Protective efficacy of a plant-produced beta variant rSARS-CoV-2 VLP vaccine in golden Syrian hamsters.

In the quest for heightened protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, we engineered a prototype vaccine utilizing the plant expression system of Nicotiana benthamiana, to produce a recombinant SARS-CoV-2 virus-like particle (VLP) vaccine presenting the S-protein from the Beta (B.1.351) variant of concern (VOC). This innovative vaccine, formulated with either a squalene oil-in-water emulsion or a synthetic CpG oligodeoxynucleotide adjuvant, demonstrated efficacy in a golden Syrian Hamster challenge model. The Beta VLP vaccine induced a robust humoral immune response, with serum exhibiting neutralization not only against SARS-CoV-2 Beta but also cross-neutralizing Delta and Omicron pseudoviruses. Protective efficacy was demonstrated, evidenced by reduced viral RNA copies and mitigated weight loss and lung damage compared to controls. This compelling data instills confidence in the creation of a versatile platform for the local manufacturing of potential pan-sarbecovirus vaccines, against evolving viral threats.

Proteomic and genetic analyses of influenza A viruses identify pan-viral host targets.

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.

LSDV-Vectored SARS-CoV-2 S and N Vaccine Protects against Severe Clinical Disease in Hamsters.

The SARS-CoV-2 pandemic demonstrated the need for potent and broad-spectrum vaccines. This study reports the development and testing of a lumpy skin disease virus (LSDV)-vectored vaccine against SARS-CoV-2, utilizing stabilized spike and conserved nucleocapsid proteins as antigens to develop robust immunogenicity. Construction of the vaccine (LSDV-SARS2-S,N) was confirmed by polymerase chain reaction (PCR) amplification and sequencing. In vitro characterization confirmed that cells infected with LSDV-SARS2-S,N expressed SARS-CoV-2 spike and nucleocapsid protein. In BALB/c mice, the vaccine elicited high magnitude IFN-γ ELISpot responses (spike: 2808 SFU/106 splenocytes) and neutralizing antibodies (ID50 = 6552). Testing in hamsters, which emulate human COVID-19 disease progression, showed the development of high titers of neutralizing antibodies against the Wuhan and Delta SARS-CoV-2 variants (Wuhan ID50 = 2905; Delta ID50 = 4648). Additionally, hamsters vaccinated with LSDV-SARS2-S,N displayed significantly less weight loss, lung damage, and reduced viral RNA copies following SARS-CoV-2 infection with the Delta variant as compared to controls, demonstrating protection against disease. These data demonstrate that LSDV-vectored vaccines display promise as an effective SARS-CoV-2 vaccine and as a potential vaccine platform for communicable diseases in humans and animals. Further efficacy testing and immune response analysis, particularly in non-human primates, are warranted.

A plant-produced SARS-CoV-2 spike protein elicits heterologous immunity in hamsters.

Molecular farming of vaccines has been heralded as a cheap, safe and scalable production platform. In reality, however, differences in the plant biosynthetic machinery, compared to mammalian cells, can complicate the production of viral glycoproteins. Remodelling the secretory pathway presents an opportunity to support key post-translational modifications, and to tailor aspects of glycosylation and glycosylation-directed folding. In this study, we applied an integrated host and glyco-engineering approach, NXS/T Generation™, to produce a SARS-CoV-2 prefusion spike trimer in Nicotiana benthamiana as a model antigen from an emerging virus. The size exclusion-purified protein exhibited a characteristic prefusion structure when viewed by transmission electron microscopy, and this was indistinguishable from the equivalent mammalian cell-produced antigen. The plant-produced protein was decorated with under-processed oligomannose N-glycans and exhibited a site occupancy that was comparable to the equivalent protein produced in mammalian cell culture. Complex-type glycans were almost entirely absent from the plant-derived material, which contrasted against the predominantly mature, complex glycans that were observed on the mammalian cell culture-derived protein. The plant-derived antigen elicited neutralizing antibodies against both the matched Wuhan and heterologous Delta SARS-CoV-2 variants in immunized hamsters, although titres were lower than those induced by the comparator mammalian antigen. Animals vaccinated with the plant-derived antigen exhibited reduced viral loads following challenge, as well as significant protection from SARS-CoV-2 disease as evidenced by reduced lung pathology, lower viral loads and protection from weight loss. Nonetheless, animals immunized with the mammalian cell-culture-derived protein were better protected in this challenge model suggesting that more faithfully reproducing the native glycoprotein structure and associated glycosylation of the antigen may be desirable.

Common and species-specific molecular signatures, networks, and regulators of influenza virus infection in mice, ferrets, and humans.

Molecular responses to influenza A virus (IAV) infections vary between mammalian species. To identify conserved and species-specific molecular responses, we perform a comparative study of transcriptomic data derived from blood cells, primary epithelial cells, and lung tissues collected from IAV-infected humans, ferrets, and mice. The molecular responses in the human host have unique functions such as antigen processing that are not observed in mice or ferrets. Highly conserved gene coexpression modules across the three species are enriched for IAV infection-induced pathways including cell cycle and interferon (IFN) signaling. TDRD7 is predicted as an IFN-inducible host factor that is up-regulated upon IAV infection in the three species. TDRD7 is required for antiviral IFN response, potentially modulating IFN signaling via the JAK/STAT/IRF9 pathway. Identification of the common and species-specific molecular signatures, networks, and regulators of IAV infection provides insights into host-defense mechanisms and will facilitate the development of novel therapeutic interventions against IAV infection.

Food and Feed Safety of NS-B5ØØ27-4 Omega-3 Canola (Brassica napus): A New Source of Long-Chain Omega-3 Fatty Acids.

DHA canola, a genetically engineered Brassica napus (OECD Unique Identifier NS-B5ØØ27-4), has been developed as one of the first land-based production systems for omega-3 long-chain polyunsaturated fatty acids (LCPUFA), whose health benefits are well-established. Yet, the marine sources of these nutrients are under high pressures due to over-fishing and increasing demand. DHA canola is a plant-based source for these essential fatty acids that produces a high level of docosahexaenoic acid (DHA). This terrestrial system allows for sustainable, scalable and stable production of omega-3 LCPUFA that addresses not only the increasing market demand, but also the complex interplay of agriculture, aquaculture, and human nutrition. The vector used to produce the desired oil profile in DHA canola contains the expression cassettes of seven genes in the DHA biosynthesis pathway and was specifically designed to convert oleic acid to DHA in canola seed. The characterization and safety evaluation of food and feed produced from DHA canola are described and supported by a detailed nutritional analysis of the seed, meal, and oil. Aside from the intended changes of the fatty acid profile, none of the other compositional analytes showed biologically meaningful differences when compared to conventional canola varieties. In addition, the meal from DHA canola is compositionally equivalent to conventional canola meal. Further evidence of nutritional value and safety of DHA canola oil have been confirmed in fish feeding studies. Given that most human populations lack sufficient daily intakes of omega-3 LCPUFA, a dietary exposure assessment is also included. In conclusion, the results from these studies demonstrate it is safe to use products derived from DHA canola in human foods, nutraceuticals, or animal feeds.

Functional landscape of SARS-CoV-2 cellular restriction.

A deficient interferon (IFN) response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been implicated as a determinant of severe coronavirus disease 2019 (COVID-19). To identify the molecular effectors that govern IFN control of SARS-CoV-2 infection, we conducted a large-scale gain-of-function analysis that evaluated the impact of human IFN-stimulated genes (ISGs) on viral replication. A limited subset of ISGs were found to control viral infection, including endosomal factors inhibiting viral entry, RNA binding proteins suppressing viral RNA synthesis, and a highly enriched cluster of endoplasmic reticulum (ER)/Golgi-resident ISGs inhibiting viral assembly/egress. These included broad-acting antiviral ISGs and eight ISGs that specifically inhibited SARS-CoV-2 and SARS-CoV-1 replication. Among the broad-acting ISGs was BST2/tetherin, which impeded viral release and is antagonized by SARS-CoV-2 Orf7a protein. Overall, these data illuminate a set of ISGs that underlie innate immune control of SARS-CoV-2/SARS-CoV-1 infection, which will facilitate the understanding of host determinants that impact disease severity and offer potential therapeutic strategies for COVID-19.

Enisamium Reduces Influenza Virus Shedding and Improves Patient Recovery by Inhibiting Viral RNA Polymerase Activity.

Infections with respiratory viruses constitute a huge burden on our health and economy. Antivirals against some respiratory viruses are available, but further options are urgently needed. Enisamium iodide (laboratory code FAV00A, trade name Amizon) is an antiviral, marketed in countries of the Commonwealth of Independent States for the treatment of viral respiratory infections, but its clinical efficacy and mode of action are not well understood. In this study, we investigated the efficacy of enisamium in patients aged between 18 and 60 years with confirmed influenza virus and other viral respiratory infections. Enisamium treatment resulted in reduced influenza virus shedding (at day 3, 71.2% in the enisamium group tested negative versus 25.0% in placebo group [P < 0.0001]), faster patient recovery (at day 14, 93.9% in the enisamium group had recovered versus 32.5% in placebo group [P < 0.0001]), and reduced disease symptoms (from 9.6 ± 0.7 to 4.6 ± 0.9 score points in enisamium group versus 9.7 ± 1.1 to 5.6 ± 1.1 score points in placebo group [P < 0.0001]) compared to those in the placebo group. Using mass spectrometry, and cell-based and cell-free viral RNA synthesis assays, we identified a hydroxylated metabolite of enisamium, VR17-04. VR17-04 is capable of inhibiting influenza virus RNA synthesis and is present in plasma of patients treated with enisamium. VR17-04 inhibits the activity of the influenza virus RNA polymerase more potently than its parent compound. Overall, these results suggest that enisamium is metabolized in humans to an inhibitor of the influenza virus RNA polymerase that reduces viral shedding and improves patient recovery in influenza patients. (This study has been registered at ClinicalTrials.gov under identifier NCT04682444.).

RADx Variant Task Force Program for Assessing the Impact of Variants on SARS-CoV-2 Molecular and Antigen Tests.

Goal: Monitoring the genetic diversity and emerging mutations of SARS-CoV-2 is crucial for understanding the evolution of the virus and assuring the performance of diagnostic tests, vaccines, and therapies against COVID-19. SARS-CoV-2 is still adapting to humans and, as illustrated by B.1.1.7 (Alpha) and B.1.617.2 (Delta), lineage dynamics are fluid, and strain prevalence may change radically in a matter of months. The National Institutes of Health's Rapid Acceleration of Diagnostics (RADxSM) initiative created a Variant Task Force to assess the impact of emerging SARS-CoV-2 variants on in vitro diagnostic testing. Working in tandem with clinical laboratories, the FDA, and the CDC, the Variant Task Force uses both in silico modeling and in vitro testing to determine the effect of SARS-CoV-2 mutations on diagnostic molecular and antigen tests. Here, we offer an overview of the approach and activities of the RADx Variant Task Force to ensure test performance against emerging SARS-CoV-2 lineages.

Functional Landscape of SARS-CoV-2 Cellular Restriction.

A deficient interferon response to SARS-CoV-2 infection has been implicated as a determinant of severe COVID-19. To identify the molecular effectors that govern interferon control of SARS-CoV-2 infection, we conducted a large-scale gain-of-function analysis that evaluated the impact of human interferon stimulated genes (ISGs) on viral replication. A limited subset of ISGs were found to control viral infection, including endosomal factors that inhibited viral entry, nucleic acid binding proteins that suppressed viral RNA synthesis, and a highly enriched cluster of ER and Golgi-resident ISGs that inhibited viral translation and egress. These included the type II integral membrane protein BST2/tetherin, which was found to impede viral release, and is targeted for immune evasion by SARS-CoV-2 Orf7a protein. Overall, these data define the molecular basis of early innate immune control of viral infection, which will facilitate the understanding of host determinants that impact disease severity and offer potential therapeutic strategies for COVID-19.

Enantioselective Enzymatic Reduction of Acrylic Acids.

An ene-reductase (ERED 36) with broad substrate specificity was identified, and optimization studies led to the development of an enzymatic protocol for the reduction of α,ß-unsaturated acids under mild, aqueous conditions. The substrate scope includes aromatic- and aliphatic-substituted acrylic acids, as well as cyclic α,ß-substituted acrylic acids, yielding chiral α-substituted acids with exquisite levels of enantioselectivity (>99% ee).

An Influenza Virus Entry Inhibitor Targets Class II PI3 Kinase and Synergizes with Oseltamivir.

Two classes of antivirals targeting the viral neuraminidase (NA) and endonuclease are currently the only clinically useful drugs for the treatment of influenza. However, resistance to both antivirals has been observed in clinical isolates, and there was widespread resistance to oseltamivir (an NA inhibitor) among H1N1 viruses prior to 2009. This potential for resistance and lack of diversity for antiviral targets highlights the need for new influenza antivirals with a higher barrier to resistance. In this study, we identified an antiviral compound, M85, that targets host kinases, epidermal growth factor receptor (EGFR), and phosphoinositide 3 class II ß (PIK3C2ß) and is not susceptible to resistance by viral mutations. M85 blocks endocytosis of influenza viruses and inhibits a broad-spectrum of viruses with minimal cytotoxicity. In vitro, we found that combinations of M85 and oseltamivir have strong synergism. In the mouse model for influenza, treatment with the combination therapy was more protective against a lethal viral challenge than oseltamivir alone, indicating that development of M85 could lead to combination therapies for influenza. Finally, through this discovery of M85 and its antiviral mechanism, we present the first description of PIK3C2ß as a necessary host factor for influenza virus entry.

Baloxavir marboxil: the new influenza drug on the market.

For the first time in nearly 20 years there is a new class of antiviral drug for influenza. The latest approved antiviral is baloxavir marboxil (trade name, Xofluza) which targets the endonuclease function of the viral PA polymerase subunit and prevents the transcription of viral mRNA. The most promising aspect of this new drug is its pharmacology which allows for effective treatment of influenza A or B virus infection with just a single dose. A clinical trial showed greater reductions in viral loads with baloxavir marboxil treatment compared with oseltamivir, although no difference in the time to alleviation of symptoms between these two drugs. With this new class of influenza drug comes exciting prospects for combination therapy with the neuraminidase inhibitors which may help to abate concerns about the development of resistance.

Nucleolar Relocalization of RBM14 by Influenza A Virus NS1 Protein.

Viruses utilize a number of host factors in order to carry out their replication cycles. Influenza A virus (IAV) and human respiratory syncytial virus (RSV) both infect the tissues of the respiratory tract, and as such we hypothesize that they might require similar host factors. Several published genome-wide screens have identified putative IAV host factors; however, there is significant discordance between their hits. In order to build on this work, we integrated a variety of "OMICS" data sources using two complementary network analyses, yielding 51 genes enriched for both IAV and RSV replication. We designed a targeted small interfering RNA (siRNA)-based assay to screen these genes against IAV under robust conditions and identified 13 genes supported by two IAV subtypes in both primary and transformed human lung cells. One of these hits, RNA binding motif 14 (RBM14), was validated as a required host factor and furthermore was shown to relocalize to the nucleolus upon IAV infection but not with other viruses. Additionally, the IAV NS1 protein is both necessary and sufficient for RBM14 relocalization, and relocalization also requires the double-stranded RNA (dsRNA) binding capacity of NS1. This work reports the discovery of a new host requirement for IAV replication and exposes a novel example of interplay between IAV NS1 and the host protein, RBM14.IMPORTANCE Influenza A virus (IAV) and respiratory syncytial virus (RSV) present major global disease burdens. There are high economic costs associated with morbidity as well as significant mortality rates, especially in developing countries, in children, and in the elderly. There are currently limited therapeutic options for these viruses, which underscores the need for novel research into virus biology that may lead to the discovery of new therapeutic approaches. This work extends existing research into host factors involved in virus replication and explores the interaction between IAV and one such host factor, RBM14. Further study to fully characterize this interaction may elucidate novel mechanisms used by the virus during its replication cycle and open new avenues for understanding virus biology.

Transcription Elongation Can Affect Genome 3D Structure.

How transcription affects genome 3D organization is not well understood. We found that during influenza A (IAV) infection, rampant transcription rapidly reorganizes host cell chromatin interactions. These changes occur at the ends of highly transcribed genes, where global inhibition of transcription termination by IAV NS1 protein causes readthrough transcription for hundreds of kilobases. In these readthrough regions, elongating RNA polymerase II disrupts chromatin interactions by inducing cohesin displacement from CTCF sites, leading to locus decompaction. Readthrough transcription into heterochromatin regions switches them from the inert (B) to the permissive (A) chromatin compartment and enables transcription factor binding. Data from non-viral transcription stimuli show that transcription similarly affects cohesin-mediated chromatin contacts within gene bodies. Conversely, inhibition of transcription elongation allows cohesin to accumulate at previously transcribed intragenic CTCF sites and to mediate chromatin looping and compaction. Our data indicate that transcription elongation by RNA polymerase II remodels genome 3D architecture.

Author Correction: SMARCA2-regulated host cell factors are required for MxA restriction of influenza A viruses.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

SMARCA2-regulated host cell factors are required for MxA restriction of influenza A viruses.

The human interferon (IFN)-induced MxA protein is a key antiviral host restriction factor exhibiting broad antiviral activity against many RNA viruses, including highly pathogenic avian influenza A viruses (IAV) of the H5N1 and H7N7 subtype. To date the mechanism for how MxA exerts its antiviral activity is unclear, however, additional cellular factors are believed to be essential for this activity. To identify MxA cofactors we performed a genome-wide siRNA-based screen in human airway epithelial cells (A549) constitutively expressing MxA using an H5N1 reporter virus. These data were complemented with a proteomic screen to identify MxA-interacting proteins. The combined data identified SMARCA2, the ATPase subunit of the BAF chromatin remodeling complex, as a crucial factor required for the antiviral activity of MxA against IAV. Intriguingly, our data demonstrate that although SMARCA2 is essential for expression of some IFN-stimulated genes (ISGs), and the establishment of an antiviral state, it is not required for expression of MxA, suggesting an indirect effect on MxA activity. Transcriptome analysis of SMARCA2-depleted A549-MxA cells identified a small set of SMARCA2-regulated factors required for activity of MxA, in particular IFITM2 and IGFBP3. These findings reveal that several virus-inducible factors work in concert to enable MxA restriction of IAV.

Paramyxovirus V Proteins Interact with the RIG-I/TRIM25 Regulatory Complex and Inhibit RIG-I Signaling.

Paramyxovirus V proteins are known antagonists of the RIG-I-like receptor (RLR)-mediated interferon induction pathway, interacting with and inhibiting the RLR MDA5. We report interactions between the Nipah virus V protein and both RIG-I regulatory protein TRIM25 and RIG-I. We also observed interactions between these host proteins and the V proteins of measles virus, Sendai virus, and parainfluenza virus. These interactions are mediated by the conserved C-terminal domain of the V protein, which binds to the tandem caspase activation and recruitment domains (CARDs) of RIG-I (the region of TRIM25 ubiquitination) and to the SPRY domain of TRIM25, which mediates TRIM25 interaction with the RIG-I CARDs. Furthermore, we show that V interaction with TRIM25 and RIG-I prevents TRIM25-mediated ubiquitination of RIG-I and disrupts downstream RIG-I signaling to the mitochondrial antiviral signaling protein. This is a novel mechanism for innate immune inhibition by paramyxovirus V proteins, distinct from other known V protein functions such as MDA5 and STAT1 antagonism.IMPORTANCE The host RIG-I signaling pathway is a key early obstacle to paramyxovirus infection, as it results in rapid induction of an antiviral response. This study shows that paramyxovirus V proteins interact with and inhibit the activation of RIG-I, thereby interrupting the antiviral signaling pathway and facilitating virus replication.