A highly efficacious live attenuated mumps virus-based SARS-CoV-2 vaccine candidate expressing a six-proline stabilized prefusion spike.

With the rapid increase in SARS-CoV-2 cases in children, a safe and effective vaccine for this population is urgently needed. The MMR (measles/mumps/rubella) vaccine has been one of the safest and most effective human vaccines used in infants and children since the 1960s. Here, we developed live attenuated recombinant mumps virus (rMuV)-based SARS-CoV-2 vaccine candidates using the MuV Jeryl Lynn (JL2) vaccine strain backbone. The soluble prefusion SARS-CoV-2 spike protein (preS) gene, stablized by two prolines (preS-2P) or six prolines (preS-6P), was inserted into the MuV genome at the P-M or F-SH gene junctions in the MuV genome. preS-6P was more efficiently expressed than preS-2P, and preS-6P expression from the P-M gene junction was more efficient than from the F-SH gene junction. In mice, the rMuV-preS-6P vaccine was more immunogenic than the rMuV-preS-2P vaccine, eliciting stronger neutralizing antibodies and mucosal immunity. Sera raised in response to the rMuV-preS-6P vaccine neutralized SARS-CoV-2 variants of concern, including the Delta variant equivalently. Intranasal and/or subcutaneous immunization of IFNAR1-/- mice and golden Syrian hamsters with the rMuV-preS-6P vaccine induced high levels of neutralizing antibodies, mucosal immunoglobulin A antibody, and T cell immune responses, and were completely protected from challenge by both SARS-CoV-2 USA-WA1/2020 and Delta variants. Therefore, rMuV-preS-6P is a highly promising COVID-19 vaccine candidate, warranting further development as a tetravalent MMR vaccine, which may include protection against SARS-CoV-2.

SARS-CoV-2 prefusion spike protein stabilized by six rather than two prolines is more potent for inducing antibodies that neutralize viral variants of concern.

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the main target for neutralizing antibodies (NAbs). The S protein trimer is anchored in the virion membrane in its prefusion (preS) but metastable form. The preS protein has been stabilized by introducing two or six proline substitutions, to generate stabilized, soluble 2P or HexaPro (6P) preS proteins. Currently, it is not known which form is the most immunogenic. Here, we generated recombinant vesicular stomatitis virus (rVSV) expressing preS-2P, preS-HexaPro, and native full-length S, and compared their immunogenicity in mice and hamsters. The rVSV-preS-HexaPro produced and secreted significantly more preS protein compared to rVSV-preS-2P. Importantly, rVSV-preS-HexaPro triggered significantly more preS-specific serum IgG antibody than rVSV-preS-2P in both mice and hamsters. Antibodies induced by preS-HexaPro neutralized the B.1.1.7, B.1.351, P.1, B.1.427, and B.1.617.2 variants approximately two to four times better than those induced by preS-2P. Furthermore, preS-HexaPro induced a more robust Th1-biased cellular immune response than preS-2P. A single dose (104 pfu) immunization with rVSV-preS-HexaPro and rVSV-preS-2P provided complete protection against challenge with mouse-adapted SARS-CoV-2 and B.1.617.2 variant, whereas rVSV-S only conferred partial protection. When the immunization dose was lowered to 103 pfu, rVSV-preS-HexaPro induced two- to sixfold higher antibody responses than rVSV-preS-2P in hamsters. In addition, rVSV-preS-HexaPro conferred 70% protection against lung infection whereas only 30% protection was observed in the rVSV-preS-2P. Collectively, our data demonstrate that both preS-2P and preS-HexaPro are highly efficacious but preS-HexaPro is more immunogenic and protective, highlighting the advantages of using preS-HexaPro in the next generation of SARS-CoV-2 vaccines.

Potent universal beta-coronavirus therapeutic activity mediated by direct respiratory administration of a Spike S2 domain-specific human neutralizing monoclonal antibody.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) marks the third novel ß-coronavirus to cause significant human mortality in the last two decades. Although vaccines are available, too few have been administered worldwide to keep the virus in check and to prevent mutations leading to immune escape. To determine if antibodies could be identified with universal coronavirus activity, plasma from convalescent subjects was screened for IgG against a stabilized pre-fusion SARS-CoV-2 spike S2 domain, which is highly conserved between human ß-coronavirus. From these subjects, several S2-specific human monoclonal antibodies (hmAbs) were developed that neutralized SARS-CoV-2 with recognition of all variants of concern (VoC) tested (Beta, Gamma, Delta, Epsilon, and Omicron). The hmAb 1249A8 emerged as the most potent and broad hmAb, able to recognize all human ß-coronavirus and neutralize SARS-CoV and MERS-CoV. 1249A8 demonstrated significant prophylactic activity in K18 hACE2 mice infected with SARS-CoV-2 lineage A and lineage B Beta, and Omicron VoC. 1249A8 delivered as a single 4 mg/kg intranasal (i.n.) dose to hamsters 12 hours following infection with SARS-CoV-2 Delta protected them from weight loss, with therapeutic activity further enhanced when combined with 1213H7, an S1-specific neutralizing hmAb. As little as 2 mg/kg of 1249A8 i.n. dose 12 hours following infection with SARS-CoV Urbani strain, protected hamsters from weight loss and significantly reduced upper and lower respiratory viral burden. These results indicate in vivo cooperativity between S1 and S2 specific neutralizing hmAbs and that potent universal coronavirus neutralizing mAbs with therapeutic potential can be induced in humans and can guide universal coronavirus vaccine development.

Identification and characterization of a marine bacterium extract from Mameliella sp. M20D2D8 with antiviral effects against influenza A and B viruses.

Despite significant improvements in vaccines and chemotherapeutic drugs, pathogenic RNA viruses continue to have a profound impact on the global economy and pose a serious threat to animal and human health through emerging and re-emerging outbreaks of diseases. To overcome the challenge of viral adaptation and evolution, increased vigilance is required. Particularly, antiviral drugs derived from new, natural sources provide an attractive strategy for controlling problematic viral diseases. In this antiviral study, we discovered a previously unknown bacterium, Mameliella sp. M20D2D8, by conducting an antiviral screening of marine microorganisms. An extract from M20D2D8 exhibited antiviral activity with low cytotoxicity and was found to be effective in vitro against multiple influenza virus strains: A/PR8 (IC50 = 2.93 µg/mL, SI = 294.85), A/Phil82 (IC50 = 1.42 µg/mL, SI = 608.38), and B/Yamagata (IC50 = 1.59 µg/mL, SI = 543.33). The antiviral action was found to occur in the post-entry stages of viral replication and to suppress viral replication by inducing apoptosis in infected cells. Moreover, it efficiently suppressed viral genome replication, protein synthesis, and infectivity in MDCK and A549 cells. Our findings highlight the antiviral capabilities of a novel marine bacterium, which could potentially be useful in the development of drugs for controlling viral diseases.

FACT subunit SUPT16H associates with BRD4 and contributes to silencing of interferon signaling.

FACT (FAcilitates Chromatin Transcription) is a heterodimeric protein complex composed of SUPT16H and SSRP1, and a histone chaperone participating in chromatin remodeling during gene transcription. FACT complex is profoundly regulated, and contributes to both gene activation and suppression. Here we reported that SUPT16H, a subunit of FACT, is acetylated in both epithelial and natural killer (NK) cells. The histone acetyltransferase TIP60 contributes to the acetylation of SUPT16H middle domain (MD) at lysine 674 (K674). Such acetylation of SUPT16H is recognized by bromodomain protein BRD4, which promotes protein stability of SUPT16H in both epithelial and NK cells. We further demonstrated that SUPT16H-BRD4 associates with histone modification enzymes (HDAC1, EZH2), and further regulates their activation status and/or promoter association as well as affects the relevant histone marks (H3ac, H3K9me3 and H3K27me3). BRD4 is known to profoundly regulate interferon (IFN) signaling, while such function of SUPT16H has never been explored. Surprisingly, our results revealed that SUPT16H genetic knockdown via RNAi or pharmacological inhibition by using its inhibitor, curaxin 137 (CBL0137), results in the induction of IFNs and interferon-stimulated genes (ISGs). Through this mechanism, depletion or inhibition of SUPT16H is shown to efficiently inhibit infection of multiple viruses, including Zika, influenza, and SARS-CoV-2. Furthermore, we demonstrated that depletion or inhibition of SUPT16H also causes the remarkable activation of IFN signaling in NK cells, which promotes the NK-mediated killing of virus-infected cells in a co-culture system using human primary NK cells. Overall, our studies unraveled the previously un-appreciated role of FACT complex in coordinating with BRD4 and regulating IFN signaling in both epithelial and NK cells, and also proposed the novel application of the FACT inhibitor CBL0137 to treat viral infections.

Analysis of SARS-CoV-2 infection dynamic in vivo using reporter-expressing viruses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the current COVID-19 pandemic, is one of the biggest threats to public health. However, the dynamic of SARS-CoV-2 infection remains poorly understood. Replication-competent recombinant viruses expressing reporter genes provide valuable tools to investigate viral infection. Low levels of reporter gene expressed from previous reporter-expressing recombinant (r)SARS-CoV-2 in the locus of the open reading frame (ORF)7a protein have jeopardized their use to monitor the dynamic of SARS-CoV-2 infection in vitro or in vivo. Here, we report an alternative strategy where reporter genes were placed upstream of the highly expressed viral nucleocapsid (N) gene followed by a porcine tescherovirus (PTV-1) 2A proteolytic cleavage site. The higher levels of reporter expression using this strategy resulted in efficient visualization of rSARS-CoV-2 in infected cultured cells and excised lungs or whole organism of infected K18 human angiotensin converting enzyme 2 (hACE2) transgenic mice. Importantly, real-time viral infection was readily tracked using a noninvasive in vivo imaging system and allowed us to rapidly identify antibodies which are able to neutralize SARS-CoV-2 infection in vivo. Notably, these reporter-expressing rSARS-CoV-2, in which a viral gene was not deleted, not only retained wild-type (WT) virus-like pathogenicity in vivo but also exhibited high stability in vitro and in vivo, supporting their use to investigate viral infection, dissemination, pathogenesis, and therapeutic interventions for the treatment of SARS-CoV-2 in vivo.

Opposite Effects of Apoptotic and Necroptotic Cellular Pathways on Rotavirus Replication.

Group A rotavirus (RVA), one of the leading pathogens causing severe acute gastroenteritis in children and a wide variety of young animals worldwide, induces apoptosis upon infecting cells. Though RVA-induced apoptosis mediated via the dual modulation of its NSP4 and NSP1 proteins is relatively well studied, the nature and signaling pathway(s) involved in RVA-induced necroptosis are yet to be fully elucidated. Here, we demonstrate the nature of RVA-induced necroptosis, the signaling cascade involved, and correlation with RVA-induced apoptosis. Infection with the bovine NCDV and human DS-1 RVA strains was shown to activate receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed-lineage kinase domain-like protein (MLKL), the key necroptosis molecules in virus-infected cells. Using an immunoprecipitation assay, RIPK1 was found to bind phosphorylated RIPK3 (pRIPK3) and pMLKL. pMLKL, the major executioner molecule in the necroptotic pathway, was translocated to the plasma membrane of RVA-infected cells to puncture the cell membrane. Interestingly, transfection of RVA NSP4 also induced necroptosis through the RIPK1/RIPK3/MLKL necroptosis pathway. Blockage of each key necroptosis molecule in the RVA-infected or NSP4-transfected cells resulted in decreased necroptosis but increased cell viability and apoptosis, thereby resulting in decreased viral yields in the RVA-infected cells. In contrast, suppression of RVA-induced apoptosis increased necroptosis and virus yields. Our findings suggest that RVA NSP4 also induces necroptosis via the RIPK1/RIPK3/MLKL necroptosis pathway. Moreover, necroptosis and apoptosis-which have proviral and antiviral effects, respectively-exhibited cross talk in RVA-infected cells. These findings significantly increase our understanding of the nature of RVA-induced necroptosis and the cross talk between RVA-induced necroptosis and apoptosis. IMPORTANCE Viral infection usually culminates in cell death through apoptosis, necroptosis, and, rarely, pyroptosis. Necroptosis is a form of programmed necrosis that is mediated by signaling complexes of the receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed-lineage kinase domain-like protein (MLKL). Although apoptosis induction by rotavirus and its NSP4 protein is well known, rotavirus-induced necroptosis is not fully understood. Here, we demonstrate that rotavirus and also its NSP4 protein can induce necroptosis in cultured cells through activation of the RIPK1/RIPK3/MLKL necroptosis pathway. Moreover, rotavirus-induced necroptosis and apoptosis have opposite effects on viral yield, i.e., they function as proviral and antiviral processes, respectively, and counterbalance each other in rotavirus-infected cells. Our findings provide important insights for understanding the nature of rotavirus-induced necroptosis and the development of novel therapeutic strategies against infection with rotavirus and other RNA viruses.

The K18-Human ACE2 Transgenic Mouse Model Recapitulates Non-severe and Severe COVID-19 in Response to an Infectious Dose of the SARS-CoV-2 Virus.

A comprehensive analysis and characterization of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection model that mimics non-severe and severe coronavirus disease 2019 (COVID-19) in humans is warranted for understating the virus and developing preventive and therapeutic agents. Here, we characterized the K18-hACE2 mouse model expressing human (h)ACE2 in mice, controlled by the human keratin 18 (K18) promoter, in the epithelia, including airway epithelial cells where SARS-CoV-2 infections typically start. We found that intranasal inoculation with higher viral doses (2 × 103 and 2 × 104 PFU) of SARS-CoV-2 caused lethality of all mice and severe damage of various organs, including lung, liver, and kidney, while lower doses (2 × 101 and 2 × 102 PFU) led to less severe tissue damage and some mice recovered from the infection. In this hACE2 mouse model, SARS-CoV-2 infection damaged multiple tissues, with a dose-dependent effect in most tissues. Similar damage was observed in postmortem samples from COVID-19 patients. Finally, the mice that recovered from infection with a low dose of virus survived rechallenge with a high dose of virus. Compared to other existing models, the K18-hACE2 model seems to be the most sensitive COVID-19 model reported to date. Our work expands the information available about this model to include analysis of multiple infectious doses and various tissues with comparison to human postmortem samples from COVID-19 patients. In conclusion, the K18-hACE2 mouse model recapitulates both severe and non-severe COVID-19 in humans being dose-dependent and can provide insight into disease progression and the efficacy of therapeutics for preventing or treating COVID-19. IMPORTANCE The pandemic of coronavirus disease 2019 (COVID-19) has reached nearly 240 million cases, caused nearly 5 million deaths worldwide as of October 2021, and has raised an urgent need for the development of novel drugs and therapeutics to prevent the spread and pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To achieve this goal, an animal model that recapitulates the features of human COVID-19 disease progress and pathogenesis is greatly needed. In this study, we have comprehensively characterized a mouse model of SARS-CoV-2 infection using K18-hACE2 transgenic mice. We infected the mice with low and high doses of SARS-CoV-2 to study the pathogenesis and survival in response to different infection patterns. Moreover, we compared the pathogenesis of the K18-hACE2 transgenic mice with that of the COVID-19 patients to show that this model could be a useful tool for the development of antiviral drugs and therapeutics.

Targeted Protein Upregulation of STING for Boosting the Efficacy of Immunotherapy.

Modulating target proteins via the ubiquitin-proteasome system has recently expanded the scope of pharmacological inventions. Stimulator of interferon genes (STING) is an auspicious target for immunotherapy. Seminal studies envisioned the importance of STING as well as the utility of its agonists in immunotherapy outcomes. Herein, we suggest UPPRIS (upregulation of target proteins by protein-protein interaction strategy) to pharmacologically increase cellular STING levels for improved immunotherapy. We discovered the small molecule SB24011 that inhibits STING-TRIM29 E3 ligase interaction, thus blocking TRIM29-induced degradation of STING. SB24011 enhanced STING immunity by upregulating STING protein levels, which robustly potentiated the immunotherapy efficacy of STING agonist and anti-PD-1 antibody via systemic anticancer immunity. Overall, we demonstrated that targeted protein upregulation of STING can be a promising approach for immuno-oncology.

Generation and Characterization of recombinant SARS-CoV-2 expressing reporter genes.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen responsible of coronavirus disease 2019 (COVID-19), has devastated public health services and economies worldwide. Despite global efforts to contain the COVID-19 pandemic, SARS-CoV-2 is now found in over 200 countries and has caused an upward death toll of over 1 million human lives as of November 2020. To date, only one Food and Drug Administration (FDA)-approved therapeutic drug (Remdesivir) and a monoclonal antibody, MAb (Bamlanivimab) are available for the treatment of SARS-CoV-2. As with other viruses, studying SARS-CoV-2 requires the use of secondary approaches to detect the presence of the virus in infected cells. To overcome this limitation, we have generated replication-competent recombinant (r)SARS-CoV-2 expressing fluorescent (Venus or mCherry) or bioluminescent (Nluc) reporter genes. Vero E6 cells infected with reporter-expressing rSARS-CoV-2 can be easily detected via fluorescence or luciferase expression and display a good correlation between reporter gene expression and viral replication. Moreover, rSARS-CoV-2 expressing reporter genes have comparable plaque sizes and growth kinetics to those of wild-type virus, rSARS-CoV-2/WT. We used these reporter-expressing rSARS-CoV-2 to demonstrate their feasibility to identify neutralizing antibodies (NAbs) or antiviral drugs. Our results demonstrate that reporter-expressing rSARS-CoV-2 represent an excellent option to identify therapeutics for the treatment of SARS-CoV-2, where reporter gene expression can be used as valid surrogates to track viral infection. Moreover, the ability to manipulate the viral genome opens the feasibility of generating viruses expressing foreign genes for their use as vaccines for the treatment of SARS-CoV-2 infection.IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen that causes coronavirus disease 2019 (COVID-19), has significantly impacted the human health and economic status worldwide. There is an urgent need to identify effective prophylactics and therapeutics for the treatment of SARS-CoV-2 infection and associated COVID-19 disease. The use of fluorescent- or luciferase-expressing reporter expressing viruses has significantly advanced viral research. Here, we generated recombinant (r)SARS-CoV-2 expressing fluorescent (Venus and mCherry) or luciferase (Nluc) reporter genes and demonstrate that they represent an excellent option to track viral infections in vitro. Importantly, reporter-expressing rSARS-CoV-2 display similar growth kinetics and plaque phenotype that their wild-type counterpart (rSARS-CoV-2/WT), demonstrating their feasibility to identify drugs and/or neutralizing antibodies (NAbs) for the therapeutic treatment of SARS-CoV-2. Henceforth, these reporter-expressing rSARS-CoV-2 can be used to interrogate large libraries of compounds and/or monoclonal antibodies (MAb), in high-throughput screening settings, to identify those with therapeutic potential against SARS-CoV-2.

Contribution of SARS-CoV-2 Accessory Proteins to Viral Pathogenicity in K18 Human ACE2 Transgenic Mice.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen responsible for the current coronavirus disease 2019 (COVID-19) pandemic. As of 19 May 2021, John Hopkins University's COVID-19 tracking platform reported 3.3 million deaths associated with SARS-CoV-2 infection. Currently, the World Health Organization has granted emergency use listing (EUL) to six COVID-19 vaccine candidates. However, much of the pathogenesis observed during SARS-CoV-2 infection remains elusive. To gain insight into the contribution of individual accessory open reading frame (ORF) proteins in SARS-CoV-2 pathogenesis, we used our recently described reverse-genetics system approach to successfully engineer recombinant SARS-CoV-2 (rSARS-CoV-2) constructs; we removed individual viral ORF3a, -6, -7a, -7b, and -8 proteins from them, and we characterized the resulting recombinant viruses in vitro and in vivo. Our results indicate differences in plaque morphology, with ORF-deficient (ΔORF) viruses producing smaller plaques than those of the wild type (rSARS-CoV-2/WT). However, growth kinetics of ΔORF viruses were like those of rSARS-CoV-2/WT. Interestingly, infection of K18 human angiotensin-converting enzyme 2 (hACE2) transgenic mice with the ΔORF rSARS-CoV-2s identified ORF3a and ORF6 as the major contributors of viral pathogenesis, while ΔORF7a, ΔORF7b, and ΔORF8 rSARS-CoV-2s induced pathology comparable to that of rSARS-CoV-2/WT. This study demonstrates the robustness of our reverse-genetics system to generate rSARS-CoV-2 constructs and the major role for ORF3a and ORF6 in viral pathogenesis, providing important information for the generation of attenuated forms of SARS-CoV-2 for their implementation as live attenuated vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19. IMPORTANCE Despite great efforts put forward worldwide to combat the current coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to be a human health and socioeconomic threat. Insights into the pathogenesis of SARS-CoV-2 and the contribution of viral proteins to disease outcome remain elusive. Our study aims (i) to determine the contribution of SARS-CoV-2 accessory open reading frame (ORF) proteins to viral pathogenesis and disease outcome and (ii) to develop a synergistic platform combining our robust reverse-genetics system to generate recombinant SARS-CoV-2 constructs with a validated rodent model of infection and disease. We demonstrate that SARS-CoV-2 ORF3a and ORF6 contribute to lung pathology and ultimately disease outcome in K18 hACE2 transgenic mice, while ORF7a, ORF7b, and ORF8 have little impact on disease outcome. Moreover, our combinatory platform serves as a foundation for generating attenuated forms of the virus to develop live attenuated vaccines for the treatment of SARS-CoV-2.

A Bifluorescent-Based Assay for the Identification of Neutralizing Antibodies against SARS-CoV-2 Variants of Concern In Vitro and In Vivo.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and has been responsible for the still ongoing coronavirus disease 2019 (COVID-19) pandemic. Prophylactic vaccines have been authorized by the U.S. Food and Drug Administration (FDA) for the prevention of COVID-19. Identification of SARS-CoV-2-neutralizing antibodies (NAbs) is important to assess vaccine protection efficacy, including their ability to protect against emerging SARS-CoV-2 variants of concern (VoC). Here, we report the generation and use of a recombinant (r)SARS-CoV-2 USA/WA1/2020 (WA-1) strain expressing Venus and an rSARS-CoV-2 strain expressing mCherry and containing mutations K417N, E484K, and N501Y found in the receptor binding domain (RBD) of the spike (S) glycoprotein of the South African (SA) B.1.351 (beta [ß]) VoC in bifluorescent-based assays to rapidly and accurately identify human monoclonal antibodies (hMAbs) able to neutralize both viral infections in vitro and in vivo. Importantly, our bifluorescent-based system accurately recapitulated findings observed using individual viruses. Moreover, fluorescent-expressing rSARS-CoV-2 strain and the parental wild-type (WT) rSARS-CoV-2 WA-1 strain had similar viral fitness in vitro, as well as similar virulence and pathogenicity in vivo in the K18 human angiotensin-converting enzyme 2 (hACE2) transgenic mouse model of SARS-CoV-2 infection. We demonstrate that these new fluorescent-expressing rSARS-CoV-2 can be used in vitro and in vivo to easily identify hMAbs that simultaneously neutralize different SARS-CoV-2 strains, including VoC, for the rapid assessment of vaccine efficacy or the identification of prophylactic and/or therapeutic broadly NAbs for the treatment of SARS-CoV-2 infection. IMPORTANCE SARS-CoV-2 is responsible of the COVID-19 pandemic that has warped daily routines and socioeconomics. There is still an urgent need for prophylactics and therapeutics to treat SARS-CoV-2 infections. In this study, we demonstrate the feasibility of using bifluorescent-based assays for the rapid identification of hMAbs with neutralizing activity against SARS-CoV-2, including VoC in vitro and in vivo. Importantly, results obtained with these bifluorescent-based assays recapitulate those observed with individual viruses, demonstrating their feasibility to rapidly advance our understanding of vaccine efficacy and to identify broadly protective human NAbs for the therapeutic treatment of SARS-CoV-2.

Identification and Characterization of Novel Compounds with Broad-Spectrum Antiviral Activity against Influenza A and B Viruses.

Influenza A (IAV) and influenza B (IBV) viruses are highly contagious pathogens that cause fatal respiratory disease every year, with high economic impact. In addition, IAV can cause pandemic infections with great consequences when new viruses are introduced into humans. In this study, we evaluated 10 previously described compounds with antiviral activity against mammarenaviruses for their ability to inhibit IAV infection using our recently described bireporter influenza A/Puerto Rico/8/34 (PR8) H1N1 (BIRFLU). Among the 10 tested compounds, eight (antimycin A [AmA], brequinar [BRQ], 6-azauridine, azaribine, pyrazofurin [PF], AVN-944, mycophenolate mofetil [MMF], and mycophenolic acid [MPA]), but not obatoclax or Osu-03012, showed potent anti-influenza virus activity under posttreatment conditions [median 50% effective concentration (EC50) = 3.80 nM to 1.73 µM; selective index SI for 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, >28.90 to 13,157.89]. AmA, 6-azauridine, azaribine, and PF also showed potent inhibitory effect in pretreatment (EC50 = 0.14 µM to 0.55 µM; SI-MTT = 70.12 to >357.14) or cotreatment (EC50 = 34.69 nM to 7.52 µM; SI-MTT = 5.24 to > 1,441.33) settings. All of the compounds tested inhibited viral genome replication and gene transcription, and none of them affected host cellular RNA polymerase II activities. The antiviral activity of the eight identified compounds against BIRFLU was further confirmed with seasonal IAVs (A/California/04/2009 H1N1 and A/Wyoming/3/2003 H3N2) and an IBV (B/Brisbane/60/2008, Victoria lineage), demonstrating their broad-spectrum prophylactic and therapeutic activity against currently circulating influenza viruses in humans. Together, our results identified a new set of antiviral compounds for the potential treatment of influenza viral infections.IMPORTANCE Influenza viruses are highly contagious pathogens and are a major threat to human health. Vaccination remains the most effective tool to protect humans against influenza infection. However, vaccination does not always guarantee complete protection against drifted or, more noticeably, shifted influenza viruses. Although U.S. Food and Drug Administration (FDA) drugs are approved for the treatment of influenza infections, influenza viruses resistant to current FDA antivirals have been reported and continue to emerge. Therefore, there is an urgent need to find novel antivirals for the treatment of influenza viral infections in humans, a search that could be expedited by repurposing currently approved drugs. In this study, we assessed the influenza antiviral activity of 10 compounds previously shown to inhibit mammarenavirus infection. Among them, eight drugs showed antiviral activities, providing a new battery of drugs that could be used for the treatment of influenza infections.

Kidney residency of VISTA-positive macrophages accelerates repair from ischemic injury.

Tissue-resident macrophages have unique tissue-specific functions in maintaining homeostasis and resolving inflammation. However, the repair role and relevant molecules of kidney-resident macrophages after ischemic injury remain unresolved. To this end, mice without kidney-resident R1 macrophages but containing infiltrating monocyte-derived R2 macrophages were generated using differential cellular kinetics following clodronate liposome treatment. When ischemia-reperfusion injury was induced in these mice, late phase repair was reduced. Transcriptomic and flow cytometric analyses identified that V-domain Ig suppressor of T cell activation (VISTA), an inhibitory immune checkpoint molecule, was constitutively expressed in kidney-resident R1 macrophages, but not in other tissue-resident macrophages. Here, VISTA functioned as a scavenger of apoptotic cells and served as a checkpoint to control kidney-infiltrating T cells upon T cell receptor-mediated stimulation. Together these functions improved the repair process after ischemia-reperfusion injury. CD14+ CD33+ mononuclear phagocytes of human kidney also expressed VISTA, which has similar functions to the mouse counterpart. Thus, VISTA is upregulated in kidney macrophages in a tissue-dependent manner and plays a repair role during ischemic injury.

Early Porcine Sapovirus Infection Disrupts Tight Junctions and Uses Occludin as a Coreceptor.

The genus Sapovirus belongs to the family Caliciviridae, and its members are common causative agents of severe acute gastroenteritis in both humans and animals. Some caliciviruses are known to use either terminal sialic acids or histo-blood group antigens as attachment factors and/or cell surface proteins, such as CD300lf, CD300ld, and junctional adhesion molecule 1 of tight junctions (TJs), as receptors. However, the roles of TJs and their proteins in sapovirus entry have not been examined. In this study, we found that porcine sapovirus (PSaV) significantly decreased transepithelial electrical resistance and increased paracellular permeability early in infection of LLC-PK cells, suggesting that PSaV dissociates TJs of cells. This led to the interaction between PSaV particles and occludin, which traveled in a complex into late endosomes via Rab5- and Rab7-dependent trafficking. Inhibition of occludin using small interfering RNA (siRNA), a specific antibody, or a dominant-negative mutant significantly blocked the entry of PSaV. Transient expression of occludin in nonpermissive Chinese hamster ovary (CHO) cells conferred susceptibility to PSaV, but only for a limited time. Although claudin-1, another TJ protein, neither directly interacted nor was internalized with PSaV particles, it facilitated PSaV entry and replication in the LLC-PK cells. We conclude that PSaV particles enter LLC-PK cells by binding to occludin as a coreceptor in PSaV-dissociated TJs. PSaV and occludin then form a complex that moves to late endosomes via Rab5- and Rab7-dependent trafficking. In addition, claudin-1 in the TJs opened by PSaV infection facilitates PSaV entry and infection as an entry factor.IMPORTANCE Sapoviruses (SaVs) cause severe acute gastroenteritis in humans and animals. Although they replicate in intestinal epithelial cells, which are tightly sealed by apical-junctional complexes, such as tight junctions (TJs), the mechanisms by which SaVs hijack TJs and their proteins for successful entry and infection remain largely unknown. Here, we demonstrate that porcine SaVs (PSaVs) induce early dissociation of TJs, allowing them to bind to the TJ protein occludin as a functional coreceptor. PSaVs then travel in a complex with occludin into late endosomes through Rab5- and Rab7-dependent trafficking. Claudin-1, another TJ protein, does not directly interact with PSaV but facilitates the entry of PSaV into cells as an entry factor. This work contributes to our understanding of the entry of SaV and other caliciviruses into cells and may aid in the development of efficient and affordable drugs to treat SaV infections.

Dual Recognition of Sialic Acid and αGal Epitopes by the VP8* Domains of the Bovine Rotavirus G6P[5] WC3 and of Its Mono-reassortant G4P[5] RotaTeq Vaccine Strains.

Group A rotaviruses, an important cause of severe diarrhea in children and young animals, initiate infection via interactions of the VP8* domain of the VP4 spike protein with cell surface sialic acids (SAs) or histo-blood group antigens (HBGAs). Although the bovine G6P[5] WC3 strain is an important animal pathogen and is also used in the bovine-human reassortant RotaTeq vaccine, the receptor(s) for the VP8* domain of WC3 and its reassortant strains have not yet been identified. In the present study, HBGA- and saliva-binding assays showed that both G6P[5] WC3 and mono-reassortant G4P[5] strains recognized the αGal HBGA. The infectivity of both P[5]-bearing strains was significantly reduced in αGal-free MA-104 cells by pretreatment with a broadly specific neuraminidase or by coincubation with the α2,6-linked SA-specific Sambucus nigra lectin, but not by the α2,3-linked specific sialidase or by Maackia amurensis lectin. Free NeuAc and the αGal trisaccharide also prevented the infectivity of both strains. This indicated that both P[5]-bearing strains utilize α2,6-linked SA as a ligand on MA104 cells. However, the two strains replicated in differentiated bovine small intestinal enteroids and in their human counterparts that lack α2,6-linked SA or αGal HBGA, suggesting that additional or alternative receptors such as integrins, hsp70, and tight-junction proteins bound directly to the VP5* domain can be used by the P[5]-bearing strains to initiate the infection of human cells. In addition, these data also suggested that P[5]-bearing strains have potential for cross-species transmission.IMPORTANCE Group A rotaviruses initiate infection through the binding of the VP8* domain of the VP4 protein to sialic acids (SAs) or histo-blood group antigens (HBGAs). Although the bovine G6P[5] WC3 strain is an important animal pathogen and is used as the backbone in the bovine-human reassortant RotaTeq vaccine, the receptor(s) for their P[5] VP8* domain has remained elusive. Using a variety of approaches, we demonstrated that the WC3 and bovine-human mono-reassortant G4P[5] vaccine strains recognize both α2,6-linked SA and αGal HBGA as ligands. Neither ligand is expressed on human small intestinal epithelial cells, explaining the absence of natural human infection by P[5]-bearing strains. However, we observed that the P[5]-bearing WC3 and G4P[5] RotaTeq vaccine strains could still infect human intestinal epithelial cells. Thus, the four P[5] RotaTeq vaccine strains potentially binding to additional alternative receptors may be efficient and effective in providing protection against severe rotavirus disease in human.

Activation of PI3K, Akt, and ERK during early rotavirus infection leads to V-ATPase-dependent endosomal acidification required for uncoating.

The cellular PI3K/Akt and/or MEK/ERK signaling pathways mediate the entry process or endosomal acidification during infection of many viruses. However, their roles in the early infection events of group A rotaviruses (RVAs) have remained elusive. Here, we show that late-penetration (L-P) human DS-1 and bovine NCDV RVA strains stimulate these signaling pathways very early in the infection. Inhibition of both signaling pathways significantly reduced production of viral progeny due to blockage of virus particles in the late endosome, indicating that neither of the two signaling pathways is involved in virus trafficking. However, immunoprecipitation assays using antibodies specific for pPI3K, pAkt, pERK and the subunit E of the V-ATPase co-immunoprecipitated the V-ATPase in complex with pPI3K, pAkt, and pERK. Moreover, Duolink proximity ligation assay revealed direct association of the subunit E of the V-ATPase with the molecules pPI3K, pAkt, and pERK, indicating that both signaling pathways are involved in V-ATPase-dependent endosomal acidification. Acidic replenishment of the medium restored uncoating of the RVA strains in cells pretreated with inhibitors specific for both signaling pathways, confirming the above results. Isolated components of the outer capsid proteins, expressed as VP4-VP8* and VP4-VP5* domains, and VP7, activated the PI3K/Akt and MEK/ERK pathways. Furthermore, psoralen-UV-inactivated RVA and CsCl-purified RVA triple-layered particles triggered activation of the PI3K/Akt and MEK/ERK pathways, confirming the above results. Our data demonstrate that multistep binding of outer capsid proteins of L-P RVA strains with cell surface receptors phosphorylates PI3K, Akt, and ERK, which in turn directly interact with the subunit E of the V-ATPase to acidify the late endosome for uncoating of RVAs. This study provides a better understanding of the RVA-host interaction during viral uncoating, which is of importance for the development of strategies aiming at controlling or preventing RVA infections.

Phosphatidylinositol 3-Kinase/Akt and MEK/ERK Signaling Pathways Facilitate Sapovirus Trafficking and Late Endosomal Acidification for Viral Uncoating in LLC-PK Cells.

Sapovirus, an important cause of acute gastroenteritis in humans and animals, travels from the early to the late endosomes and requires late endosomal acidification for viral uncoating. However, the signaling pathways responsible for these viral entry processes remain unknown. Here we demonstrate the receptor-mediated early activation of phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein extracellular signal-regulated kinase/extracellular signal-regulated kinase (MEK/ERK) signaling pathways involved in sapovirus entry processes. Both signaling pathways were activated during the early stage of porcine sapovirus (PSaV) infection. However, depletion of the cell surface carbohydrate receptors by pretreatment with sodium periodate or neuraminidase reduced the PSaV-induced early activation of these signaling pathways, indicating that PSaV binding to the cell surface carbohydrate receptors triggered these cascades. Addition of bile acid, known to be essential for PSaV escape from late endosomes, was also found to exert a stiffening effect to stimulate both pathways. Inhibition of these signaling pathways by use of inhibitors specific for PI3K or MEK or small interfering RNAs (siRNAs) against PI3K or MEK resulted in entrapment of PSaV particles in early endosomes and prevented their trafficking to late endosomes. Moreover, phosphorylated PI3K and ERK coimmunoprecipitated subunit E of the V-ATPase proton pump that is important for endosomal acidification. Based on our data, we conclude that receptor binding of PSaV activates both PI3K/Akt and MEK/ERK signaling pathways, which in turn promote PSaV trafficking from early to late endosomes and acidification of late endosomes for PSaV uncoating. These signaling cascades may provide a target for potent therapeutics against infections by PSaV and other caliciviruses.IMPORTANCE Sapoviruses cause acute gastroenteritis in both humans and animals. However, the host signaling pathway(s) that facilitates host cell entry by sapoviruses remains largely unknown. Here we demonstrate that porcine sapovirus (PSaV) activates both PI3K/Akt and MEK/ERK cascades at an early stage of infection. Removal of cell surface receptors decreased PSaV-induced early activation of both cascades. Moreover, blocking of PI3K/Akt and MEK/ERK cascades entrapped PSaV particles in early endosomes and prevented their trafficking to the late endosomes. PSaV-induced early activation of PI3K and ERK molecules further mediated V-ATPase-dependent late endosomal acidification for PSaV uncoating. This work unravels a new mechanism by which receptor-mediated early activation of both cascades may facilitate PSaV trafficking from early to late endosomes and late endosomal acidification for PSaV uncoating, which in turn can be a new target for treatment of sapovirus infection.

Bovine Nebovirus Interacts with a Wide Spectrum of Histo-Blood Group Antigens.

Some viruses within the Caliciviridae family initiate their replication cycle by attachment to cell surface carbohydrate moieties, histo-blood group antigens (HBGAs), and/or terminal sialic acids (SAs). Although bovine nebovirus (BNeV), one of the enteric caliciviruses, is an important causative agent of acute gastroenteritis in cattle, its attachment factors and possibly other cellular receptors remain unknown. Using a comprehensive series of protein-ligand biochemical assays, we sought to determine whether BNeV recognizes cell surface HBGAs and/or SAs as attachment factors. It was found that BNeV virus-like particles (VLPs) bound to A type/H type 2/Ley HBGAs expressed in the bovine digestive tract and are related to HBGAs expressed in humans and other host species, suggesting a wide spectrum of HBGA recognition by BNeV. BNeV VLPs also bound to a large variety of different bovine and human saliva samples of all ABH and Lewis types, supporting previously obtained results and suggesting a zoonotic potential of BNeV transmission. Removal of α1,2-linked fucose and α1,3/4-linked fucose epitopes of target HBGAs by confirmation-specific enzymes reduced the binding of BNeV VLPs to synthetic HBGAs, bovine and human saliva, cultured cell lines, and bovine small intestine mucosa, further supporting a wide HBGA binding spectrum of BNeV through recognition of α1,2-linked fucose and α1,3/4-linked fucose epitopes of targeted HBGAs. However, removal of terminal α2,3- and α2,6-linked SAs by their specific enzyme had no inhibitory effects on binding of BNeV VLPs, indicating that BNeV does not use terminal SAs as attachment factors. Further details of the binding specificity of BNeV remain to be explored.IMPORTANCE Enteric caliciviruses such as noroviruses, sapoviruses, and recoviruses are the most important etiological agents of severe acute gastroenteritis in humans and many other mammalian host species. They initiate infection by attachment to cell surface carbohydrate moieties, HBGAs, and/or terminal SAs. However, the attachment factor(s) for BNeV, a recently classified enteric calicivirus genus/type species, remains unexplored. Here, we demonstrate that BNeV VLPs have a wide spectrum of binding to synthetic HBGAs, bovine and human saliva samples, and bovine duodenal sections. We further discovered that α1,2-linked fucose and α1,3/4-linked fucose epitopes are essential for binding of BNeV VLPs. However, BNeV VLPs do not bind to terminal SAs on cell carbohydrates. Continued investigation regarding the proteinaceous receptor(s) will be necessary for better understanding of the tropism, pathogenesis, and host range of this important viral genus.

Development of a live attenuated trivalent porcine rotavirus A vaccine against disease caused by recent strains most prevalent in South Korea.

Porcine rotaviruses cause severe economic losses in the Korean swine industry due to G- and P-genotype mismatches between the predominant field and vaccine strains. Here, we developed a live attenuated trivalent porcine group A rotavirus vaccine using 80 cell culture passages of the representative Korean predominant strains G8P[7] 174-1, G9P[23] PRG942, and G5P[7] K71. Vaccination with the trivalent vaccine or its individual components induced no diarrhea during the first 2 weeks post-vaccination, i.e., the vaccines were attenuated. Challenge of trivalent-vaccinated or component-vaccinated piglets with homologous virulent strain(s) did not induce diarrhea for 2 weeks post-challenge. Immunization with the trivalent vaccine or its individual components also alleviated the histopathological lesions in the small intestines caused by challenge with the corresponding original virulent strain(s). Fecal secretory IgAs specific for each of vaccine strains were detected starting at 14 days post-vaccination (dpv), and IgA levels gradually increased up to 28 dpv. Oral immunization with the trivalent vaccine or its individual components induced high levels of serum virus-neutralizing antibody by 7 dpv. No diarrhea was observed in any experimental piglets during five consecutive passages of each vaccine strain. Our data indicated that the live attenuated trivalent vaccine was safe and effective at protecting piglets from diarrhea induced by challenge exposure of homologous virulent strains. This trivalent vaccine will potentially contribute toward controlling porcine rotavirus disease in South Korea and other countries where rotavirus infections with similar G and P genotypes are problematic.