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Abstract Influenza viruses pose a threat to public health as evidenced by severe morbidity and mortality in humans on a yearly basis. Given the constant changes in the viral glycoproteins owing to antigenic drift, seasonal influenza vaccines need to be updated periodically and effectiveness often drops due to mismatches between vaccine and circulating strains. In addition, seasonal influenza vaccines are not protective against antigenically shifted influenza viruses with pandemic potential. Here, we have developed a highly immunogenic vaccination regimen based on live-attenuated influenza vaccines (LAIVs) comprised of an attenuated virus backbone lacking non-structural protein 1 (ΔNS1), the primary host interferon antagonist of influenza viruses, with chimeric hemagglutinins (cHA) composed of exotic avian head domains with a highly conserved stalk domain, to redirect the humoral response towards the HA stalk. In this study, we showed that cHA-LAIV vaccines induce robust serum and mucosal responses against group 1 stalk and confer antibody-dependent cell cytotoxicity activity. Mice that intranasally received cH8/1-ΔNS1 followed by a cH11/1-ΔNS1 heterologous booster had robust humoral responses for influenza A virus group 1 HAs and were protected from seasonal H1N1 influenza virus and heterologous highly pathogenic avian H5N1 lethal challenges. When compared with mice immunized with the standard of care or cold-adapted cHA-LAIV, cHA-ΔNS1 immunized mice had robust antigen-specific CD8+ T-cell responses which also correlated with markedly reduced lung pathology post-challenge. These observations support the development of a trivalent universal influenza vaccine for the protection against group 1 and group 2 influenza A viruses and influenza B viruses.

Abstract Zika and dengue virus nonstructural protein 5 antagonism of STAT2, a critical interferon signaling transcription factor, to suppress the host interferon response is required for viremia and pathogenesis in a vertebrate host. This affects viral species tropism, as mouse STAT2 resistance renders only immunocompromised or humanized STAT2 mice infectable. Here, we explore how STAT2 evolution impacts antagonism. By measuring the susceptibility of 38 diverse STAT2 proteins, we demonstrate that resistance arose numerous times in mammalian evolution. In four species, resistance requires distinct sets of multiple amino acid changes that often individually disrupt STAT2 signaling. This reflects an evolutionary ridge where progressive resistance is balanced by the need to maintain STAT2 function. Furthermore, resistance may come with a fitness cost, as resistance that arose early in lemur evolution was subsequently lost in some lemur lineages. These findings underscore that while it is possible to evolve resistance to antagonism, complex evolutionary trajectories are required to avoid detrimental host fitness consequences.

Current influenza vaccine strategies have yet to overcome significant obstacles, including rapid antigenic drift of seasonal influenza viruses, in generating efficacious long-term humoral immunity. Due to the necessity of germinal center formation in generating long-lived high affinity antibodies, the germinal center has increasingly become a target for the development of novel or improvement of less-efficacious vaccines. However, there remains a major gap in current influenza research to effectively target T follicular helper cells during vaccination to alter the germinal center reaction. In this study, we used a heterologous infection or immunization priming strategy to seed an antigen-specific memory CD4+ T cell pool prior to influenza infection in mice to evaluate the effect of recalled memory T follicular helper cells in increased help to influenza-specific primary B cells and enhanced generation of neutralizing antibodies. We found that heterologous priming with intranasal infection with acute lymphocytic choriomeningitis virus (LCMV) or intramuscular immunization with adjuvanted recombinant LCMV glycoprotein induced increased antigen-specific effector CD4+ T and B cellular responses following infection with a recombinant influenza strain that expresses LCMV glycoprotein. Heterologously primed mice had increased expansion of secondary Th1 and Tfh cell subsets, including increased CD4+ TRM cells in the lung. However, the early enhancement of the germinal center cellular response following influenza infection did not impact influenza-specific antibody generation or B cell repertoires compared to primary influenza infection. Overall, our study suggests that while heterologous infection or immunization priming of CD4+ T cells is able to enhance the early germinal center reaction, further studies to understand how to target the germinal center and CD4+ T cells specifically to increase long-lived antiviral humoral immunity are needed.

Abstract The 2023 monkeypox (mpox) epidemic was caused by a subclade IIb descendant of a monkeypox virus (MPXV) lineage traced back to Nigeria in 1971. Person-to-person transmission appears higher than for clade I or subclade IIa MPXV, possibly caused by genomic changes in subclade IIb MPXV. Key genomic changes could occur in the genome’s low-complexity regions (LCRs), which are challenging to sequence and are often dismissed as uninformative. Here, using a combination of highly sensitive techniques, we determine a high-quality MPXV genome sequence of a representative of the current epidemic with LCRs resolved at unprecedented accuracy. This reveals significant variation in short tandem repeats within LCRs. We demonstrate that LCR entropy in the MPXV genome is significantly higher than that of single-nucleotide polymorphisms (SNPs) and that LCRs are not randomly distributed. In silico analyses indicate that expression, translation, stability, or function of MPXV orthologous poxvirus genes (OPGs), including OPG153, OPG204, and OPG208, could be affected in a manner consistent with the established “genomic accordion” evolutionary strategies of orthopoxviruses. We posit that genomic studies focusing on phenotypic MPXV differences should consider LCR variability.

Further development of direct-acting antiviral agents against human SARS-CoV-2 infections remains a public health priority. Here, we report that an antisense peptide-conjugated morpholino oligomer (PPMO) named 5′END-2, targeting a highly conserved sequence in the 5′ UTR of SARS-CoV-2 genomic RNA, potently suppressed SARS-CoV-2 growth in vitro and in vivo. In HeLa-ACE 2 cells, 5′END-2 produced IC50 values of between 40 nM and 1.15 μM in challenges using six genetically disparate strains of SARS-CoV-2, including JN.1. In vivo, using K18-hACE2 mice and the WA-1/2020 virus isolate, two doses of 5′END-2 at 10 mg/kg, administered intranasally on the day before and the day after infection, produced approximately 1.4 log10 virus titer reduction in lung tissue at 3 days post-infection. Under a similar dosing schedule, intratracheal administration of 1.0–2.0 mg/kg 5′END-2 produced over 3.5 log10 virus growth suppression in mouse lungs. Electrophoretic mobility shift assays characterized specific binding of 5′END-2 to its complementary target RNA. Furthermore, using reporter constructs containing SARS-CoV-2 5′ UTR leader sequence, in an in-cell system, we observed that 5′END-2 could interfere with translation in a sequence-specific manner. The results demonstrate that direct pulmonary delivery of 5′END-2 PPMO is a promising antiviral strategy against SARS-CoV-2 infections and warrants further development.

The vast majority of data obtained from sequence analysis of influenza A viruses (IAVs) have revealed that nonstructural 1 (NS1) proteins from H1N1 swine, H3N8 equine, H3N2 avian and the correspondent subtypes from dogs have a conserved four C-terminal amino acid motif when independent cross-species transmission occurs between these species. To test the influence of the C-terminal amino acid motifs of NS1 protein on the replication and virulence of IAVs, we systematically generated 7 recombinants, which carried naturally truncated NS1 proteins, and their last four C-terminal residues were replaced with PEQK and SEQK (for H1N1), EPEV and KPEI (for H3N8) and ESEV and ESEI (for H3N2) IAVs. Another recombinant was generated by removing the C-terminal residues by reverse genetics. Remarkably, the ESEI and KPEI motifs circulating in canines largely contributed efficient replication in cultured cells and these had enhanced virulence. In contrast, the avian ESEV motif was only responsible for high pathogenicity in mice. We examined the effects of these motifs upon interferon (IFN) induction. The 7 mutant viruses replicated in vitro in an IFN-independent manner, and the canine SEQK motif was able to induced higher levels of IFN-β in human cell lines. These findings shed further new light on the role of the four C-terminal residues in replication and virulence of IAVs and suggest that these motifs can modulate viral replication in a species-specific manner.

ABSTRACTInfluenza A viruses (IAVs) pose a persistent potential threat to human health because of the spillover from avian and swine infections. Extensive surveillance was performed in 12 cities of Guangxi, China, during 2018 and 2023. A total of 2540 samples (including 2353 nasal swabs and 187 lung tissues) were collected from 18 pig farms with outbreaks of respiratory disease. From these, 192 IAV-positive samples and 19 genomic sequences were obtained. We found that the H1 and H3 swine influenza A viruses (swIAVs) of multiple lineages and genotypes have continued to co-circulate during that time in this region. Genomic analysis revealed the Eurasian avian-like H1N1 swIAVs (G4) still remained predominant in pig populations. Strikingly, the novel multiple H3N2 genotypes were found to have been generated through the repeated introduction of the early H3N2 North American triple reassortant viruses (TR H3N2 lineage) that emerged in USA and Canada in 1998 and 2005, respectively. Notably, when the matrix gene segment derived from the H9N2 avian influenza virus was introduced into endemic swIAVs, this produced a novel quadruple reassortant H1N2 swIAV that could pose a potential risk for zoonotic infection.

ABSTRACTSARS-CoV-2 Omicron subvariants are still emerging and spreading worldwide. These variants contain a high number of polymorphisms in the spike (S) glycoprotein that could potentially impact their pathogenicity and transmission. We have previously shown that the S:655Y and P681H mutations enhance S protein cleavage and syncytia formation. Interestingly, these polymorphisms are present in Omicron S protein. Here, we characterized the cleavage efficiency and fusogenicity of the S protein of different Omicron sublineages. Our results showed that Omicron BA.1 subvariant is efficiently cleaved but it is poorly fusogenic compared to previous SARS-CoV-2 strains. To understand the basis of this phenotype, we generated chimeric S protein using combinations of the S1 and S2 domains from WA1, Delta and Omicron BA.1 variants. We found that the S2 domain of Omicron BA.1 hindered efficient cell–cell fusion. Interestingly, this domain only contains six unique polymorphisms never detected before in ancestral SARS-CoV-2 variants. WA1614G S proteins containing the six individuals S2 Omicron mutations were assessed for their fusogenicity and S surface expression after transfection in cells. Results showed that the S:N856K and N969K substitutions decreased syncytia formation and impacted S protein cell surface levels. However, we observed that “first-generation” Omicron sublineages that emerged subsequently, had convergently evolved to an enhanced fusogenic activity and S expression on the surface of infected cells while “second-generation” Omicron variants have highly diverged and showed lineage-specific fusogenic properties. Importantly, our findings could have potential implications in the improvement and redesign of COVID-19 vaccines.

ABSTRACTCurrent clinical guidelines support the concomitant administration of seasonal influenza vaccines and COVID-19 mRNA boosters vaccine. Whether dual vaccination may impact vaccine immunogenicity due to an interference between influenza or SARS-CoV-2 antigens is unknown. We aimed to understand the impact of mRNA COVID-19 vaccines administered concomitantly on the immune response to influenza vaccines. For this, 128 volunteers were vaccinated during the 22-23 influenza season. Three groups of vaccination were assembled: FLU vaccine only (46, 35%) versus volunteers that received the mRNA bivalent COVID-19 vaccines concomitantly to seasonal influenza vaccines, FluCOVID vaccine in the same arm (42, 33%) or different arm (40, 31%), respectively. Sera and whole blood were obtained the day of vaccination, +7, and +28 days after for antibody and T cells response quantification. As expected, side effects were increased in individuals who received the FluCOVID vaccine as compared to FLU vaccine only based on the known reactogenicity of mRNA vaccines. In general, antibody levels were high at 4 weeks post-vaccination and differences were found only for the H3N2 virus when administered in different arms compared to the other groups at day 28 post-vaccination. Additionally, our data showed that subjects that received the FluCOVID vaccine in different arm tended to have better antibody induction than those receiving FLU vaccines for H3N2 virus in the absence of pre-existing immunity. Furthermore, no notable differences in the influenza-specific cellular immune response were found for any of the vaccination groups. Our data supports the concomitant administration of seasonal influenza and mRNA COVID-19 vaccines.

ABSTRACTSince emerging in late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has repeatedly crossed the species barrier with natural infections reported in various domestic and wild animal species. The emergence and global spread of SARS-CoV-2 variants of concern (VOCs) has expanded the range of susceptible host species. Previous experimental infection studies in cattle using Wuhan-like SARS-CoV-2 isolates suggested that cattle were not likely amplifying hosts for SARS-CoV-2. However, SARS-CoV-2 sero- and RNA-positive cattle have since been identified in Europe, India, and Africa. Here, we investigated the susceptibility and transmission of the Delta and Omicron SARS-CoV-2 VOCs in cattle. Eight Holstein calves were co-infected orally and intranasally with a mixed inoculum of SARS-CoV-2 VOCs Delta and Omicron BA.2. Twenty-four hours post-challenge, two sentinel calves were introduced to evaluate virus transmission. The co-infection resulted in a high proportion of calves shedding SARS-CoV-2 RNA at 1- and 2-days post-challenge (DPC). Extensive tissue distribution of SARS-CoV-2 RNA was observed at 3 and 7 DPC and infectious virus was recovered from two calves at 3 DPC. Next-generation sequencing revealed that only the SARS-CoV-2 Delta variant was detected in clinical samples and tissues. Similar to previous experimental infection studies in cattle, we observed only limited seroconversion and no clear evidence of transmission to sentinel calves. Together, our findings suggest that cattle are more permissive to infection with SARS-CoV-2 Delta than Omicron BA.2 and Wuhan-like isolates but, in the absence of horizontal transmission, are not likely to be reservoir hosts for currently circulating SARS-CoV-2 variants.

Summary: Background: In order to prevent the emergence and spread of future variants of concern of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), developing vaccines capable of stopping transmission is crucial. The SARS-CoV-2 vaccine NDV-HXP-S can be administered live intranasally (IN) and thus induce protective immunity in the upper respiratory tract. The vaccine is based on Newcastle disease virus (NDV) expressing a stabilised SARS-CoV-2 spike protein. NDV-HXP-S can be produced as influenza virus vaccine at low cost in embryonated chicken eggs. Methods: The NDV-HXP-S vaccine was genetically engineered to match the Omicron variants of concern (VOC) BA.1 and BA.5 and tested as an IN two or three dose vaccination regimen in female mice. Furthermore, female mice intramuscularly (IM) vaccinated with mRNA-lipid nanoparticles (LNPs) were IN boosted with NDV-HXP-S. Systemic humoral immunity, memory T cell responses in the lungs and spleens as well as immunoglobulin A (IgA) responses in distinct mucosal tissues were characterised. Findings: NDV-HXP-S Omicron variant vaccines elicited high mucosal IgA and serum IgG titers against respective SARS-CoV-2 VOC in female mice following IN administration and protected against challenge from matched variants. Additionally, antigen-specific memory B cells and local T cell responses in the lungs were induced. Host immunity against the NDV vector did not interfere with boosting. Intramuscular vaccination with mRNA-LNPs was enhanced by IN NDV-HXP-S boosting resulting in improvement of serum neutralization titers and induction of mucosal immunity. Interpretation: We demonstrate that NDV-HXP-S Omicron variant vaccines utilised for primary immunizations or boosting efficiently elicit humoral and cellular immunity. The described induction of systemic and mucosal immunity has the potential to reduce infection and transmission. Funding: This work was partially funded by the NIAID Centers of Excellence for Influenza Research and Response (CEIRR) and by the NIAID Collaborative Vaccine Innovation Centers and by institutional funding from the Icahn School of Medicine at Mount Sinai. See under Acknowledgements for details.

Summary: Background: The development of a universal influenza virus vaccine, to protect against both seasonal and pandemic influenza A viruses, is a long-standing public health goal. The conserved stalk domain of haemagglutinin (HA) is a promising vaccine target. However, the stalk is immunosubdominant. As such, innovative approaches are required to elicit robust immunity against this domain. In a previously reported observer-blind, randomised placebo-controlled phase I trial (NCT03300050), immunisation regimens using chimeric HA (cHA)-based immunogens formulated as inactivated influenza vaccines (IIV) −/+ AS03 adjuvant, or live attenuated influenza vaccines (LAIV), elicited durable HA stalk-specific antibodies with broad reactivity. In this study, we sought to determine if these vaccines could also boost T cell responses against HA stalk, and nucleoprotein (NP). Methods: We measured interferon-γ (IFN-γ) responses by Enzyme-Linked ImmunoSpot (ELISpot) assay at baseline, seven days post-prime, pre-boost and seven days post-boost following heterologous prime:boost regimens of LAIV and/or adjuvanted/unadjuvanted IIV-cHA vaccines. Findings: Our findings demonstrate that immunisation with adjuvanted cHA-based IIVs boost HA stalk-specific and NP-specific T cell responses in humans. To date, it has been unclear if HA stalk-specific T cells can be boosted in humans by HA-stalk focused universal vaccines. Therefore, our study will provide valuable insights for the design of future studies to determine the precise role of HA stalk-specific T cells in broad protection. Interpretation: Considering that cHA-based vaccines also elicit stalk-specific antibodies, these data support the further clinical advancement of cHA-based universal influenza vaccine candidates. Funding: This study was funded in part by the Bill and Melinda Gates Foundation (BMGF).

Abstract Background The eukaryotic translation initiation protein eIF5A is a highly conserved and essential factor that plays a critical role in different physiological and pathological processes including stress response and cancer. Different proteomic studies suggest that eIF5A may be a small ubiquitin-like modifier (SUMO) substrate, but whether eIF5A is indeed SUMOylated and how relevant is this modification for eIF5A activities are still unknown. Methods SUMOylation was evaluated using in vitro SUMOylation assays, Histidine-tagged proteins purification from His6–SUMO2 transfected cells, and isolation of endogenously SUMOylated proteins using SUMO-binding entities (SUBES). Mutants were engineered by site-directed mutagenesis. Protein stability was measured by a cycloheximide chase assay. Protein localization was determined using immunofluorescence and cellular fractionation assays. The ability of eIF5A1 constructs to complement the growth of Saccharomyces cerevisiae strains harboring thermosensitive mutants of a yeast EIF5A homolog gene (HYP2) was analyzed. The polysome profile and the formation of stress granules in cells expressing Pab1–GFP (a stress granule marker) by immunofluorescence were determined in yeast cells subjected to heat shock. Cell growth and migration of pancreatic ductal adenocarcinoma PANC-1 cells overexpressing different eIF5A1 constructs were evaluated using crystal violet staining and transwell inserts, respectively. Statistical analysis was performed with GraphPad Software, using unpaired Student’s t-test, or one-way or two-way analysis of variance (ANOVA). Results We found that eIF5A is modified by SUMO2 in vitro, in transfected cells and under endogenous conditions, revealing its physiological relevance. We identified several SUMO sites in eIF5A and found that SUMOylation modulates both the stability and the localization of eIF5A in mammalian cells. Interestingly, the SUMOylation of eIF5A responds to specific stresses, indicating that it is a regulated process. SUMOylation of eIF5A is conserved in yeast, the eIF5A SUMOylation mutants are unable to completely suppress the defects of HYP2 mutants, and SUMOylation of eIF5A is important for both stress granules formation and disassembly of polysomes induced by heat-shock. Moreover, mutation of the SUMOylation sites in eIF5A abolishes its promigratory and proproliferative activities in PANC-1 cells. Conclusions SUMO2 conjugation to eIF5A is a stress-induced response implicated in the adaptation of yeast cells to heat-shock stress and required to promote the growth and migration of pancreatic ductal adenocarcinoma cells. Graphical Abstract

IntroductionSeveral effective vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been developed and implemented in the population. However, the current production capacity falls short of meeting global demand. Therefore, it is crucial to further develop novel vaccine platforms that can bridge the distribution gap. AVX/COVID-12 is a vector-based vaccine that utilizes the Newcastle Disease virus (NDV) to present the SARS-CoV-2 spike protein to the immune system.MethodsThis study aims to analyze the antigenicity of the vaccine candidate by examining antibody binding and T-cell activation in individuals infected with SARS-CoV-2 or variants of concern (VOCs), as well as in healthy volunteers who received coronavirus disease 2019 (COVID-19) vaccinations.ResultsOur findings indicate that the vaccine effectively binds antibodies and activates T-cells in individuals who received 2 or 3 doses of BNT162b2 or AZ/ChAdOx-1-S vaccines. Furthermore, the stimulation of T-cells from patients and vaccine recipients with AVX/COVID-12 resulted in their proliferation and secretion of interferon-gamma (IFN-γ) in both CD4+ and CD8+ T-cells.DiscussionThe AVX/COVID-12 vectored vaccine candidate demonstrates the ability to stimulate robust cellular responses and is recognized by antibodies primed by the spike protein present in SARS-CoV-2 viruses that infected patients, as well as in the mRNA BNT162b2 and AZ/ChAdOx-1-S vaccines. These results support the inclusion of the AVX/COVID-12 vaccine as a booster in vaccination programs aimed at addressing COVID-19 caused by SARS-CoV-2 and its VOCs.

There are considerable avenues through which currently licensed influenza vaccines could be optimized. We tested influenza vaccination in a mouse model with two adjuvants: Sendai virus-derived defective interfering (SDI) RNA, a RIG-I agonist; and an amphiphilic imidazoquinoline (IMDQ-PEG-Chol), a TLR7/8 agonist. The negatively charged SDI RNA was formulated into lipid nanoparticles (LNPs) facilitating direct delivery of SDI RNA to the cytosol, where RIG-I sensing induces inflammatory and type I interferon responses. We previously tested SDI RNA and IMDQ-PEG-Chol as standalone and combination adjuvants for influenza and SARS-CoV-2 vaccines. Here, we tested two different ionizable lipids, K-Ac7-Dsa and S-Ac7-Dog, for LNP formulations. The LNPs were incorporated with SDI RNA to determine its potential as a combination adjuvant with IMDQ-PEG-Chol by evaluating the host immune response to vaccination and infection in immunized BALB/c mice. Adjuvanticity of IMDQ-PEG-Chol with and without empty or SDI-loaded LNPs was validated with quadrivalent inactivated influenza vaccine (QIV), showing robust induction of antibody titers and T-cell responses. Depending on the adjuvant combination and LNP formulation, humoral and cellular vaccine responses could be tailored towards type 1 or type 2 host responses with specific cytokine profiles that correlated with the protective responses to viral infection. The extent of protection conferred by different vaccine/LNP/adjuvant combinations was tested by challenging mice with a vaccine-matched strain of influenza A virus A/Singapore/gp1908/2015 IVR-180 (H1N1). Groups that received either LNP formulated with SDI or IMDQ-PEG-Chol, or both, showed very low levels of viral replication in their lungs at 5 days post-infection (DPI). These studies provide evidence that the combination of vaccines with LNPs and/or adjuvants promote antigen-specific cellular responses that can contribute to protection upon infection. Interestingly, we observed differences in humoral and cellular responses to vaccination between different groups receiving K-Ac7-Dsa or S-Ac7-Dog lipids in LNP formulations. The differences were also reflected in inflammatory responses in lungs of vaccinated animals to infection, depending on LNP formulations. Therefore, this study suggests that the composition of the LNPs, particularly the ionizable lipid, plays an important role in inducing inflammatory responses in vivo, which is important for vaccine safety and to prevent adverse effects upon viral exposure.

Summary: Despite multiple research efforts to characterize coronavirus disease 2019 (COVID-19) in humans, there is no clear data on the specific role of mucosal immunity on COVID-19 disease. Here, we longitudinally profile the antibody response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and seasonal HCoV-OC43 S proteins in serum and nasopharyngeal swabs from COVID-19 patients. Results showed that specific antibody responses against SARS-CoV-2 and HCoV-OC43 S proteins can be detected in the upper respiratory tract. We found that COVID-19 patients mounted a robust mucosal antibody response against SARS-CoV-2 S with specific secretory immunoglobulin A (sIgA), IgA, IgG, and IgM antibody subtypes detected in the nasal swabs. Additionally, COVID-19 patients showed IgG, IgA, and sIgA responses against HCoV-OC43 S in the local mucosa, whereas no specific IgM was detected. Interestingly, mucosal antibody titers against SARS-CoV-2 peaked at day 7, whereas HCoV-OC43 titers peaked earlier at day 3 post-recruitment, suggesting an immune memory recall to conserved epitopes of beta-HCoVs in the upper respiratory tract.

Abstract Influenza A Virus (IAV) is a recurring respiratory virus with limited availability of antiviral therapies. Understanding host proteins essential for IAV infection can identify targets for alternative host-directed therapies (HDTs). Using affinity purification-mass spectrometry and global phosphoproteomic and protein abundance analyses using three IAV strains (pH1N1, H3N2, H5N1) in three human cell types (A549, NHBE, THP-1), we map 332 IAV-human protein-protein interactions and identify 13 IAV-modulated kinases. Whole exome sequencing of patients who experienced severe influenza reveals several genes, including scaffold protein AHNAK, with predicted loss-of-function variants that are also identified in our proteomic analyses. Of our identified host factors, 54 significantly alter IAV infection upon siRNA knockdown, and two factors, AHNAK and coatomer subunit COPB1, are also essential for productive infection by SARS-CoV-2. Finally, 16 compounds targeting our identified host factors suppress IAV replication, with two targeting CDK2 and FLT3 showing pan-antiviral activity across influenza and coronavirus families. This study provides a comprehensive network model of IAV infection in human cells, identifying functional host targets for pan-viral HDT.

In patients with lung cancer (LC), understanding factors that impact the dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) anti-spike antibody (SAb) titers over time is critical, but challenging, due to evolving treatments, infections, vaccinations, and health status. The objective was to develop a time-dependent regression model elucidating individual contributions of factors influencing SAb levels in LC patients using a prospective, longitudinal, multi-institutional cohort study initiated in January 2021. The study evaluated 296 LC patients—median age 69; 55% female; 50% stage IV. Blood samples were collected every three months to measure SAb levels using FDA-approved ELISA. Asymptomatic and unreported infections were documented through measurement of anti-nucleocapsid Ab levels (Meso Scale Discovery). Associations between clinical characteristics and titers were evaluated using a time-dependent linear regression model with a generalized estimating equation (GEE), considering time-independent variables (age, sex, ethnicity, smoking history, histology, and stage) and time-dependent variables (booster vaccinations, SARS-CoV-2 infections, cancer treatment, steroid use, and influenza vaccination). Significant time-dependent effects increasing titer levels were observed for prior SARS-CoV-2 infection (p < 0.001) and vaccination/boosters (p < 0.001). Steroid use (p = 0.043) and chemotherapy (p = 0.033) reduced titer levels. Influenza vaccination was associated with increased SAb levels (p < 0.001), independent of SARS-CoV-2 vaccine boosters. Prior smoking significantly decreased titers in females (p = 0.001). Age showed no association with titers. This GEE-based linear regression model unveiled the nuanced impact of multiple variables on patient anti-spike Ab levels over time. After controlling for the major influences of vaccine and SARS-CoV-2 infections, chemotherapy and steroid use were found to have negatively affected titers. Smoking in females significantly decreased titers. Surprisingly, influenza vaccinations were also significantly associated, likely indirectly, with improved SARS-CoV-2 titers.

Abstract Multiple FDA-approved SARS-CoV-2 vaccines currently provide excellent protection against severe disease. Despite this, immunity can wane relatively fast, particularly in the elderly and novel viral variants capable of evading infection- and vaccination-induced immunity continue to emerge. Intranasal (IN) vaccination more effectively induces mucosal immune responses than parenteral vaccines, which would improve protection and reduce viral transmission. Here, we developed a rationally designed IN adjuvant consisting of a combined nanoemulsion (NE)-based adjuvant and an RNA-based RIG-I agonist (IVT DI) to drive more robust, broadly protective antibody and T cell responses. We previously demonstrated this combination adjuvant (NE/IVT) potently induces protective immunity through synergistic activation of an array of innate receptors. We now demonstrate that NE/IVT with the SARS-CoV-2 receptor binding domain (RBD), induces robust and durable humoral, mucosal, and cellular immune responses of equivalent magnitude and quality in young and aged mice. This contrasted with the MF59-like intramuscular adjuvant, Addavax, which showed a decrease in immunogenicity with age. Robust antigen-specific IFN-γ/IL-2/TNF-α was induced in both young and aged NE/IVT-immunized animals, which is significant as their reduced production is associated with suboptimal protective immunity in the elderly. These findings highlight the potential of adjuvanted mucosal vaccines for improving protection against COVID-19.

ABSTRACT Serum titers of SARS-CoV-2-neutralizing antibodies (nAbs) correlate well with protection from symptomatic COVID-19 but decay rapidly in the months following vaccination or infection. In contrast, measles-protective nAb titers are lifelong after measles vaccination, possibly due to persistence of the live-attenuated virus in lymphoid tissues. We, therefore, sought to generate a live recombinant measles vaccine capable of driving high SARS-CoV-2 nAb responses. Since previous clinical testing of a live measles vaccine encoding a SARS-CoV-2 spike glycoprotein resulted in suboptimal anti-spike antibody titers, our new vectors were designed to encode prefusion-stabilized SARS-CoV-2 spike glycoproteins, trimerized via an inserted peptide domain, and displayed on a dodecahedral miniferritin scaffold. Additionally, to circumvent the blunting of vaccine efficacy by preformed anti-measles antibodies, we extensively modified the measles surface glycoproteins. Comprehensive in vivo mouse testing demonstrated the potent induction of high titer nAbs in measles-immune mice and confirmed the significant contributions to overall potency afforded by prefusion stabilization, trimerization, and miniferritin display of the SARS-CoV-2 spike glycoprotein. In animals primed and boosted with a measles virus (MeV) vaccine encoding the ancestral SARS-CoV-2 spike, high-titer nAb responses against ancestral virus strains were only weakly cross-reactive with the Omicron variant. However, in primed animals that were boosted with a MeV vaccine encoding the Omicron BA.1 spike, antibody titers to both ancestral and Omicron strains were robustly elevated, and the passive transfer of serum from these animals protected K18-ACE2 mice from infection and morbidity after exposure to BA.1 and WA1/2020 strains. Our results demonstrate that by engineering the antigen, we can develop potent measles-based vaccine candidates against SARS-CoV-2.IMPORTANCEAlthough the live-attenuated measles virus (MeV) is one of the safest and most efficacious human vaccines, a measles-vectored COVID-19 vaccine candidate expressing the SARS-CoV-2 spike failed to elicit neutralizing antibody (nAb) responses in a phase-1 clinical trial, especially in measles-immune individuals. Here, we constructed a comprehensive panel of MeV-based COVID-19 vaccine candidates using a MeV with extensive modifications on the envelope glycoproteins (MeV-MR). We show that artificial trimerization of the spike is critical for the induction of nAbs and that their magnitude can be significantly augmented when the spike protein is synchronously fused to a dodecahedral scaffold. Furthermore, preexisting measles immunity did not abolish heterologous immunity elicited by our vector. Our results highlight the importance of antigen optimization in the development of spike-based COVID-19 vaccines and therapies.

ABSTRACTA wide range of animal species show variable susceptibility to SARS-CoV-2; however, host factors associated with varied susceptibility remain to be defined. Here, we examined whether susceptibility to SARS-CoV-2 and virus tropism in different animal species are dependent on the expression and distribution of the virus receptor angiotensin-converting enzyme 2 (ACE2) and the host cell factor transmembrane serine protease 2 (TMPRSS2). We cataloged the upper and lower respiratory tract of multiple animal species and humans in a tissue-specific manner and quantitatively evaluated the distribution and abundance of ACE2 and TMPRSS2 mRNA in situ. Our results show that: (i) ACE2 and TMPRSS2 mRNA are abundant in the conduction portion of the respiratory tract, (ii) ACE2 mRNA occurs at a lower abundance compared to TMPRSS2 mRNA, (iii) co-expression of ACE2-TMPRSS2 mRNAs is highest in those species with the highest susceptibility to SARS-CoV-2 infection (i.e., cats, Syrian hamsters, and white-tailed deer), and (iv) expression of ACE2 and TMPRSS2 mRNA was not altered following SARS-CoV-2 infection. Our results demonstrate that while specific regions of the respiratory tract are enriched in ACE2 and TMPRSS2 mRNAs in different animal species, this is only a partial determinant of susceptibility to SARS-CoV-2 infection.IMPORTANCESARS-CoV-2 infects a wide array of domestic and wild animals, raising concerns regarding its evolutionary dynamics in animals and potential for spillback transmission of emerging variants to humans. Hence, SARS-CoV-2 infection in animals has significant public health relevance. Host factors determining animal susceptibility to SARS-CoV-2 are vastly unknown, and their characterization is critical to further understand susceptibility and viral dynamics in animal populations and anticipate potential spillback transmission. Here, we quantitatively assessed the distribution and abundance of the two most important host factors, angiotensin-converting enzyme 2 and transmembrane serine protease 2, in the respiratory tract of various animal species and humans. Our results demonstrate that while specific regions of the respiratory tract are enriched in these two host factors, they are only partial determinants of susceptibility. Detailed analysis of additional host factors is critical for our understanding of the underlying mechanisms governing viral susceptibility and reservoir hosts.

Hybrid immunity (vaccination + natural infection) to SARS-CoV-2 provides superior protection to re-infection. We performed immune profiling studies during breakthrough infections in mRNA-vaccinated hamsters to evaluate hybrid immunity induction. The mRNA vaccine, BNT162b2, was dosed to induce binding antibody titers against ancestral spike, but inefficient serum virus neutralization of ancestral SARS-CoV-2 or variants of concern (VoCs). Vaccination reduced morbidity and controlled lung virus titers for ancestral virus and Alpha but allowed breakthrough infections in Beta, Delta and Mu-challenged hamsters. Vaccination primed for T cell responses that were boosted by infection. Infection back-boosted neutralizing antibody responses against ancestral virus and VoCs. Hybrid immunity resulted in more cross-reactive sera, reflected by smaller antigenic cartography distances. Transcriptomics post-infection reflects both vaccination status and disease course and suggests a role for interstitial macrophages in vaccine-mediated protection. Therefore, protection by vaccination, even in the absence of high titers of neutralizing antibodies in the serum, correlates with recall of broadly reactive B- and T-cell responses.

Summary: Primary human lung organoid-derived air-liquid interface (ALI) cultures serve as a physiologically relevant model to study human airway epithelium in vitro. Here, we present a protocol for establishing these cultures from cryopreserved human lung tissue. We describe steps for lung tissue cryostorage, tissue dissociation, lung epithelial organoid generation, and ALI culture differentiation. We also include quality control steps and technical readouts for monitoring virus response. This protocol demonstrates severe acute respiratory syndrome coronavirus 2 infection in these cultures as an example of their utility.For complete details on the use and execution of this protocol, please refer to Diana Cadena Castaneda et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

ABSTRACTMonkeypox (MPOX) is a zoonotic disease endemic to regions of Central/Western Africa. The geographic endemicity of MPV has expanded, broadening the human-monkeypox virus interface and its potential for spillover. Since May 2022, a large multi-country MPV outbreak with no proven links to endemic countries has originated in Europe and has rapidly expanded around the globe, setting off genomic surveillance efforts. Here, we conducted a genomic analysis of 23 MPV-infected patients from New York City during the early outbreak, assessing the phylogenetic relationship of these strains against publicly available MPV genomes. Additionally, we compared the genomic sequences of clinical isolates versus culture-passaged samples from a subset of samples. Phylogenetic analysis revealed that MPV genomes included in this study cluster within the B.1 lineage (Clade IIb), with some of the samples displaying further differentiation into five different sub-lineages of B.1. Mutational analysis revealed 55 non-synonymous polymorphisms throughout the genome, with some of these mutations located in critical regions required for viral multiplication, structural and assembly functions, as well as the target region for antiviral treatment. In addition, we identified a large majority of polymorphisms associated with GA > AA and TC > TT nucleotide replacements, suggesting the action of human APOBEC3 enzyme. A comparison between clinical isolates and cell culture-passaged samples failed to reveal any difference. Our results provide a first glance at the mutational landscape of early MPV-2022 (B.1) circulating strains in NYC.

Abstract There is still a need for safe, efficient, and low-cost coronavirus disease 2019 (COVID-19) vaccines that can stop transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we evaluated a vaccine candidate based on a live recombinant Newcastle disease virus (NDV) that expresses a stable version of the spike protein in infected cells as well as on the surface of the viral particle (AVX/COVID-12-HEXAPRO, also known as NDV-HXP-S). This vaccine candidate can be grown in embryonated eggs at a low cost, similar to influenza virus vaccines, and it can also be administered intranasally, potentially to induce mucosal immunity. We evaluated this vaccine candidate in prime-boost regimens via intramuscular, intranasal, or intranasal followed by intramuscular routes in an open-label non-randomized non-placebo-controlled phase I clinical trial in Mexico in 91 volunteers. The primary objective of the trial was to assess vaccine safety, and the secondary objective was to determine the immunogenicity of the different vaccine regimens. In the interim analysis reported here, the vaccine was found to be safe, and the higher doses tested were found to be immunogenic when given intramuscularly or intranasally followed by intramuscular administration, providing the basis for further clinical development of the vaccine candidate. The study is registered under ClinicalTrials.gov identifier NCT04871737.

Abstract Nuclear export of influenza A virus (IAV) mRNAs occurs through the nuclear pore complex (NPC). Using the Auxin-Induced Degron (AID) system to rapidly degrade proteins, we show that among the nucleoporins localized at the nucleoplasmic side of the NPC, TPR is the key nucleoporin required for nuclear export of influenza virus mRNAs. TPR recruits the TRanscription and EXport complex (TREX)−2 to the NPC for exporting a subset of cellular mRNAs. By degrading components of the TREX-2 complex (GANP, Germinal-center Associated Nuclear Protein; PCID2, PCI domain containing 2), we show that influenza mRNAs require the TREX-2 complex for nuclear export and replication. Furthermore, we found that cellular mRNAs whose export is dependent on GANP have a small number of exons, a high mean exon length, long 3’ UTR, and low GC content. Some of these features are shared by influenza virus mRNAs. Additionally, we identified a 45 nucleotide RNA signal from influenza virus HA mRNA that is sufficient to mediate GANP-dependent mRNA export. Thus, we report a role for the TREX-2 complex in nuclear export of influenza mRNAs and identified RNA determinants associated with the TREX-2-dependent mRNA export.

ABSTRACT Single-cell RNA sequencing (scRNA-Seq) studies have provided critical insight into the pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19). scRNA-Seq library preparation methods and data processing workflows are generally designed for the detection and quantification of eukaryotic host mRNAs and not viral RNAs. Here, we compare different scRNA-Seq library preparation methods for their ability to quantify and detect SARS-CoV-2 RNAs with a focus on subgenomic mRNAs (sgmRNAs). We show that compared to 10X Genomics Chromium Next GEM Single Cell 3′ (10X 3′) libraries or 10X Genomics Chromium Next GEM Single Cell V(D)J (10X 5′) libraries sequenced with standard read configurations, 10X 5′ libraries sequenced with an extended length read 1 (R1) that covers both cell barcode and transcript sequence (termed “10X 5′ with extended R1”) increase the number of unambiguous reads spanning leader-sgmRNA junction sites. We further present a data processing workflow, single-cell coronavirus sequencing (scCoVseq), which quantifies reads unambiguously assigned to viral sgmRNAs or viral genomic RNA (gRNA). We find that combining 10X 5′ with extended R1 library preparation/sequencing and scCoVseq data processing maximizes the number of viral UMIs per cell quantified by scRNA-Seq. Corresponding sgmRNA expression levels are highly correlated with expression in matched bulk RNA-Seq data sets quantified with established tools for SARS-CoV-2 analysis. Using this scRNA-Seq approach, we find that SARS-CoV-2 gene expression is highly correlated across individual infected cells, which suggests that the proportion of viral sgmRNAs remains generally consistent throughout infection. Taken together, these results and corresponding data processing workflow enable robust quantification of coronavirus sgmRNA expression at single-cell resolution, thereby supporting high-resolution studies of viral RNA processes in individual cells. IMPORTANCE Single-cell RNA sequencing (scRNA-Seq) has emerged as a valuable tool to study host-virus interactions, especially for coronavirus disease 2019 (COVID-19). Here we compare the performance of different scRNA-Seq library preparation methods and sequencing strategies to detect SARS-CoV-2 RNAs and develop a data processing workflow to quantify unambiguous sequence reads derived from SARS-CoV-2 genomic RNA and subgenomic mRNAs. After establishing a workflow that maximizes the detection of SARS-CoV-2 subgenomic mRNAs, we explore patterns of SARS-CoV-2 gene expression across cells with variable levels of total viral RNA, assess host gene expression differences between infected and bystander cells, and identify non-canonical and lowly abundant SARS-CoV-2 RNAs. The sequencing and data processing strategies developed here can enhance studies of coronavirus RNA biology at single-cell resolution and thereby contribute to our understanding of viral pathogenesis.

Abstract Influenza B virus (IBV) strains are one of the components of seasonal influenza vaccines in both trivalent and quadrivalent formulations. The vast majority of these vaccines are produced in embryonated chickens’ eggs. While optimized backbones for vaccine production in eggs exist and are in use for influenza A viruses, no such backbones exist for IBVs, resulting in unpredictable production yields. To generate an optimal vaccine seed virus backbone, we have compiled a panel of 71 IBV strains from 1940 to present day, representing the known temporal and genetic variability of IBV circulating in humans. This panel contains strains from the B/Victoria/2/87-like lineage, B/Yamagata/16/88-like lineage and the ancestral lineage that preceded their split to provide a diverse set that would help to identify a suitable backbone which can be used in combination with hemagglutinin (HA) and neuraminidase (NA) glycoproteins from any IBV strain to be incorporated into the seasonal vaccine. We have characterized and ranked the growth profiles of the 71 IBV strains and the best performing strains were used for co-infection of eggs, followed by serial passaging to select for high-growth reassortant viruses. After serial passaging, we selected 10 clonal isolates based on their growth profiles assessed by hemagglutination and plaque-forming units. We then generated reverse genetics systems for the three clones that performed best in growth curves. The selected backbones were then used to generate different reassortant viruses with HA/NA combinations from high and low titer yielding wild type IBV. When the growth profiles of the recombinant reassortant viruses were tested, the low titer yielding HA/NA viruses with the selected backbones yielded higher titers similar to those from high titer yielding HA/NA combinations. The use of these IBV backbones with improved replication in eggs might increase yields for the influenza B virus components of seasonal influenza virus vaccines.

Abstract Human respiratory syncytial virus (HRSV) is a major cause of severe lower respiratory tract disease in infants and the elderly, yet no safe, effective vaccine is commercially available. Closely related bovine RSV (BRSV) causes respiratory disease in young calves, with many similar features to those seen in HRSV. We previously showed that a Newcastle disease virus (NDV)-vectored vaccine expressing the F glycoprotein of HRSV reduced viral loads in lungs of mice and cotton rats and protected from HRSV. However, clinical signs and pathogenesis of disease in laboratory animals following HRSV infection differs from that observed in human infants. Thus, we examined whether a similar vaccine would protect neonatal calves from BRSV infection. Codon-optimized rNDV vaccine (rNDV-BRSV Fopt) was constructed and administered to colostrum-deprived calves. The rNDV-BRSV Fopt vaccine was well-tolerated and there was no evidence of vaccine-enhanced disease in the upper airways or lungs of these calves compared to the non-vaccinated calves. We found two intranasal doses reduces severity of gross and microscopic lesions and decreases viral load in the lungs. Furthermore, serum neutralizing antibodies were generated in vaccinated calves. Finally, reduced lung CXC chemokine levels were observed in vaccinated calves after BRSV challenge. In summary, we have shown that rNDV-BRSV Fopt vaccine is safe in colostrum-deprived calves, and is effective in reducing lung lesions, and decreasing viral load in upper respiratory tract and lungs after challenge.

Abstract A phase 1 clinical trial to test the immunogenicity of a chimeric group 1 HA (cHA) universal influenza virus vaccine targeting the conserved stalk domain of the hemagglutinin of influenza viruses was carried out. Vaccination with adjuvanted-inactivated vaccines induced high anti-stalk antibody titers. We sought to identify gene expression signatures that correlate with such induction. Messenger-RNA sequencing in whole blood was performed on the peripheral blood of 53 vaccinees. We generated longitudinal data on the peripheral blood of 53 volunteers, at early (days 3 and 7) and late (28 days) time points after priming and boosting with cHAs. Differentially expressed gene analysis showed no differences between placebo and live-attenuated vaccine groups. However, an upregulation of genes involved in innate immune responses and type I interferon signaling was found at day 3 after vaccination with inactivated adjuvanted formulations. Cell type deconvolution analysis revealed a significant enrichment for monocyte markers and different subsets of dendritic cells as mediators for optimal B cell responses and significant increase of anti-stalk antibodies in sera. A significant upregulation of immunoglobulin-related genes was only observed after administration of adjuvanted vaccines (either as primer or booster) with specific induction of anti-stalk IGVH1-69. This approach informed of specific immune signatures that correlate with robust anti-stalk antibody responses, while also helping to understand the regulation of gene expression induced by cHA proteins under different vaccine regimens.

Current influenza vaccines elicit humoral immune responses against the haemagglutinin (HA) protein of influenza viruses. Different antigenic sites have been identified in the HA head as the main target of haemagglutination inhibition (HAI) antibodies (Sb, Sa, Cb, Ca1 and Ca2). To determine immunodominance (ID) of each site, we performed HAI assays against a panel of mutant viruses, each one lacking one of the classically defined antigenic sites and compared it to wild type (Wt). Agglutinating antibodies were measured before and after vaccination in two different regimens: Quadrivalent Influenza Vaccine (QIV) in young adults; or Adjuvanted Trivalent influenza Vaccine (ATIV) in elderly. Our results showed abs before vaccination were significantly reduced against all antigenic sites in the elderly and only against Sb and Ca2 in young adults compared to the Wt. Humoral response to vaccination was significantly reduced against all viruses compared to the Wt for the ATIV and only against Sb and Ca2 for the QIV. The strongest reduction was observed in all cases against Sb followed by Ca2. We concluded that ID profile was clearly dominated by Sb followed by Ca2. Additionally, the antibody response evolved with age, increasing the response towards less immunodominant epitopes of HA head. Adjuvants can positively influence ID hierarchy broadening responses towards multiple antigenic sites of HA head.

SARS-CoV-2 was first reported circulating in human populations in December 2019 and has since become a global pandemic. Recent history involving SARS-like coronavirus outbreaks have demonstrated the significant role of intermediate hosts in viral maintenance and transmission. Evidence of SARS-CoV-2 natural infection and experimental infections of a wide variety of animal species has been demonstrated, and in silico and in vitro studies have indicated that deer are susceptible to SARS-CoV-2 infection. White-tailed deer (WTD) are amongst the most abundant and geographically widespread wild ruminant species in the US. Recently, WTD fawns were shown to be susceptible to SARS-CoV-2. In the present study, we investigated the susceptibility and transmission of SARS-CoV-2 in adult WTD. In addition, we examined the competition of two SARS-CoV-2 isolates, representatives of the ancestral lineage A and the alpha variant of concern (VOC) B.1.1.7 through co-infection of WTD. Next-generation sequencing was used to determine the presence and transmission of each strain in the co-infected and contact sentinel animals. Our results demonstrate that adult WTD are highly susceptible to SARS-CoV-2 infection and can transmit the virus through direct contact as well as vertically from doe to fetus. Additionally, we determined that the alpha VOC B.1.1.7 isolate of SARS-CoV-2 outcompetes the ancestral lineage A isolate in WTD, as demonstrated by the genome of the virus shed from nasal and oral cavities from principal infected and contact animals, and from the genome of virus present in tissues of principal infected deer, fetuses and contact animals.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for a global pandemic that has had significant impacts on human health and economies worldwide. SARS-CoV-2 is highly transmissible and the cause of coronavirus disease 2019 in humans. A wide range of animal species have also been shown to be susceptible to SARS-CoV-2 by experimental and/or natural infections. Sheep are a commonly farmed domestic ruminant that have not been thoroughly investigated for their susceptibility to SARS-CoV-2. Therefore, we performed in vitro and in vivo studies which consisted of infection of ruminant-derived cells and experimental challenge of sheep to investigate their susceptibility to SARS-CoV-2. Our results showed that sheep-derived kidney cells support SARS-CoV-2 replication. Furthermore, the experimental challenge of sheep demonstrated limited infection with viral RNA shed in nasal and oral swabs at 1 and 3-days post challenge (DPC); viral RNA was also detected in the respiratory tract and lymphoid tissues at 4 and 8 DPC. Sero-reactivity was observed in some of the principal infected sheep but not the contact sentinels, indicating that transmission to co-mingled naïve sheep was not highly efficient; however, viral RNA was detected in respiratory tract tissues of sentinel animals at 21 DPC. Furthermore, we used a challenge inoculum consisting of a mixture of two SARS-CoV-2 isolates, representatives of the ancestral lineage A and the B.1.1.7-like alpha variant of concern, to study competition of the two virus strains. Our results indicate that sheep show low susceptibility to SARS-CoV-2 infection and that the alpha variant outcompeted the lineage A strain.

Strategies to advance T cell based immune therapies are mostly focusing on the improvement of CD8 T cell effector functions, such as cytotoxicity or recruitment to the tumor. Here authors show that by combining in situ vaccination with oncolytic Newcastle Disease Virus and Flt3L-driven dendritic cell expansion, the anti-tumor T cell response is amplified via increased antigen cross-presentation.

Summary: Individuals with Down syndrome (DS) clinically manifest severe respiratory illnesses; however, there is a paucity of data on how DS influences homeostatic physiology of lung airway, and its reactive responses to pulmonary pathogens. We generated well-differentiated ciliated airway epithelia using tracheas from wild-type and Dp(16)1/Yey mice in vitro, and discovered that Dp(16)1/Yey epithelia have significantly lower abundance of ciliated cells, an altered ciliary beating profile, and reduced mucociliary transport. Interestingly, both sets of differentiated epithelia released similar quantities of viral particles after infection with influenza A virus (IAV). However, RNA-sequencing and proteomic analyses revealed an immune hyperreactive phenotype particularly for monocyte-recruiting chemokines in Dp(16)1/Yey epithelia. Importantly, when we challenged mice in vivo with IAV, we observed immune hyper-responsiveness in Dp(16)1/Yey mice, evidenced by higher quantities of lung airway infiltrated monocytes, and elevated levels of pro-inflammatory cytokines in bronchoalveolar lavage fluid. Our findings illuminate mechanisms underlying DS-mediated pathophysiological changes in airway epithelium.

Summary: The COVID-19 pandemic continues to be a health crisis with major unmet medical needs. The early responses from airway epithelial cells, the first target of the virus regulating the progression toward severe disease, are not fully understood. Primary human air-liquid interface cultures representing the broncho-alveolar epithelia were used to study the kinetics and dynamics of SARS-CoV-2 variants infection. The infection measured by nucleoprotein expression, was a late event appearing between day 4–6 post infection for Wuhan-like virus. Other variants demonstrated increasingly accelerated timelines of infection. All variants triggered similar transcriptional signatures, an “early” inflammatory/immune signature preceding a “late” type I/III IFN, but differences in the quality and kinetics were found, consistent with the timing of nucleoprotein expression. Response to virus was spatially organized: CSF3 expression in basal cells and CCL20 in apical cells. Thus, SARS-CoV-2 virus triggers specific responses modulated over time to engage different arms of immune response.

SARS-CoV-2 infection remains a global burden. Despite intensive research, the mechanism and dynamics of early viral replication are not completely understood, such as the kinetics of the formation of genomic RNA (gRNA), sub-genomic RNA (sgRNA), and replication centers/organelles (ROs). We employed single-molecule RNA-fluorescence in situ hybridization (smRNA-FISH) to simultaneously detect viral gRNA and sgRNA and immunofluorescence to detect nsp3 protein, a marker for the formation of RO, and carried out a time-course analysis. We found that single molecules of gRNA are visible within the cytoplasm at 30 min post infection (p.i.). Starting from 2 h p.i., most of the viral RNA existed in clusters/speckles, some of which were surrounded by single molecules of sgRNA. These speckles associated with nsp3 protein starting at 3 h p.i., indicating that these were precursors to ROs. Furthermore, RNA replication was asynchronous, as cells with RNA at all stages of replication were found at any given time point. Our probes detected the SARS-CoV-2 variants of concern, and also suggested that the BA.1 strain exhibited a slower rate of replication kinetics than the WA1 strain. Our results provide insights into the kinetics of SARS-CoV-2 early post-entry events, which will facilitate identification of new therapeutic targets for early-stage replication to combat COVID-19.

Here, Rathnasinghe et al. characterize the pathogenesis of a mouse-adapted SARS-CoV-2 in infected obese and aged mice, and identify mutations that are present in SARS-CoV-2 variants of concern, associated with higher transmissibility.

ObjectiveAntibodies elicited by seasonal influenza vaccines mainly target the head of hemagglutinin (HA). However, antibodies against the stalk domain are cross-reactive and have been proven to play a role in reducing influenza disease severity. We investigated the induction of HA stalk-specific antibodies after seasonal influenza vaccination, considering the age of the cohorts.MethodsA total of 166 individuals were recruited during the 2018 influenza vaccine campaign (IVC) and divided into groups:

Vaccine-induced immune thrombotic thrombocytopenia (VITT), also known as thrombosis with thrombocytopenia syndrome, is a catastrophic and life-threatening reaction to coronavirus disease 2019 (COVID-19) vaccines, which occurs disproportionately in response to vaccination with non-replicating adenovirus vector (AV) vaccines. The mechanism of VITT is not well defined and it has not been resolved why cases of VITT are predominated by vaccination with AV vaccines. However, virtually all VITT patients have positive platelet-activating anti-platelet factor 4 (PF4) antibody titers. Subsequently, platelets are activated and depleted in an Fcγ-receptor IIa (FcγRIIa or CD32a)-dependent manner, but it is not clear why or how the anti-PF4 response is mounted. This review describes the pathogenesis of VITT and provides insight into possible mechanisms that prompt the formation of a PF4/polyanion complex, which drives VITT pathology, as an amalgam of current experimental data or hypotheses.

ABSTRACT Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns – conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we – a broad group of working virologists – seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology.

Alongside the development and progress in cancer immunotherapy, research in oncolytic viruses (OVs) continues advancing novel treatment strategies to the clinic. With almost 50 clinical trials carried out over the last decade, the opportunities for intervention using OVs are expanding beyond the old-fashioned concept of “lytic killers”, with promising breakthrough therapeutic strategies focused on leveraging the immunostimulatory potential of different viral platforms. This review presents an overview of non-human-adapted RNA viruses engineered for cancer therapy. Moreover, we describe the diverse strategies employed to manipulate the genomes of these viruses to optimize their therapeutic capabilities. By focusing on different aspects of this particular group of viruses, we describe the insights into the promising advancements in the field of virotherapy and its potential to revolutionize cancer treatment.

The rapid evolution of SARS-CoV-2 since its pandemic outbreak has underscored the need for improved SARS-CoV-2 vaccines that efficiently reduce not only hospitalizations and deaths, but also infections and transmission. This might be achieved by a new generation of intranasally administered SARS-CoV-2 vaccines to stimulate protective mucosal immunity. Among all different approaches, preclinical and clinical information using Newcastle Disease Virus (NDV)-vectors expressing S of SARS-CoV2 as a COVID-19 vaccine show the potential of this vaccine platform as an affordable, highly immunogenic, safe strategy to intranasally vaccinate humans against SARS-CoV-2 and other infectious diseases. These vaccine vectors consist on the use of a harmless avian negative strand RNA virus to deliver intranasally a self-replicating RNA expressing the vaccine antigen in the cells of the respiratory mucosa. The vector also incorporates the antigen in the virus particle used for RNA delivery, thus combining the properties of nanoparticle-based and RNA-based vaccines. Other advantages of NDV-based vectors include the worldwide availability of manufacturing facilities for their production and their stability at non-freezing temperatures. While phase 3 clinical studies to evaluate efficacy are still pending, phase 1 and 2 clinical studies have demonstrated the safety and immunogenicity of NDV-S vaccines against SARS-CoV-2.

TIPiCO is an annual expert meeting and workshop on infectious diseases and vaccination. The edition of 2020 changed its name and format to aTIPiCO, the first series and podcasts on infectious diseases and vaccines. A total of 13 prestigious experts from different countries participated in this edition launched on the 26 November 2020. The state of the art of coronavirus disease-2019 (COVID-19) and the responsible pathogen, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), and the options to tackle the pandemic situation were discussed in light of the knowledge in November 2020. Despite COVID-19, the status of other infectious diseases, including influenza infections, respiratory syncytial virus disease, human papillomavirus infection, measles, pertussis, tuberculosis, meningococcal disease, and pneumococcal disease, were also addressed. The essential lessons that can be learned from these diseases and their vaccines to use in the COVID-19 pandemic were also commented with the experts.

Abstract The influenza A non-structural protein 1 (NS1) is known for its ability to hinder the synthesis of type I interferon (IFN) during viral infection. Influenza viruses lacking NS1 (ΔNS1) are under clinical development as live attenuated human influenza virus vaccines and induce potent influenza virus-specific humoral and cellular adaptive immune responses. Attenuation of ΔNS1 influenza viruses is due to their high IFN inducing properties, that limit their replication in vivo. This study demonstrates that pre-treatment with a ΔNS1 virus results in an antiviral state which prevents subsequent replication of homologous and heterologous viruses, preventing disease from virus respiratory pathogens, including SARS-CoV-2. Our studies suggest that ΔNS1 influenza viruses could be used for the prophylaxis of influenza, SARS-CoV-2 and other human respiratory viral infections, and that an influenza virus vaccine based on ΔNS1 live attenuated viruses would confer broad protection against influenza virus infection from the moment of administration, first by non-specific innate immune induction, followed by specific adaptive immunity.

Monoclonal antibodies show great promise in treating Covid-19 patients. Here, Maisonnasse, Aldon and colleagues report pre-clinical results for COVA1-18 and demonstrate that it reduces viral infectivity in three animal models with over 95% efficacy in macaques upper respiratory tract.

The 3CL protease of SARS-CoV-2 is inhibited by PF-00835231 in vitro. Here, the authors show that the prodrug PF-07304814 has broad spectrum activity, inhibiting SARS-CoV and SARS-CoV-2 in mice and its ADME and safety profile support clinical development.

Here the authors show that a Newcastle disease virus based COVID-19 vaccine expressing a stabilized spike protein induces protective immunity in small animal models and reduces replication of variants of concerns. This vaccine candidate can be produced by influenza virus vaccine manufactures around the world.

Abstract Particulate respirators such as N95s are an essential component of personal protective equipment (PPE) for front-line workers. This study describes a rapid and effective UVC irradiation system that would facilitate the safe re-use of N95 respirators and provides supporting information for deploying UVC for decontamination of SARS-CoV-2 during the COVID-19 pandemic. To assess the inactivation potential of the proposed UVC germicidal device as a function of time by using 3 M 8211-N95 particulate respirators inoculated with SARS-CoV-2. A germicidal UVC device to deliver tailored UVC dose was developed and test coupons (2.5 cm2) of the 3 M-N95 respirator were inoculated with 106 plaque-forming units (PFU) of SARS-CoV-2 and were UV irradiated. Different exposure times were tested (0–164 s) by fixing the distance between the lamp and the test coupon to 15.2 cm while providing an exposure of at least 5.43 mWcm−2. Primary measure of outcome was titration of infectious virus recovered from virus-inoculated respirator test coupons after UVC exposure. Other measures included the method validation of the irradiation protocol, using lentiviruses (biosafety level-2 agent) and establishment of the germicidal UVC exposure protocol. An average of 4.38 × 103 PFU ml−1 (SD 772.68) was recovered from untreated test coupons while 4.44 × 102 PFU ml−1 (SD 203.67), 4.00 × 102 PFU ml−1 (SD 115.47), 1.56 × 102 PFU ml−1 (SD 76.98) and 4.44 × 101 PFU ml−1 (SD 76.98) was recovered in exposures 2, 6, 18 and 54 s per side respectively. The germicidal device output and positioning was monitored and a minimum output of 5.43 mW cm−2 was maintained. Infectious SARS-CoV-2 was not detected by plaque assays (minimal level of detection is 67 PFU ml−1) on N95 respirator test coupons when irradiated for 120 s per side or longer suggesting 3.5 log reduction in 240 s of irradiation, 1.3 J cm−2. A scalable germicidal UVC device to deliver tailored UVC dose for rapid decontamination of SARS-CoV-2 was developed. UVC germicidal irradiation of N95 test coupons inoculated with SARS-CoV-2 for 120 s per side resulted in 3.5 log reduction of virus. These data support the reuse of N95 particle-filtrate apparatus upon irradiation with UVC and supports use of UVC-based decontamination of SARS-CoV-2 during the COVID-19 pandemic.

Infectious Bursal Disease Virus (IBDV) is the causative agent of an immunosuppressive disease that affects domestic chickens (Gallus gallus) severely affecting poultry industry worldwide. IBDV infection is characterized by a rapid depletion of the bursal B cell population by apoptosis and the atrophy of this chief lymphoid organ. Previous results from our laboratory have shown that exposure of infected cells to type I IFN leads to an exacerbated apoptosis, indicating an important role of IFN in IBDV pathogenesis. It has been described that recognition of the dsRNA IBDV genome by MDA5, the only known cytoplasmic pattern recognition receptor for viral RNA in chickens, leads to type I IFN production. Here, we confirm that TRIM25, an E3 ubiquitin ligase that leads to RIG-I activation in mammalian cells, significantly contributes to positively regulate MDA5-mediated activation of the IFN-inducing pathway in chicken DF-1 cells. Ectopic expression of chTRIM25 together with chMDA5 or a deletion mutant version exclusively harboring the CARD domains (chMDA5 2CARD) enhances IFN-β and NF-ĸB promoter activation. Using co-immunoprecipitation assays, we show that chMDA5 interacts with chTRIM25 through the CARD domains. Moreover, chTRIM25 co-localizes with both chMDA5 and chMDA5 2CARD, but not with chMDA5 mutant proteins partially or totally lacking these domains. On the other hand, ablation of endogenous chTRIM25 expression reduces chMDA5-induced IFN-β and NF-ĸB promoter activation. Interestingly, ectopic expression of either wild-type chTRIM25, or a mutant version (chTRIM25 C59S/C62S) lacking the E3 ubiquitin ligase activity, restores the co-stimulatory effect of chMDA5 in chTRIM25 knockout cells, suggesting that the E3-ubiquitin ligase activity of chTRIM25 is not required for its downstream IFN-β and NF-ĸB activating function. Also, IBDV-induced expression of IFN-β, Mx and OAS genes was reduced in chTRIM25 knockout as compared to wild-type cells, hence contributing to the enhancement of IBDV replication. Enhanced permissiveness to replication of other viruses, such as avian reovirus, Newcastle disease virus and vesicular stomatitis virus was also observed in chTRIM25 knockout cells. Additionally, chTRIM25 knockout also results in reduced MAVS-induced IFN-β promoter stimulation. Nonetheless, similarly to its mammalian counterpart, chTRIM25 overexpression in wild-type DF-1 cells causes the degradation of ectopically expressed chMAVS.