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Antiviral responses are often accompanied by formation of stress granules (SG) in infected cells. However, the triggers for SG formation and the function of SG during infection remain subjects of active investigation. Copy-back viral genomes (cbVGs) are the primary inducers of the Mitochondrial Antiviral Signaling (MAVS) pathway and antiviral immunity during Sendai Virus (SeV) and Respiratory Syncytial virus (RSV) infections. The relationship between cbVGs and SG formation is unknown. Here we show that SG form during infections containing high levels of cbVGs, and not during infections with low levels of cbVGs. Moreover, using RNA fluorescent in situ hybridization to differentiate accumulation of standard viral genomes from cbVGs at a single-cell level during infection, we show that SG form exclusively in cells that accumulate high levels of cbVGs. As expected, virus-induced SG depend on signaling by Protein Kinase R (PKR), however, they form independent of MAVS signaling. Furthermore, inhibition of SG does not affect the induction of antiviral immunity, demonstrating that cbVGs induce antiviral immunity and SG formation through two independent mechanisms. Using live-cell imaging, we show that SG formation is highly dynamic and correlates with a drastic reduction of viral protein expression even in cells infected for several weeks. Together, our data demonstrate that SG are important agents of cbVG-induced interference by reducing viral protein expression independently of the antiviral response and suggest that SG contribute to sustain persistent infections.One Sentence SummarycbVGs trigger SG independent of the antiviral response during RSV and parainfluenza virus infection leading to a reduction of virus protein expression in acute and persisten infections

Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) antibody responses in children remain poorly characterized. Here, we show that pediatric patients with multisystem inflammatory syndrome in children (MIS-C) possess higher SARS-CoV-2 spike immunoglobulin G (IgG) titers compared with those with severe coronavirus disease 2019, likely reflecting a longer time since the onset of infection in MIS-C patients.

Chronic obstructive pulmonary disease (COPD) and asthma remain prevalent human lung diseases. Variability in epithelial and inflammatory components that results in pathologic heterogeneity complicates the development of treatments for these diseases. Early childhood infection with parainfluenza virus or respiratory syncytial virus is strongly associated with the development of asthma and COPD later in life, and exacerbations of these diseases correlate with the presence of viral RNA in the lung. Well-characterized animal models of postviral chronic lung diseases are necessary to study the underlying mechanisms of viral-related COPD and asthma and to develop appropriate therapies. In this study, we cross-analyzed chronic lung disease caused by infection with Sendai virus (SeV) or influenza A virus in mice. Differences were observed in lesion composition and inflammatory profiles between SeV- and influenza A virus–induced long-term lung disease. In addition, a primary SeV infection led to worsened pathologic findings on secondary heterologous viral challenge, whereas the reversed infection scheme protected against disease in response to a secondary viral challenge >1 month after the primary infection. These data demonstrate the differential effect of primary viral infections in the susceptibility to disease exacerbation in response to a different secondary viral infection and highlight the usefulness of these viral models as tools to understand the underlying mechanisms that mediate distinct chronic postviral lung diseases.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a recently emerged tickborne virus in east Asia with over 8,000 confirmed cases. With a high case fatality ratio, SFTSV has been designated a high priority pathogen by the WHO and the NIAID. Despite this, there are currently no approved therapies or vaccines to treat or prevent SFTS. Vesicular stomatitis virus (VSV) represents an FDA-approved vaccine platform that has been considered for numerous viruses due to its low sero-prevalence in humans, ease in genetic manipulation and promiscuity in incorporating foreign glycoproteins into its virions. In this study, we developed a recombinant VSV (rVSV) expressing the SFTSV glycoproteins Gn/Gc (rVSV-SFTSV) and assessed its safety, immunogenicity and efficacy in mice. We demonstrate that rVSV-SFTSV is safe when given to immunocompromised animals and is not neuropathogenic when injected intracranially into young immunocompetent mice. Immunization of Ifnar-/- mice with rVSV-SFTSV resulted in high levels of neutralizing antibodies and protection against lethal SFTSV challenge. Additionally, passive transfer of sera from immunized Ifnar-/- mice into naïve animals was protective when given pre- or post-exposure. Finally, we demonstrate that immunization with rVSV-SFTSV cross protects mice against challenge with the closely related Heartland virus despite low neutralizing titers to the virus. Taken together, these data suggest that rVSV-SFTSV is a promising vaccine candidate.ImportanceTick borne diseases are a growing threat to human health. Severe fever with thrombocytopenia syndrome (SFTS) and Heartland viruses are recently recognized, highly-pathogenic, tick-transmitted viruses. The fatality rates for individuals infected with SFTSV or HRTV are high and there are no therapeutics or vaccines available. The recent introduction of the tick vector for SFTSV (Haemaphysalis longicornis) to the eastern half of the United States and Austrailia raises concerns for SFTSV outbreaks outside East Asia. Here we report the development of a potential vaccine for SFTSV and HRTV based on the viral vector platform that has been successfully used for an Ebola vaccine. We demonstrate that the rVSV-SFTSV protects from lethal SFTSV or HRTV challenge when given as a single dose. We evaluated possible pathogenic effects of the vaccine and show that it is safe in immune compromised animlas and when introduced into the central nervous system.

Concerns with current mRNA Lipid Nanoparticle (LNP) systems include dose-limiting reactogenicity, adverse events that may be partly due to systemic off target expression of the immunogen, and a very limited understanding of the mechanisms responsible for the frozen storage requirement. We applied a new rational design process to identify a novel multiprotic ionizable lipid, called C24, as the key component of the mRNA LNP delivery system. We show that the resulting C24 LNP has a multistage protonation behavior resulting in greater endosomal protonation and greater translation of an mRNA-encoded luciferase reporter after intramuscular (IM) administration compared to the standard reference MC3 LNP. Off-target expression in liver after IM administration was reduced 6 fold for the C24 LNP compared to MC3. Neutralizing titers in immunogenicity studies delivering a nucleoside-modified mRNA encoding for the diproline stabilized spike protein immunogen were 10 fold higher for the C24 LNP versus MC3, and protection against viral challenge in a SARS-CoV-2 mouse model occurred at a very low 0.25 µg prime/boost dose of the same immunogen in the C24 LNP. Injection site inflammation was notably reduced for C24 compared to MC3. In addition, we found the C24 LNP to be entirely stable in bioactivity and mRNA integrity when stored at 4 ºC for at least 19 days. Storage at higher temperatures reduced both bioactivity and mRNA integrity, but less so for C24 than MC3, and in a manner consistent with the phosphodiester transesterification reaction mechanism of mRNA cleavage. The higher potency, lower injection site inflammation, and higher stability of the C24 LNP present important advancements in the evolution mRNA vaccine delivery.

Adjuvants are critical for improving the quality and magnitude of adaptive immune responses to vaccination. Lipid nanoparticle (LNP)-encapsulated nucleoside-modified mRNA vaccines have shown great efficacy against SARS-CoV-2, but the mechanism of action of this vaccine platform is not well-characterized. Using influenza virus and SARS-CoV-2 mRNA and protein subunit vaccines, we demonstrated that our LNP formulation has intrinsic adjuvant activity that promotes the induction of strong T follicular helper cell, germinal center B cell, long-lived plasma cell and memory B cell responses that are associated with durable and protective antibodies in mice. Comparative experiments demonstrated that this LNP outperformed a widely used MF59-like adjuvant, AddaVax™. The adjuvant activity of the LNP relies on the ionizable lipid component and on IL-6 cytokine induction, but not on MyD88- or MAVS-dependent sensing of LNPs. Our study identified LNPs as a versatile adjuvant that enhances the efficacy of both traditional and next-generation vaccine platforms., Graphical Abstract, The mechanism of action of nucleoside-modified mRNA-LNP vaccines is unknown. Alameh, Tombácz, Bettini et al. demonstrate that LNPs can possess adjuvant activity and promote a robust induction of Tfh cell, B cell and humoral responses when utilized in mRNA and protein subunit vaccines in mice. IL-6 induction and the ionizable lipid component are critical for the adjuvant activity of LNPs.

Summary SARS-CoV-2 infection has emerged as a serious global pandemic. Because of the high transmissibility of the virus and the high rate of morbidity and mortality associated with COVID-19, developing effective and safe vaccines is a top research priority. Here, we provide a detailed evaluation of the immunogenicity of lipid nanoparticle-encapsulated, nucleoside-modified mRNA (mRNA-LNP) vaccines encoding the full length SARS-CoV-2 spike protein or the spike receptor binding domain in mice. We demonstrate that a single dose of these vaccines induces strong type 1 CD4+ and CD8+ T cell responses, as well as long-lived plasma and memory B cell responses. Additionally, we detect robust and sustained neutralizing antibody responses and the antibodies elicited by nucleoside-modified mRNA vaccines do not show antibody-dependent enhancement of infection in vitro. Our findings suggest that the nucleoside-modified mRNA-LNP vaccine platform can induce robust immune responses and is a promising candidate to combat COVID-19., Highlights • mRNA vaccines induce robust type 1 CD4+ and CD8+ T cells in the spleen and lung • Vaccine-induced T cells readily exit the vasculature and enter the lung parenchyma • mRNA vaccines elicit strong long-lived plasma cell and memory B cell responses • mRNA vaccines induce antibodies with potent anti-SARS-CoV-2 neutralization activity, SARS-CoV-2 mRNA-based vaccines are among the most promising vaccine candidates against COVID-19. Laczkó et al. evaluate two nucleoside-modified mRNA vaccine candidates in mice and demonstrate that they induce potent T and B cell immune responses and high levels of neutralizing antibodies after administration of a single vaccine dose.

The deployment of effective vaccines against SARS-CoV-2 is critical to eradicate the COVID-19 pandemic. Many licensed vaccines confer protection by inducing long-lived plasma cells (LLPC) and memory B cells (MBC), cell types canonically generated during germinal center (GC) reactions. Here, we directly compared two vaccine platforms −mRNA vaccines and a recombinant protein formulated with an MF59-like adjuvant− for their ability to quantitatively and qualitatively shape SARS-CoV-2-specific primary GC responses over time. We demonstrated that a single immunization with SARS-CoV-2 mRNA, but not with the recombinant protein vaccine, elicited potent SARS-CoV-2-specific GC B and T follicular helper (Tfh) cell responses as well as LLPC and MCB. Importantly, GC responses strongly correlated with neutralizing antibody production. mRNA vaccines more efficiently induced key regulators of the Tfh cell program and influenced the functional properties of Tfh cells. Overall, this study identifies SARS-CoV-2 mRNA vaccines as strong candidates for promoting robust GC-derived immune responses., Graphical Abstract, Highlights • SARS-CoV-2 mRNA vaccines elicit potent germinal center (GC) B cell responses • GC responses are associated with a robust development of neutralizing antibodies • SARS-CoV-2 mRNA vaccines promote antigen-specific T follicular helper (Tfh) cells • Key elements of the Tfh cell program are modulated by SARS-CoV-2 mRNA vaccines, Herein, Lederer et al. show a nucleic acid-based vaccine platform for SARS-CoV-2 that potently induces germinal center (GC) responses. GC are microanatomical sites harboring the formation of high-quality, protective antibody responses. Such vaccine platforms can be promising candidates to mitigate the COVID-19 pandemic.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread within the human population. Although SARS-CoV-2 is a novel coronavirus, most humans had been previously exposed to other antigenically distinct common seasonal human coronaviruses (hCoVs) before the COVID-19 pandemic. Here, we quantified levels of SARS-CoV-2-reactive antibodies and hCoV-reactive antibodies in serum samples collected from 431 humans before the COVID-19 pandemic. We then quantified pre-pandemic antibody levels in serum from a separate cohort of 251 individuals who became PCR-confirmed infected with SARS-CoV-2. Finally, we longitudinally measured hCoV and SARS-CoV-2 antibodies in the serum of hospitalized COVID-19 patients. Our studies indicate that most individuals possessed hCoV-reactive antibodies before the COVID-19 pandemic. We determined that ∼20% of these individuals possessed non-neutralizing antibodies that cross-reacted with SARS-CoV-2 spike and nucleocapsid proteins. These antibodies were not associated with protection against SARS-CoV-2 infections or hospitalizations, but they were boosted upon SARS-CoV-2 infection., Analysis of human serum samples before and after the onset of the COVID-19 pandemic show that antibodies against common seasonal human coronaviruses are cross-reactive against SARS-CoV-2 but do not confer cross-protection against infection or hospitalization.

Defective viral genomes (DVGs) are natural products of virus replication that occur in many positive and negative sense RNA viruses, including Ebola, dengue and respiratory syncytial virus. DVGs, which have severe genomic truncations and require a helper virus to replicate, have three well-described functions: interference with standard virus replication, immunostimulation, and establishment of virus persistence. These functions of DVGs were first described almost 50 years ago, yet only recent studies have shown the molecular intersection between their immunostimulatory and pro-persistence activities. Here, we review more than half a century of scientific literature on the immunostimulatory and pro-persistence functions of DVGs. We highlight recent advances in the field and the critical role DVGs have in both the acute and long-term virus–host interactions.

Humans encode seven APOBEC3 proteins (A–H), with A3G, 3F and 3H as the major factors restricting HIV-1 replication. HIV-1, however, encodes Vif, which counteracts A3 proteins by chaperoning them to the proteasome where they are degraded. Vif polymorphisms found in HIV-1s isolated from infected patients have varying anti-A3G potency when assayed in vitro, but there are few studies demonstrating this in in vivo models. Here, we created Friend murine leukemia viruses encoding vif alleles that were previously shown to differentially neutralize A3G in cell culture or that were originally found in primary HIV-1 isolates. Infection of transgenic mice expressing different levels of human A3G showed that these naturally occurring Vif variants differed in their ability to counteract A3G during in vivo infection, although the effects on viral replication were not identical to those seen in cultured cells. We also found that the polymorphic Vifs that attenuated viral replication reverted to wild type only in A3G transgenic mice. Finally, we found that the level of A3G-mediated deamination was inversely correlated with the level of viral replication. This animal model should be useful for studying the functional significance of naturally occurring vif polymorphisms, as well as viral evolution in the presence of A3G.