GPR183 antagonism reduces macrophage infiltration in influenza and SARS-CoV-2 infection.

RATIONALE: Severe viral respiratory infections are often characterised by extensive myeloid cell infiltration and activation and persistent lung tissue injury. However, the immunological mechanisms driving excessive inflammation in the lung remain poorly understood. OBJECTIVES: To identify the mechanisms that drive immune cell recruitment in the lung during viral respiratory infections and identify novel drug targets to reduce inflammation and disease severity. METHODS: Preclinical murine models of influenza virus and severe acute respiratory coronavirus 2 (SARS-CoV-2) infection. RESULTS: Oxidised cholesterols and the oxysterol-sensing receptor GPR183 were identified as drivers of monocyte-macrophage infiltration to the lung during influenza virus (IAV) and SARS-CoV-2 infection. Both IAV and SARS-CoV-2 infection upregulated the enzymes cholesterol 25-hydroxylase (CH25H) and cytochrome P450 family 7 subfamily member B1 (CYP7B1) in the lung, resulting in local production of the oxidised cholesterols 25-hydroxycholesterol (25-OHC) and 7α,25-dihydroxycholesterol (7α,25-OHC). Loss-of-function mutation of GPR183, or treatment with a GPR183 antagonist, reduced macrophage infiltration and inflammatory cytokine production in the lungs of IAV- or SARS-CoV-2-infected mice. The GPR183 antagonist significantly attenuated the severity of SARS-CoV-2 infection and viral loads. Analysis of single cell RNASeq data on bronchoalveolar lavage samples from healthy controls and COVID-19 patients with moderate and severe disease revealed that CH25H, CYP7B1 and GPR183 are significantly upregulated in macrophages during COVID-19. CONCLUSION: This study demonstrates that oxysterols drive inflammation in the lung via GPR183 and provides the first preclinical evidence for therapeutic benefit of targeting GPR183 during severe viral respiratory infections.

Oral Administration of Universal Bacterium-Vectored Nucleocapsid-Expressing COVID-19 Vaccine is Efficacious in Hamsters.

Oral delivery of an inexpensive COVID-19 (coronavirus disease 2019) vaccine could dramatically improve immunization rates, especially in low- and middle-income countries. Previously, we described a potential universal COVID-19 vaccine, rLVS ΔcapB/MN, comprising a replicating bacterial vector, LVS (live vaccine strain) ΔcapB, expressing the highly conserved SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) membrane and nucleocapsid (N) proteins, which, when administered intradermally or intranasally, protects hamsters from severe COVID-19-like disease after high-dose SARS-CoV-2 respiratory challenge. Here, we show that oral administration of the vaccine also protects against high-dose SARS-CoV-2 respiratory challenge; its protection is comparable to that of intradermal, intranasal, or subcutaneous administration. Hamsters were protected against severe weight loss and lung pathology and had reduced oropharyngeal and lung virus titers. Protection against weight loss and histopathology by the vaccine, which in mice induces splenic and lung cell interferon gamma in response to N protein stimulation, was correlated in hamsters with pre-challenge serum anti-N TH1-biased IgG (IgG2/3). Thus, rLVS ΔcapB/MN has potential as an oral universal COVID-19 vaccine. IMPORTANCE The COVID-19 pandemic continues to rage into its fourth year worldwide. To protect the world's population most effectively from severe disease, hospitalization, and death, a vaccine is needed that is resistant to rapidly emerging viral variants of the causative agent SARS-CoV-2, inexpensive to manufacture, store, and transport, and easy to administer. Ideally, such a vaccine would be capable of oral administration, especially in resource-poor countries of the world where there are shortages of needles, syringes and trained personnel to administer injectable vaccines. Here, we show that oral administration of a bacterium-vectored vaccine meeting all these criteria protects naturally susceptible Syrian hamsters from severe COVID-19-like disease, including severe weight loss and lung pathology, after high-dose SARS-CoV-2 respiratory challenge. As the vaccine is based upon inducing immunity to highly conserved SARS-CoV-2 membrane and nucleocapsid proteins, as opposed to the rapidly mutating Spike protein, it should remain resistant to newly emerging SARS-CoV-2 variants.

Immunogenicity and Efficacy of Monovalent and Bivalent Formulations of a Virus-Like Particle Vaccine against SARS-CoV-2.

Virus-like particles (VLPs) offer great potential as a safe and effective vaccine platform against SARS-CoV-2, the causative agent of COVID-19. Here, we show that SARS-CoV-2 VLPs can be generated by expression of the four viral structural proteins in a mammalian expression system. Immunization of mice with a monovalent VLP vaccine elicited a potent humoral response, showing neutralizing activity against multiple variants of SARS-CoV-2. Subsequent immunogenicity and efficacy studies were performed in the Golden Syrian hamster model, which closely resembles the pathology and progression of COVID-19 in humans. Hamsters immunized with a bivalent VLP vaccine were significantly protected from infection with the Beta or Delta variant of SARS-CoV-2. Vaccinated hamsters showed reduced viral load, shedding, replication, and pathology in the respiratory tract. Immunized hamsters also showed variable levels of cross-neutralizing activity against the Omicron variant. Overall, the VLP vaccine elicited robust protective efficacy against SARS-CoV-2. These promising results warrant further study of multivalent VLP vaccines in Phase I clinical trials in humans.

Ancestral SARS-CoV-2, but not Omicron, replicates less efficiently in primary pediatric nasal epithelial cells.

Children typically experience more mild symptoms of Coronavirus Disease 2019 (COVID-19) when compared to adults. There is a strong body of evidence that children are also less susceptible to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection with the ancestral viral isolate. However, the emergence of SARS-CoV-2 variants of concern (VOCs) has been associated with an increased number of pediatric infections. Whether this is the result of widespread adult vaccination or fundamental changes in the biology of SARS-CoV-2 remain to be determined. Here, we use primary nasal epithelial cells (NECs) from children and adults, differentiated at an air-liquid interface to show that the ancestral SARS-CoV-2 replicates to significantly lower titers in the NECs of children compared to those of adults. This was associated with a heightened antiviral response to SARS-CoV-2 in the NECs of children. Importantly, the Delta variant also replicated to significantly lower titers in the NECs of children. This trend was markedly less pronounced in the case of Omicron. It is also striking to note that, at least in terms of viral RNA, Omicron replicated better in pediatric NECs compared to both Delta and the ancestral virus. Taken together, these data show that the nasal epithelium of children supports lower infection and replication of ancestral SARS-CoV-2, although this may be changing as the virus evolves.

Susceptibility of Wild Canids to SARS-CoV-2.

We assessed 2 wild canid species, red foxes (Vulpes vulpes) and coyotes (Canis latrans), for susceptibility to SARS-CoV-2. After experimental inoculation, red foxes became infected and shed infectious virus. Conversely, experimentally challenged coyotes did not become infected; therefore, coyotes are unlikely to be competent hosts for SARS-CoV-2.

Covax-19/Spikogen® vaccine based on recombinant spike protein extracellular domain with Advax-CpG55.2 adjuvant provides single dose protection against SARS-CoV-2 infection in hamsters.

COVID-19 presents an ongoing global health crisis. Protein-based COVID-19 vaccines that are well-tolerated, safe, highly-protective and convenient to manufacture remain of major interest. We therefore sought to compare the immunogenicity and protective efficacy of a number of recombinant SARS-CoV-2 spike protein candidates expressed in insect cells. By comparison to a full length (FL) spike protein detergent-extracted nanoparticle antigen, the soluble secreted spike protein extracellular domain (ECD) generated higher protein yields per liter of culture and when formulated with either Alum-CpG55.2 or Advax-CpG55.2 combination adjuvants elicited robust antigen-specific humoral and cellular immunity in mice. In hamsters, the spike ECD when formulated with either adjuvant induced high serum neutralizing antibody titers even after a single dose. When challenged with the homologous SARS-CoV-2 virus, hamsters immunized with the adjuvanted spike ECD exhibited reduced viral load in day 1-3 oropharyngeal swabs and day 3 nasal turbinate tissue and had no recoverable infectious virus in day 3 lung tissue. The reduction in lung viral load correlated with less weight loss and lower lung pathology scores. The formulations of spike ECD with Alum-CpG55.2 or Advax-CpG55.2 were protective even after just a single dose, although the 2-dose regimen performed better overall and required only half the total amount of antigen. Pre-challenge serum neutralizing antibody levels showed a strong correlation with lung protection, with a weaker correlation seen with nasal or oropharyngeal protection. This suggests that serum neutralizing antibody levels may correlate more closely with systemic, rather than mucosal, protection. The spike protein ECD with Advax-CpG55.2 formulation (Covax-19® vaccine) was selected for human clinical development.

Nebulized delivery of a broadly neutralizing SARS-CoV-2 RBD-specific nanobody prevents clinical, virological, and pathological disease in a Syrian hamster model of COVID-19.

There remains an unmet need for globally deployable, low-cost therapeutics for the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Previously, we reported on the isolation and in vitro characterization of a potent single-domain nanobody, NIH-CoVnb-112, specific for the receptor-binding domain (RBD) of SARS-CoV-2. Here, we report on the molecular basis for the observed broad in vitro neutralization capability of NIH-CoVnb-112 against variant SARS-CoV-2 pseudoviruses. The structure of NIH-CoVnb-112 bound to SARS-CoV-2 RBD reveals a large contact surface area overlapping the angiotensin converting enzyme 2 (ACE2) binding site, which is largely unencumbered by the common RBD mutations. In an in vivo pilot study, we demonstrate effective reductions in weight loss, viral burden, and lung pathology in a Syrian hamster model of COVID-19 following nebulized delivery of NIH-CoVnb-112. These findings support the further development of NIH-CoVnb-112 as a potential adjunct preventative therapeutic for the treatment of SARS-CoV-2 infection.Abbreviations: ACE2 - angiotensin converting enzyme 2BSA - buried surface areaCDR - complementary determining regionRBD - receptor binding domainRBM - receptor-binding motifSARS-CoV-2 - severe acute respiratory syndrome coronavirus 2.

Activity of Galidesivir in a Hamster Model of SARS-CoV-2.

Coronavirus disease 2019 (COVID-19) has claimed the lives of millions of people worldwide since it first emerged. The impact of the COVID-19 pandemic on public health and the global economy has highlighted the medical need for the development of broadly acting interventions against emerging viral threats. Galidesivir is a broad-spectrum antiviral compound with demonstrated in vitro and in vivo efficacy against several RNA viruses of public health concern, including those causing yellow fever, Ebola, Marburg, and Rift Valley fever. In vitro studies have shown that the antiviral activity of galidesivir also extends to coronaviruses. Herein, we describe the efficacy of galidesivir in the Syrian golden hamster model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Treatment with galidesivir reduced lung pathology in infected animals compared with untreated controls when treatment was initiated 24 h prior to infection. These results add to the evidence of the applicability of galidesivir as a potential medical intervention for a range of acute viral illnesses, including coronaviruses.

Susceptibility of livestock to SARS-CoV-2 infection.

We report pilot studies to evaluate the susceptibility of common domestic livestock (cattle, sheep, goat, alpaca, rabbit, and horse) to intranasal infection with SARS-CoV-2. None of the infected animals shed infectious virus via nasal, oral, or faecal routes, although viral RNA was detected in several animals. Further, neutralizing antibody titres were low or non-existent one month following infection. These results suggest that domestic livestock are unlikely to contribute to SARS-CoV-2 epidemiology.

Complete protection by a single-dose skin patch-delivered SARS-CoV-2 spike vaccine.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 160 million people and resulted in more than 3.3 million deaths, and despite the availability of multiple vaccines, the world still faces many challenges with their rollout. Here, we use the high-density microarray patch (HD-MAP) to deliver a SARS-CoV-2 spike subunit vaccine directly to the skin. We show that the vaccine is thermostable on the patches, with patch delivery enhancing both cellular and antibody immune responses. Elicited antibodies potently neutralize clinically relevant isolates including the Alpha and Beta variants. Last, a single dose of HD-MAP­delivered spike provided complete protection from a lethal virus challenge in an ACE2-transgenic mouse model. Collectively, these data show that HD-MAP delivery of a SARS-CoV-2 vaccine was superior to traditional needle-and-syringe vaccination and may be a significant addition to the ongoing COVID-19 (coronavirus disease 2019) pandemic.

Peridomestic Mammal Susceptibility to Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

Wild animals have been implicated as the origin of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but it is largely unknown how the virus affects most wildlife species and if wildlife could ultimately serve as a reservoir for maintaining the virus outside the human population. We show that several common peridomestic species, including deer mice, bushy-tailed woodrats, and striped skunks, are susceptible to infection and can shed the virus in respiratory secretions. In contrast, we demonstrate that cottontail rabbits, fox squirrels, Wyoming ground squirrels, black-tailed prairie dogs, house mice, and racoons are not susceptible to SARS-CoV-2 infection. Our results expand the knowledge base of susceptible species and provide evidence that human-wildlife interactions could result in continued transmission of SARS-CoV-2.

Replicating bacterium-vectored vaccine expressing SARS-CoV-2 Membrane and Nucleocapsid proteins protects against severe COVID-19-like disease in hamsters.

To generate an inexpensive readily manufactured COVID-19 vaccine, we employed the LVS ΔcapB vector platform, previously used to generate potent candidate vaccines against Select Agent diseases tularemia, anthrax, plague, and melioidosis. Vaccines expressing SARS-CoV-2 structural proteins are constructed using the LVS ΔcapB vector, a highly attenuated replicating intracellular bacterium, and evaluated for efficacy in golden Syrian hamsters, which develop severe COVID-19-like disease. Hamsters immunized intradermally or intranasally with a vaccine co-expressing the Membrane and Nucleocapsid proteins and challenged 5 weeks later with a high dose of SARS-CoV-2 are protected against severe weight loss and lung pathology and show reduced viral loads in the oropharynx and lungs. Protection correlates with anti-Nucleocapsid antibody. This potent vaccine should be safe; inexpensive; easily manufactured, stored, and distributed; and given the high homology between Membrane and Nucleocapsid proteins of SARS-CoV and SARS-CoV-2, potentially serve as a universal vaccine against the SARS subset of pandemic causing ß-coronaviruses.