment of prevalent infectious diseases and tumors should benefit from improvements in the induction of antigen-specific T cell immunity. To assess the potential of antigen targeting to dendritic cells to improve immunity, we incorporated ovalbumin protein into a monoclonal antibody to the DEC-205 receptor, an endocytic receptor that is abundant on these cells in lymphoid tissues. Simultaneously, we injected agonistic α-CD40 antibody to mature the dendritic cells. We found that a single low dose of antibody-conjugated ovalbumin initiated immunity from the naive CD4+ and CD8+ T cell repertoire. Unexpectedly, the αDEC-205 antigen conjugates, given s.c., targeted to dendritic cells systemically and for long periods, and ovalbumin peptide was presented on MHC class I for 2 weeks. This was associated with stronger CD8+ T cell–mediated immunity relative to other forms of antigen delivery, even when the latter was given at a thousand times higher doses. In parallel, the mice showed enhanced resistance to an established rapidly growing tumor and to viral infection at a mucosal site. By better harnessing the immunizing functions of maturing dendritic cells, antibody-mediated antigen targeting via the DEC-205 receptor increases the efficiency of vaccination for T cell immunity, including systemic and mucosal resistance in disease models.

Identification of immune effectors and the post-translational modifications that control their activity is essential for dissecting mechanisms of immunity. Here we demonstrate that the antiviral activity of interferon-induced transmembrane protein 3 (IFITM3) is post-translationally regulated by S-palmitoylation. Large-scale profiling of palmitoylated proteins in a dendritic cell line using a chemical reporter strategy revealed over 150 lipid-modified proteins with diverse cellular functions, including innate immunity. We discovered that S-palmitoylation of IFITM3 on membrane-proximal cysteines controls its clustering in membrane compartments and its antiviral activity against influenza virus. The sites of S-palmitoylation are highly conserved among the IFITM family of proteins in vertebrates, which suggests that S-palmitoylation of these immune effectors may be an ancient post-translational modification that is crucial for host resistance to viral infections. The S-palmitoylation and clustering of IFITM3 will be important for elucidating its mechanism of action and for the design of antiviral therapeutics.Vertebrates have evolved sophisticated innate and adaptive mechanisms of immunity to combat microbial pathogens 1 . In response, viruses and pathogenic bacteria have acquired virulence factors that subvert or disarm host defenses 1 . For example, cellular membranes provide a simple barrier to infection, but viruses such as influenza virus have evolved specific proteins that fuse with membranes to allow viral replication inside host cells 2 . Alternatively, intracellular bacterial pathogens are taken up by phagocytic cells, but they then remodel cellular membranes to prevent their own degradation inside lysosomal compartments 3 . Cellular membranes are key interfaces for host resistance and prime targets for microbial virulence factors. We therefore performed large-scale profiling of palmitoylated proteins in phagocytic cells to identify membrane-associated proteins that contribute to immunity against microbial pathogens. We found that S-palmitoylation of interferon-induced transmembrane protein 3 (IFITM3) enhances its clustering in membranes * Correspondence and requests for materials should be addressed to H.C.H. hhang@rockefeller.edu. Author contributionsJ.S.Y. conceived the study, designed and performed experiments, interpreted data and co-wrote the manuscript; Y.-Y.Y. and G.C. synthesized reagents for palmitoylome profiling studies; B.M., C.B.L. and T.M.M. provided reagents and expertise on influenza virus infections; H.C.H. conceived the study, designed experiments, interpreted data and co-wrote the manuscript. Competing financial interestsThe authors declare no competing financial interests. Additional information RESULTS Proteomic analysis of palmitoylated proteins in DC2.4 cellsTo identify lipid-modified and membrane-associated proteins that may contribute to immune responses to microbial infections, we performed large-scale profiling of fatty-acylated proteins in the mouse DC line DC2.4 (ref. ...

After the emergence of pandemic influenza viruses in 1957, 1968, and 2009, existing seasonal viruses were observed to be replaced in the human population by the novel pandemic strains. We have previously hypothesized that the replacement of seasonal strains was mediated, in part, by a population-scale boost in antibodies specific for conserved regions of the hemagglutinin stalk and the viral neuraminidase. Numerous recent studies have shown the role of stalk-specific antibodies in neutralization of influenza viruses; the finding that stalk antibodies can effectively neutralize virus alters the existing dogma that influenza virus neutralization is mediated solely by antibodies that react with the globular head of the viral hemagglutinin. The present study explores the possibility that stalkspecific antibodies were boosted by infection with the 2009 H1N1 pandemic virus and that those antibodies could have contributed to the disappearance of existing seasonal H1N1 influenza virus strains. To study stalk-specific antibodies, we have developed chimeric hemagglutinin constructs that enable the measurement of antibodies that bind the hemagglutinin protein and neutralize virus but do not have hemagglutination inhibition activity. Using these chimeric hemagglutinin reagents, we show that infection with the 2009 pandemic H1N1 virus elicited a boost in titer of virus-neutralizing antibodies directed against the hemagglutinin stalk. In addition, we describe assays that can be used to measure influenza virus-neutralizing antibodies that are not detected in the traditional hemagglutination inhibition assay.cross-reactivity | cross-protection | subtype