Intranasal vaccination with an NDV-vectored SARS-CoV-2 vaccine protects against Delta and Omicron challenges.

The rapid development and deployment of vaccines following the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been estimated to have saved millions of lives. Despite their immense success, there remains a need for next-generation vaccination approaches for SARS-CoV-2 and future emerging coronaviruses and other respiratory viruses. Here we utilized a Newcastle Disease virus (NDV) vectored vaccine expressing the ancestral SARS-CoV-2 spike protein in a pre-fusion stabilized chimeric conformation (NDV-PFS). When delivered intranasally, NDV-PFS protected both Syrian hamsters and K18 mice against Delta and Omicron SARS-CoV-2 variants of concern. Additionally, intranasal vaccination induced robust, durable protection that was extended to 6 months post-vaccination. Overall, our data provide evidence that NDV-vectored vaccines represent a viable next-generation mucosal vaccination approach.

Mucosal Vaccination with a Newcastle Disease Virus-Vectored Vaccine Reduces Viral Loads in SARS-CoV-2-Infected Cynomolgus Macaques.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged following an outbreak of unexplained viral illness in China in late 2019. Since then, it has spread globally causing a pandemic that has resulted in millions of deaths and has had enormous economic and social consequences. The emergence of SARS-CoV-2 saw the rapid and widespread development of a number of vaccine candidates worldwide, and this never-before-seen pace of vaccine development led to several candidates progressing immediately through clinical trials. Many countries have now approved vaccines for emergency use, with large-scale vaccination programs ongoing. Despite these successes, there remains a need for ongoing pre-clinical and clinical development of vaccine candidates against SARS-CoV-2, as well as vaccines that can elicit strong mucosal immune responses. Here, we report on the efficacy of a Newcastle disease virus-vectored vaccine candidate expressing SARS-CoV-2 spike protein (NDV-FLS) administered to cynomolgus macaques. Macaques given two doses of the vaccine via respiratory immunization developed robust immune responses and had reduced viral RNA levels in nasal swabs and in the lower airway. Our data indicate that NDV-FLS administered mucosally provides significant protection against SARS-CoV-2 infection, resulting in reduced viral burden and disease manifestation, and should be considered as a viable candidate for clinical development.

Extracellular vesicle storm during the course of Ebola virus infection in primates.

Introduction: Ebola virus (EBOV) is an RNA virus of the Filoviridae family that is responsible for outbreaks of hemorrhagic fevers in primates with a lethality rate as high as 90%. EBOV primarily targets host macrophages leading to cell activation and systemic cytokine storm, and fatal infection is associated with an inhibited interferon response, and lymphopenia. The EBOV surface glycoprotein (GP) has been shown to directly induce T cell depletion and can be secreted outside the virion via extracellular vesicles (EVs), though most studies are limited to epithelial cells and underlying mechanisms remain poorly elucidated. Methods: To assess the role of GP on EBOV-induced dysregulation of host immunity, we first utilized EBOV virus-like particles (VLPs) expressing VP40 and NP either alone (Bald-VLP) or in conjunction with GP (VLP-GP) to investigate early inflammatory responses in THP-1 macrophages and in a murine model. We then sought to decipher the role of non-classical inflammatory mediators such as EVs over the course of EBOV infection in two EBOV-infected rhesus macaques by isolating and characterizing circulatory EVs throughout disease progression using size exclusion chromatography, nanoparticle tracking-analysis, and LC-MS/MS. Results: While all VLPs could induce inflammatory mediators and recruit small peritoneal macrophages, pro-inflammatory cytokine and chemokine gene expression was exacerbated by the presence of GP. Further, quantification of EVs isolated from infected rhesus macaques revealed that the concentration of vesicles peaked in circulation at the terminal stage, at which time EBOV GP could be detected in host-derived exosomes. Moreover, comparative proteomics conducted across EV populations isolated from serum at various time points before and after infection revealed differences in host-derived protein content that were most significantly pronounced at the endpoint of infection, including significant expression of mediators of TLR4 signaling. Discussion: These results suggest a dynamic role for EVs in the modification of disease states in the context of EBOV. Overall, our work highlights the importance of viral factors, such as the GP, and host derived EVs in the inflammatory cascade and pathogenesis of EBOV, which can be collectively further exploited for novel antiviral development.

Oral Immunization with rVSV Bivalent Vaccine Elicits Protective Immune Responses, Including ADCC, against Both SARS-CoV-2 and Influenza A Viruses.

COVID-19 and influenza both cause enormous disease burdens, and vaccines are the primary measures for their control. Since these viral diseases are transmitted through the mucosal surface of the respiratory tract, developing an effective and convenient mucosal vaccine should be a high priority. We previously reported a recombinant vesicular stomatitis virus (rVSV)-based bivalent vaccine (v-EM2/SPΔC1Delta) that protects animals from both SARS-CoV-2 and influenza viruses via intramuscular and intranasal immunization. Here, we further investigated the immune response induced by oral immunization with this vaccine and its protective efficacy in mice. The results demonstrated that the oral delivery, like the intranasal route, elicited strong and protective systemic immune responses against SARS-CoV-2 and influenza A virus. This included high levels of neutralizing antibodies (NAbs) against SARS-CoV-2, as well as strong anti-SARS-CoV-2 spike protein (SP) antibody-dependent cellular cytotoxicity (ADCC) and anti-influenza M2 ADCC responses in mice sera. Furthermore, it provided efficient protection against challenge with influenza H1N1 virus in a mouse model, with a 100% survival rate and a significantly low lung viral load of influenza virus. All these findings provide substantial evidence for the effectiveness of oral immunization with the rVSV bivalent vaccine.

Adipose Tissues from Human and Bat-Derived Cell Lines Support Ebola Virus Infection.

Ebola virus is a zoonotic pathogen with a geographic range covering diverse ecosystems that are home to many potential reservoir species. Although researchers have detected Ebola virus RNA and serological evidence of previous infection in different rodents and bats, the infectious virus has not been isolated. The field is missing critical knowledge about where the virus is maintained between outbreaks, either because the virus is rarely encountered, overlooked during sampling, and/or requires specific unknown conditions that regulate viral expression. This study assessed adipose tissue as a previously overlooked tissue capable of supporting Ebola virus infection. Adipose tissue is a dynamic endocrine organ helping to regulate and coordinate homeostasis, energy metabolism, and neuroendocrine and immune functions. Through in vitro infection of human and bat (Eptesicus fuscus) brown adipose tissue cultures using wild-type Ebola virus, this study showed high levels of viral replication for 28 days with no qualitative indicators of cytopathic effects. In addition, alterations in adipocyte metabolism following long-term infection were qualitatively observed through an increase in lipid droplet number while decreasing in size, a harbinger of lipolysis or adipocyte browning. The finding that bat and human adipocytes are susceptible to Ebola virus infection has important implications for potential tissue tropisms that have not yet been investigated. Additionally, the findings suggest how the metabolism of this tissue may play a role in pathogenesis, viral transmission, and/or zoonotic spillover events.

The Nucleocapsid Proteins of SARS-CoV-2 and Its Close Relative Bat Coronavirus RaTG13 Are Capable of Inhibiting PKR- and RNase L-Mediated Antiviral Pathways.

Coronaviruses (CoVs), including severe acute respiratory syndrome CoV (SARS-CoV), Middle East respiratory syndrome CoV (MERS-CoV), and SARS-CoV-2, produce double-stranded RNA (dsRNA) that activates antiviral pathways such as PKR and OAS/RNase L. To successfully replicate in hosts, viruses must evade such antiviral pathways. Currently, the mechanism of how SARS-CoV-2 antagonizes dsRNA-activated antiviral pathways is unknown. In this study, we demonstrate that the SARS-CoV-2 nucleocapsid (N) protein, the most abundant viral structural protein, is capable of binding to dsRNA and phosphorylated PKR, inhibiting both the PKR and OAS/RNase L pathways. The N protein of the bat coronavirus (bat-CoV) RaTG13, the closest relative of SARS-CoV-2, has a similar ability to inhibit the human PKR and RNase L antiviral pathways. Via mutagenic analysis, we found that the C-terminal domain (CTD) of the N protein is sufficient for binding dsRNA and inhibiting RNase L activity. Interestingly, while the CTD is also sufficient for binding phosphorylated PKR, the inhibition of PKR antiviral activity requires not only the CTD but also the central linker region (LKR). Thus, our findings demonstrate that the SARS-CoV-2 N protein is capable of antagonizing the two critical antiviral pathways activated by viral dsRNA and that its inhibition of PKR activities requires more than dsRNA binding mediated by the CTD. IMPORTANCE The high transmissibility of SARS-CoV-2 is an important viral factor defining the coronavirus disease 2019 (COVID-19) pandemic. To transmit efficiently, SARS-CoV-2 must be capable of disarming the innate immune response of its host efficiently. Here, we describe that the nucleocapsid protein of SARS-CoV-2 is capable of inhibiting two critical innate antiviral pathways, PKR and OAS/RNase L. Moreover, the counterpart of the closest animal coronavirus relative of SARS-CoV-2, bat-CoV RaTG13, can also inhibit human PKR and OAS/RNase L antiviral activities. Thus, the importance of our discovery for understanding the COVID-19 pandemic is 2-fold. First, the ability of SARS-CoV-2 N to inhibit innate antiviral activity is likely a factor contributing to the transmissibility and pathogenicity of the virus. Second, the bat relative of SARS-CoV-2 has the capacity to inhibit human innate immunity, which thus likely contributed to the establishment of infection in humans. The findings described in this study are valuable for developing novel antivirals and vaccines.

Sphingosine Kinases Promote Ebola Virus Infection and Can Be Targeted to Inhibit Filoviruses, Coronaviruses, and Arenaviruses Using Late Endocytic Trafficking to Enter Cells.

Entry of enveloped viruses in host cells requires the fusion of viral and host cell membranes, a process that is facilitated by viral fusion proteins protruding from the viral envelope. These viral fusion proteins need to be triggered by host factors, and for some viruses, this event occurs inside endosomes and/or lysosomes. Consequently, these 'late-penetrating viruses' must be internalized and delivered to entry-conducive intracellular vesicles. Because endocytosis and vesicular trafficking are tightly regulated cellular processes, late-penetrating viruses also depend on specific host proteins for efficient delivery to the site of fusion, suggesting that these could be targeted for antiviral therapy. In this study, we investigated a role for sphingosine kinases (SKs) in viral entry and found that chemical inhibition of sphingosine kinase 1 (SK1) and/or SK2 and knockdown of SK1/2 inhibited entry of Ebola virus (EBOV) into host cells. Mechanistically, inhibition of SK1/2 prevented EBOV from reaching late-endosomes and lysosomes that contain the EBOV receptor, Niemann Pick C1 (NPC1). Furthermore, we present evidence that suggests that the trafficking defect caused by SK1/2 inhibition occurs independently of sphingosine-1-phosphate (S1P) signaling through cell-surface S1P receptors. Lastly, we found that chemical inhibition of SK1/2 prevents entry of other late-penetrating viruses, including arenaviruses and coronaviruses, and inhibits infection by replication-competent EBOV and SARS-CoV-2 in Huh7.5 cells. In sum, our results highlight an important role played by SK1/2 in endocytic trafficking, which can be targeted to inhibit entry of late-penetrating viruses and could serve as a starting point for the development of broad-spectrum antiviral therapeutics.

Adenosine deaminase augments SARS-CoV-2 specific cellular and humoral responses in aged mouse models of immunization and challenge.

Despite numerous clinically available vaccines and therapeutics, aged patients remain at increased risk for COVID-19 morbidity. Furthermore, various patient populations, including the aged can have suboptimal responses to SARS-CoV-2 vaccine antigens. Here, we characterized vaccine-induced responses to SARS-CoV-2 synthetic DNA vaccine antigens in aged mice. Aged mice exhibited altered cellular responses, including decreased IFNγ secretion and increased TNFα and IL-4 secretion suggestive of TH2-skewed responses. Aged mice exhibited decreased total binding and neutralizing antibodies in their serum but significantly increased TH2-type antigen-specific IgG1 antibody compared to their young counterparts. Strategies to enhance vaccine-induced immune responses are important, especially in aged patient populations. We observed that co-immunization with plasmid-encoded adenosine deaminase (pADA)enhanced immune responses in young animals. Ageing is associated with decreases in ADA function and expression. Here, we report that co-immunization with pADA enhanced IFNγ secretion while decreasing TNFα and IL-4 secretion. pADA expanded the breadth and affinity SARS-CoV-2 spike-specific antibodies while supporting TH1-type humoral responses in aged mice. scRNAseq analysis of aged lymph nodes revealed that pADA co-immunization supported a TH1 gene profile and decreased FoxP3 gene expression. Upon challenge, pADA co-immunization decreased viral loads in aged mice. These data support the use of mice as a model for age-associated decreased vaccine immunogenicity and infection-mediated morbidity and mortality in the context of SARS-CoV-2 vaccines and provide support for the use of adenosine deaminase as a molecular adjuvant in immune-challenged populations.

Significance of Conserved Regions in Coronavirus Spike Protein for Developing a Novel Vaccine against SARS-CoV-2 Infection.

Over the years, several distinct pathogenic coronaviruses have emerged, including the pandemic SARS-CoV-2, which is difficult to curtail despite the availability of licensed vaccines. The difficulty in managing SARS-CoV-2 is linked to changes in the variants' proteins, especially in the spike protein (SP) used for viral entry. These mutations, especially in the SP, enable the virus to evade immune responses induced by natural infection or vaccination. However, some parts of the SP in the S1 subunit and the S2 subunit are considered conserved among coronaviruses. In this review, we will discuss the epitopes in the SARS-CoV-2 S1 and S2 subunit proteins that have been demonstrated by various studies to be conserved among coronaviruses and may be immunogenic for the development of a vaccine. Considering the higher conservancy of the S2, we will further discuss the likely challenges that could limit the S2 subunit from inducing robust immune responses and the promising approaches to increase its immunogenicity.

Development and laboratory evaluation of a competitive ELISA for serodiagnosis of Nipah and Hendra virus infection using recombinant Nipah glycoproteins and a monoclonal antibody.

Introduction: Nipah virus (NiV) and Hendra virus (HeV), of the genus Henipavirus, family Paramyxoviridae, are classified as Risk Group 4 (RG4) pathogens that cause respiratory disease in pigs and acute/febrile encephalitis in humans with high mortality. Methods: A competitive enzyme-linked immunosorbent assay (cELISA) using a monoclonal antibody (mAb) and recombinant NiV glycoprotein (G) was developed and laboratory evaluated using sera from experimental pigs, mini pigs and nonhuman primates. The test depends on competition between specific antibodies in positive sera and a virus-specific mAb for binding to NiV-G. Results: Based on 1,199 negative and 71 NiV positive serum test results, the cutoff value was determined as 35% inhibition. The diagnostic sensitivity and specificity of the NiV cELISA was 98.58 and 99.92%, respectively. When testing sera from animals experimentally infected with NiV Malaysia, the cELISA detected antibodies from 14 days post-infection (dpi) and remained positive until the end of the experiment (28 dpi). Comparisons using the Kappa coefficient showed strong agreement (100%) between the cELISA and a plaque reduction neutralization test (PRNT). Discussion: Because our cELISA is simpler, faster, and gives comparable or better results than PRNT, it would be an adequate screening test for suspect NiV and HeV cases, and it would also be useful for epidemiological surveillance of Henipavirus infections in different animal species without changing reagents.

Cell-Free Dot Blot: an Ultra-Low-Cost and Practical Immunoassay Platform for Detection of Anti-SARS-CoV-2 Antibodies in Human and Animal Sera.

Since its emergence in late 2019, the coronavirus disease 2019 (COVID-19) pandemic has caused severe disruption to key aspects of human life globally and highlighted the need for timely, adaptive, and accessible pandemic response strategies. Here, we introduce the cell-free dot blot (CFDB) method, a practical and ultra-low-cost immune diagnostic platform capable of rapid response and mass immunity screening for the current and future pandemics. Similar in mechanism to the widely used enzyme-linked immunosorbent assays (ELISAs), our method is novel and advantageous in that (i) it uses linear DNA to produce the target viral antigen fused to a SpyTag peptide in a cell-free expression system without the need for traditional cloning and antigen purification, (ii) it uses SpyCatcher2-Apex2, an Escherichia coli-produced peroxidase conjugate as a universal secondary detection reagent, obviating the need for commercial or sophisticated enzyme conjugates, and (iii) sera are spotted directly on a nitrocellulose membrane, enabling a simple "dipping" mechanism for downstream incubation and washing steps, as opposed to individual processing of wells in a multiwell plate. To demonstrate the utility of our method, we performed CFDB to detect anti-severe acute respiratory syndrome coronavirus 2 nucleocapsid protein antibodies in precharacterized human sera (23 negative and 36 positive for COVID-19) and hamster sera (16 negative and 36 positive for COVID-19), including independent testing at a collaborating laboratory, and we show assay performance comparable to that of conventional ELISAs. At a similar capacity to 96-well plate ELISA kits, one CFDB assay costs only ~$3 USD. We believe that CFDB can become a valuable pandemic response tool for adaptive and accessible sero-surveillance in human and animal populations. IMPORTANCE The recent COVID-19 pandemic has highlighted the need for diagnostic platforms that are rapidly adaptable, affordable, and accessible globally, especially for low-resource settings. To address this need, we describe the development and functional validation of a novel immunoassay technique termed the cell-free dot blot (CFDB) method. Based on the principles of cell-free synthetic biology and alternative dot blotting procedures, our CFDB immunoassay is designed to provide for timely, practical, and low-cost responses to existing and emerging public health threats, such as the COVID-19 pandemic, at a similar throughput and comparable performance as conventional ELISAs. Notably, the molecular detection reagents used in CFDB can be produced rapidly in-house, using established protocols and basic laboratory infrastructure, minimizing reliance on strained commercial reagents. In addition, the materials and imaging instruments required for CFDB are the same as those used for common Western blotting experiments, further expanding the reach of CFDB in decentralized facilities.

In vitro and in vivo efficacy of tecovirimat against a recently emerged 2022 monkeypox virus isolate.

The recent emergence of the monkeypox virus (MPXV) in non-endemic countries has been designated a Public Health Emergency of International Concern by the World Health Organization. There are currently no approved treatments for MPXV infection in the United States or Canada. The antiviral drug tecovirimat (commonly called TPOXX), previously approved for smallpox treatment, is currently being deployed for treatment of MPXV infections where available based on previously accrued data. We tested the efficacy of TPOXX both in vitro and in vivo against a clade 2 Canadian 2022 isolate of MPXV isolated during the current outbreak. TPOXX prevented MPXV replication in vitro with an effective concentration in the nanomolar range. To evaluate TPOXX efficacy in vivo, we first characterized the CAST/EiJ mouse model with the same 2022 Canadian isolate. Unlike previous descriptions of this model, the Canadian isolate was not lethal in CAST/EiJ mice, although it replicated efficiently in the respiratory tract after intranasal infection. Subsequent experiments demonstrated that daily oral TPOXX treatment markedly reduced viral titers in the tissues 1 and 2 weeks after infection. Our data indicate that TPOXX is highly effective against currently circulating MPXV strains and could be an important contributor to curbing the ongoing outbreak.

DNA-delivered antibody cocktail exhibits improved pharmacokinetics and confers prophylactic protection against SARS-CoV-2.

Monoclonal antibody therapy has played an important role against SARS-CoV-2. Strategies to deliver functional, antibody-based therapeutics with improved in vivo durability are needed to supplement current efforts and reach underserved populations. Here, we compare recombinant mAbs COV2-2196 and COV2-2130, which compromise clinical cocktail Tixagevimab/Cilgavimab, with optimized nucleic acid-launched forms. Functional profiling of in vivo-expressed, DNA-encoded monoclonal antibodies (DMAbs) demonstrated similar specificity, broad antiviral potency and equivalent protective efficacy in multiple animal challenge models of SARS-CoV-2 prophylaxis compared to protein delivery. In PK studies, DNA-delivery drove significant serum antibody titers that were better maintained compared to protein administration. Furthermore, cryo-EM studies performed on serum-derived DMAbs provide the first high-resolution visualization of in vivo-launched antibodies, revealing new interactions that may promote cooperative binding to trimeric antigen and broad activity against VoC including Omicron lineages. These data support the further study of DMAb technology in the development and delivery of valuable biologics.

More tools for our toolkit: The application of HEL-299 cells and dsRNA-nanoparticles to study human coronaviruses in vitro.

Human coronaviruses (HCoVs) are important human pathogens, as exemplified by the current SARS-CoV-2 pandemic. While the ability of type I interferons (IFNs) to limit coronavirus replication has been established, the ability of double-stranded (ds)RNA, a potent IFN inducer, to inhibit coronavirus replication when conjugated to a nanoparticle is largely unexplored. Additionally, the number of IFN competent cell lines that can be used to study coronaviruses in vitro are limited. In the present study, we show that poly inosinic: poly cytidylic acid (pIC), when conjugated to a phytoglycogen nanoparticle (pIC+NDX) is able to protect IFN-competent human lung fibroblasts (HEL-299 cells) from infection with different HCoV species. HEL-299 was found to be permissive to HCoV-229E, -OC43 and MERS-CoV-GFP but not to HCoV-NL63 or SARS-CoV-2. Further investigation revealed that HEL-299 does not contain the required ACE2 receptor to enable propagation of both HCoV-NL63 and SARS-CoV-2. Following 24h exposure, pIC+NDX was observed to stimulate a significant, prolonged increase in antiviral gene expression (IFNß, CXCL10 and ISG15) when compared to both NDX alone and pIC alone. This antiviral response translated into complete protection against virus production, for 4 days or 7 days post treatment with HCoV-229E or -OC43 when either pre-treated for 6h or 24h respectively. Moreover, the pIC+NDX combination also provided complete protection for 2d post infection when HEL-299 cells were infected with MERS-CoV-GFP following a 24h pretreatment with pIC+NDX. The significance of this study is two-fold. Firstly, it was revealed that HEL-299 cells can effectively be used as an IFN-competent model system for in vitro analysis of MERS-CoV. Secondly, pIC+NDX acts as a powerful inducer of type I IFNs in HEL-299, to levels that provide complete protection against coronavirus replication. This suggests an exciting and novel area of investigation for antiviral therapies that utilize innate immune stimulants. The results of this study will help to expand the range of available tools scientists have to investigate, and thus further understand, human coronaviruses.

A Recombinant VSV-Based Bivalent Vaccine Effectively Protects against Both SARS-CoV-2 and Influenza A Virus Infection.

COVID-19 and influenza are both highly contagious respiratory diseases that have been serious threats to global public health. It is necessary to develop a bivalent vaccine to control these two infectious diseases simultaneously. In this study, we generated three attenuated replicating recombinant vesicular stomatitis virus (rVSV)-based vaccine candidates against both SARS-CoV-2 and influenza viruses. These rVSV-based vaccines coexpress SARS-CoV-2 Delta spike protein (SP) bearing the C-terminal 17 amino acid (aa) deletion (SPΔC) and I742A point mutation, or the SPΔC with a deletion of S2 domain, or the RBD domain, and a tandem repeat harboring four copies of the highly conserved influenza M2 ectodomain (M2e) that fused with the Ebola glycoprotein DC-targeting/activation domain. Animal immunization studies have shown that these rVSV bivalent vaccines induced efficient humoral and cellular immune responses against both SARS-CoV-2 SP and influenza M2 protein, including high levels of neutralizing antibodies against SARS-CoV-2 Delta and other variant SP-pseudovirus infections. Importantly, immunization of the rVSV bivalent vaccines effectively protected hamsters or mice against the challenges of SARS-CoV-2 Delta variant and lethal H1N1 and H3N2 influenza viruses and significantly reduced respiratory viral loads. Overall, this study provides convincing evidence for the high efficacy of this bivalent vaccine platform to be used and/or easily adapted to produce new vaccines against new or reemerging SARS-CoV-2 variants and influenza A virus infections. IMPORTANCE Given that both COVID-19 and influenza are preferably transmitted through respiratory droplets during the same seasons, it is highly advantageous to develop a bivalent vaccine that could simultaneously protect against both COVID-19 and influenza. In this study, we generated the attenuated replicating recombinant vesicular stomatitis virus (rVSV)-based vaccine candidates that target both spike protein of SARS-Cov-2 Delta variant and the conserved influenza M2 domain. Importantly, these vaccine candidates effectively protected hamsters or mice against the challenges of SARS-CoV-2 Delta variant and lethal H1N1 and H3N2 influenza viruses and significantly reduced respiratory viral loads.

Detection of Nipah and Hendra Viruses Using Recombinant Human Ephrin B2 Capture Virus in Immunoassays.

Nipah virus (NiV) and Hendra virus (HeV) are classified as high-consequence zoonotic viruses characterized by high pathogenicity and high mortality in animals and humans. Rapid diagnosis is essential to containing the outbreak. In this study, the henipavirus receptor ephrin B2 was examined to determine whether it could be used as a universal ligand for henipavirus detection in immunoassays. Enzyme-linked immunosorbent assays (ELISAs) were developed using recombinant ephrin B2 as the capture ligand and two monoclonal antibodies (mAbs) as detection reagents. Using mAb F27NiV-34, which cross-reacts with NiV and HeV, we were able to detect NiV and HeV, while mAb F20NiV-65 was used to detect NiV. Therefore, using these two ELISAs, we were able to differentiate between NiV and HeV. Furthermore, we developed a rapid lateral flow strip test for NiV detection using ephrin B2 as the capture ligand combined with mAb F20NiV-65 as the detector. Taken together, our results show that the combination of ephrin B2 and a specific mAb provides an excellent pairing for NiV and HeV detection.

Development and characterization of influenza M2 ectodomain and/or hemagglutinin stalk-based dendritic cell-targeting vaccines.

A universal influenza vaccine is required for broad protection against influenza infection. Here, we revealed the efficacy of novel influenza vaccine candidates based on Ebola glycoprotein dendritic cell (DC)-targeting domain (EΔM) fusion protein technology. The four copies of ectodomain matrix protein of influenza (tM2e) or M2e hemagglutinin stalk (HA stalk) peptides (HM2e) were fused with EΔM to generate EΔM-tM2e or EΔM-HM2e, respectively. We demonstrated that EΔM-HM2e- or EΔM-tM2e-pseudotyped viral particles can efficiently target DC/macrophages in vitro and induced significantly high titers of anti-HA and/or anti-M2e antibodies in mice. Significantly, the recombinant vesicular stomatitis virus (rVSV)-EΔM-tM2e and rVSV-EΔM-HM2e vaccines mediated rapid and potent induction of M2 or/and HA antibodies in mice sera and mucosa. Importantly, vaccination of rVSV-EΔM-tM2e or rVSV-EΔM-HM2e protected mice from influenza H1N1 and H3N2 challenges. Taken together, our study suggests that rVSV-EΔM-tM2e and rVSV-EΔM-HM2e are promising candidates that may lead to the development of a universal vaccine against different influenza strains.

Experimental Infection of Peromyscus Species Rodents with Sin Nombre Virus.

We demonstrate that 6 distinct Peromyscus rodent species are permissive to experimental infection with Sin Nombre orthohantavirus (SNV). Viral RNA and SNV antibodies were detected in members of all 6 species. P. leucopus mice demonstrated markedly higher viral and antibody titers than P. maniculatus mice, the established primary hosts for SNV.

Pandemic 1918 Influenza Virus Does Not Cause Lethal Infection in Rhesus or Cynomolgus Macaques.

The 1918 H1N1 influenza pandemic was among the most severe in history, taking the lives of approximately 50 million people worldwide, and novel prophylactic vaccines are urgently needed to prevent another pandemic. Given that macaques are physiologically relevant preclinical models of human immunology that have advanced the clinical treatment of infectious diseases, a lethal pandemic influenza challenge model would provide a stringent platform for testing new influenza vaccine concepts. To this end, we infected rhesus macaques and Mauritian cynomolgus macaques with highly pathogenic 1918 H1N1 influenza virus and assessed pathogenesis and disease severity. Despite infection with a high dose of 1918 influenza delivered via multiple routes, rhesus macaques demonstrated minimal signs of disease, with only intermittent viral shedding. Cynomolgus macaques infected via intrabronchial instillation demonstrated mild symptoms, with disease severity depending on the infection dose. Cynomolgus macaques infected with a high dose of 1918 influenza delivered via multiple routes experienced moderate disease characterized by consistent viral shedding, pulmonary infiltrates, and elevated inflammatory cytokine levels. However, 1918 influenza was uniformly nonlethal in these two species, demonstrating that this isolate is insufficiently pathogenic in rhesus and Mauritian cynomolgus macaques to support testing novel prophylactic influenza approaches where protection from severe disease combined with a lethal outcome is desired as a highly stringent indication of vaccine efficacy. IMPORTANCE The world remains at risk of an influenza pandemic, and the development of new therapeutic and preventative modalities is critically important for minimizing human death and suffering during the next influenza pandemic. Animal models are central to the development of new therapies and vaccine approaches. In particular, nonhuman primates like rhesus and cynomolgus macaques are highly relevant preclinical models given their physiological and immunological similarities to humans. Unfortunately, there remains a scarcity of macaque models of pandemic influenza with which to test novel antiviral modalities. Here, we demonstrate that even at the highest doses tested, 1918 influenza was not lethal in these two macaque species, suggesting that they are not ideal for the development and testing of novel pandemic influenza-specific vaccines and therapies. Therefore, other physiologically relevant nonhuman primate models of pandemic influenza are needed.

Mucosal chemokine adjuvant enhances synDNA vaccine-mediated responses to SARS-CoV-2 and provides heterologous protection in vivo.

The global coronavirus disease 2019 (COVID-19) pandemic has claimed more than 5 million lives. Emerging variants of concern (VOCs) continually challenge viral control. Directing vaccine-induced humoral and cell-mediated responses to mucosal surfaces may enhance vaccine efficacy. Here we investigate the immunogenicity and protective efficacy of optimized synthetic DNA plasmids encoding wild-type severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (pS) co-formulated with the plasmid-encoded mucosal chemokine cutaneous T cell-attracting chemokine (pCTACK; CCL27). pCTACK-co-immunized animals exhibit increased spike-specific antibodies at the mucosal surface and increased frequencies of interferon gamma (IFNγ)+ CD8+ T cells in the respiratory mucosa. pCTACK co-immunization confers 100% protection from heterologous Delta VOC challenge. This study shows that mucosal chemokine adjuvants can direct vaccine-induced responses to specific immunological sites and have significant effects on heterologous challenge. Further study of this unique chemokine-adjuvanted vaccine approach in the context of SARS-CoV-2 vaccines is likely important.