The emergence of SARS-CoV-1 in 2003 followed by MERS-CoV and now SARS-CoV-2 has proven the latent threat these viruses pose to humanity. While the SARS-CoV-2 pandemic has shifted to a stage of endemicity, the threat of new coronaviruses emerging from animal reservoirs remains. To address this issue, the global community must develop small molecule drugs targeting highly conserved structures in the coronavirus proteome. Here, we characterized existing drugs for their ability to inhibit the endoribonuclease activity of the SARS-CoV-2 non-structural protein 15 (nsp15) via in silico, in vitro, and in vivo techniques. We have identified nsp15 inhibition by the drugs pibrentasvir and atovaquone which effectively inhibit SARS-CoV-2 and HCoV-OC43 at low micromolar concentrations in cell cultures. Furthermore, atovaquone, but not pibrentasvir, is observed to modulate HCoV-OC43 dsRNA and infection in a manner consistent with nsp15 inhibition. Although neither pibrentasvir nor atovaquone translate to clinical efficacy in a murine prophylaxis model of SARS-CoV-2 infection, atovaquone may serve as a basis for the design of future nsp15 inhibitors.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a clear threat to humanity. It has infected over 200 million and killed 4 million people worldwide, and infections continue with no end in sight. To control the pandemic, multiple effective vaccines have been developed, and global vaccinations are in progress. However, the virus continues to mutate. Even when full vaccine coverage is achieved, vaccine-resistant mutants will likely emerge, thus requiring new annual vaccines against drifted variants analogous to influenza. A complimentary solution to this problem could be developing antiviral drugs that inhibit SARS CoV-2 and its drifted variants. Host defense peptides represent a potential source for such an antiviral as they possess broad antimicrobial activity and significant diversity across species. We screened the cathelicidin family of peptides from 16 different species for antiviral activity and identified a wild boar peptide derivative that inhibits SARS CoV-2. This peptide, which we named Yongshi and means warrior in Mandarin, acts as a viral entry inhibitor. Following the binding of SARS-CoV-2 to its receptor, the spike protein is cleaved, and heptad repeats 1 and 2 multimerize to form the fusion complex that enables the virion to enter the cell. A deep learning-based protein sequence comparison algorithm and molecular modeling suggest that Yongshi acts as a mimetic to the heptad repeats of the virus, thereby disrupting the fusion process. Experimental data confirm the binding of Yongshi to the heptad repeat 1 with a fourfold higher affinity than heptad repeat 2 of SARS-CoV-2. Yongshi also binds to the heptad repeat 1 of SARS-CoV-1 and MERS-CoV. Interestingly, it inhibits all drifted variants of SARS CoV-2 that we tested, including the alpha, beta, gamma, delta, kappa and omicron variants.

The association of seasonal trivalent influenza vaccine (TIV) with increased infection by 2009 pandemic H1N1 (A(H1N1)pdm09) virus, initially observed in Canada, has elicited numerous investigations on the possibility of vaccine-associated enhanced disease, but the potential mechanisms remain largely unresolved. Here, we investigated if prior immunization with TIV enhanced disease upon A(H1N1)pdm09 infection in mice. We found that A(H1N1)pdm09 infection in TIV-immunized mice did not enhance the disease, as measured by morbidity and mortality. Instead, TIV-immunized mice cleared A(H1N1)pdm09 virus and recovered at an accelerated rate compared to control mice. Prior TIV immunization was associated with potent inflammatory mediators and virus-specific CD8 T cell activation, but efficient immune regulation, partially mediated by IL-10R-signaling, prevented enhanced disease. Furthermore, in contrast to suggested pathological roles, pre-existing non-neutralizing antibodies (NNAbs) were not associated with enhanced virus replication, but rather with promoted antigen presentation through FcR-bearing cells that led to potent activation of virus-specific CD8 T cells. These findings provide new insights into interactions between pre-existing immunity and pandemic viruses.

Long-lived plasma cells are critical to humoral immunity as a lifelong source of protective antibodies. Antigen-activated B cells-with T-cell help-undergo affinity maturation within germinal centres and persist as long-lived IgG plasma cells in the bone marrow. Here we show that antigen-specific, induced IgM plasma cells also persist for a lifetime. Unlike long-lived IgG plasma cells, which develop in germinal centres and then home to the bone marrow, IgM plasma cells are primarily retained within the spleen and can develop even in the absence of germinal centres. Interestingly, their expressed IgV loci exhibit somatic mutations introduced by the activation-induced cytidine deaminase (AID). However, these IgM plasma cells are probably not antigen-selected, as replacement mutations are spread through the variable segment and not enriched within the CDRs. Finally, antibodies from long-lived IgM plasma cells provide protective host immunity against a lethal virus challenge.

The squalene-based oil-in-water emulsion (SE) vaccine adjuvant MF59 has been administered to more than 100 million people in more than 30 countries, in both seasonal and pandemic influenza vaccines. Despite its wide use and efficacy, its mechanisms of action remain unclear. In this study we demonstrate that immunization of mice with MF59 or its mimetic AddaVax (AV) plus soluble antigen results in robust antigen-specific antibody and CD8 T cell responses in lymph nodes and non-lymphoid tissues. Immunization triggered rapid RIPK3-kinase dependent necroptosis in the lymph node which peaked at 6 hr, followed by a sequential wave of apoptosis. Immunization with alum plus antigen did not induce RIPK3-dependent signaling. RIPK3-dependent signaling induced by MF59 or AV was essential for cross-presentation of antigen to CD8 T cells by Batf3-dependent CD8+ DCs. Consistent with this, RIPK3 deficient or Batf3 deficient mice were impaired in their ability to mount adjuvant-enhanced CD8 T cell responses. However, CD8 T cell responses were unaffected in mice deficient in MLKL, a downstream mediator of necroptosis. Surprisingly, antibody responses were unaffected in RIPK3-kinase or Batf3 deficient mice. In contrast, antibody responses were impaired by in vivo administration of the pan-caspase inhibitor Z-VAD-FMK, but normal in caspase-1 deficient mice, suggesting a contribution from apoptotic caspases, in the induction of antibody responses. These results demonstrate that squalene emulsion-based vaccine adjuvants induce antigen-specific CD8 T cell and antibody responses, through RIPK3-dependent and-independent pathways, respectively.

Therapeutic use and function of recombinant molecules can be studied by the expression of foreign genes in mice. In this study, we have expressed human Fcgamma receptor –Ig fusion molecules (FcγR-Igs) in mice by administering FcγR-Ig plasmid DNAs hydrodynamically and compared their effectiveness to purified molecules in blocking immune-complex (IC) mediated inflammation in mice. The concentration of hydrodynamically expressed FcγR-Igs (CD16AF-Ig, CD32AR-Ig and CD32AH-Ig) reached a maximum of 130 μg/ml of blood within 24 h after plasmid DNA administration. The in vivo half-life of FcγR-Igs was found to be 9-16 days and Western blot analysis showed that the FcγR-Igs were expressed as a homodimer. The hydrodynamically expressed FcγR-Igs blocked 50-80% of IC-mediated inflammation up to 3 days in a reverse passive Arthus reaction model. Comparative analysis with purified molecules showed that hydrodynamically expressed FcγR-Igs are more efficient than purified molecules in blocking IC-mediated inflammation and had a higher half-life. In summary, these results suggest that the administration of a plasmid vector with a FcγR-Ig gene can be used to study the consequences of blocking IC-binding to FcγRs during the development of inflammatory diseases. This approach may have potential therapeutic value in treating IC-mediated inflammatory autoimmune diseases such as lupus, arthritis and autoimmune vasculitis.

Human gammaherpesviruses are associated with the development of lymphoproliferative diseases and B cell lymphomas, particularly in immunosuppressed hosts. Understanding the molecular mechanisms by which human gammaherpesviruses cause disease is hampered by the lack of convenient small animal models to study them. However, infection of laboratory strains of mice with the rodent virus murine gammaherpesvirus 68 (MHV68) has been useful in gaining insights into how gammaherpesviruses contribute to the genesis and progression of lymphoproliferative lesions. In this report we make the novel observation that MHV68 infection of murine day 15 fetal liver cells results in their immortalization and differentiation into B plasmablasts that can be propagated indefinitely in vitro, and can establish metastasizing lymphomas in mice lacking normal immune competence. The phenotype of the MHV68 immortalized B cell lines is similar to that observed in lymphomas caused by KSHV and resembles the favored phenotype observed during MHV68 infection in vivo. All established cell lines maintained the MHV68 genome, with limited viral gene expression and little or no detectable virus production - although virus reactivation could be induced upon crosslinking surface Ig. Notably, transcription of the genes encoding the MHV68 viral cyclin D homolog (v-cyclin) and the homolog of the KSHV latency-associated nuclear antigen (LANA), both of which are conserved among characterized γ2-herpesviruses, could consistently be detected in the established B cell lines. Furthermore, we show that the v-cyclin and LANA homologs are required for MHV68 immortalization of murine B cells. In contrast the M2 gene, which is unique to MHV68 and plays a role in latency and virus reactivation in vivo, was dispensable for B cell immortalization. This new model of gammaherpesvirus-driven B cell immortalization and differentiation in a small animal model establishes an experimental system for detailed investigation of the role of gammaherpesvirus gene products and host responses in the genesis and progression of gammaherpesvirus-associated lymphomas, and presents a convenient system to evaluate therapeutic modalities.

Detection of immunoglobulin M (IgM) antibodies has long been used as an important diagnostic tool for identifying active viral infections, but their relevance in later stages has not been clearly defined in vivo. In this study, we followed the kinetics, longevity, and function of influenza virus-specific IgM antibodies for 2 years following sublethal infection of mice with live mouseadapted A/PR/8/34 virus or immunization with formalin-inactivated virus. These groups mounted robust protective immune responses and survived lethal challenges with 50 × 50% lethal dose (LD50) mouse-adapted A/PR/8/34 virus 600 days after the primary exposure. Surprisingly, the virus-specific IgM antibodies persisted along with IgG antibodies, and we found a significantly higher number of IgM-positive (IgM+) virus-specific plasma cells than IgG+plasma cells that persisted for at least 9 months postexposure. The IgM antibodies were functional as they neutralized influenza virus in the presence of complement just as well as IgG antibodies did.

Fluorescent proteins are increasingly being used to analyze cellular gene expression and to facilitate tracking of cell lineages in vivo. One of these, enhanced yellow fluorescent protein (EYFP) has several properties such as intense fluorescence and little to no toxicity in cells, which makes it an excellent molecule to label proteins and cells of interest. In live cells, visualization of EYFP has been highly successful; however, detection of EYFP in lymphoid tissue sections, particularly in combination with other markers of interest has been difficult. This is because of the enhanced solubility of EYFP in the absence of fixation. When extended fixation protocols are employed, EYFP is preserved but detection of other cellular antigens becomes problematic due to over fixation. Here we demonstrate that EYFP-expressing T and B cells can be efficiently visualized in lymphoid tissue sections without compromising the ability to detect other cellular markers.

The viability of long-lived plasma cells is enhanced by the expression of inducible nitric oxide synthase, which relieves endoplasmic reticulum stress by triggering a response dependent on cGMP and protein kinase G.