4'-Fluorouridine mitigates lethal infection with pandemic human and highly pathogenic avian influenza viruses.

Influenza outbreaks are associated with substantial morbidity, mortality and economic burden. Next generation antivirals are needed to treat seasonal infections and prepare against zoonotic spillover of avian influenza viruses with pandemic potential. Having previously identified oral efficacy of the nucleoside analog 4'-Fluorouridine (4'-FlU, EIDD-2749) against SARS-CoV-2 and respiratory syncytial virus (RSV), we explored activity of the compound against seasonal and highly pathogenic influenza (HPAI) viruses in cell culture, human airway epithelium (HAE) models, and/or two animal models, ferrets and mice, that assess IAV transmission and lethal viral pneumonia, respectively. 4'-FlU inhibited a panel of relevant influenza A and B viruses with nanomolar to sub-micromolar potency in HAE cells. In vitro polymerase assays revealed immediate chain termination of IAV polymerase after 4'-FlU incorporation, in contrast to delayed chain termination of SARS-CoV-2 and RSV polymerase. Once-daily oral treatment of ferrets with 2 mg/kg 4'-FlU initiated 12 hours after infection rapidly stopped virus shedding and prevented transmission to untreated sentinels. Treatment of mice infected with a lethal inoculum of pandemic A/CA/07/2009 (H1N1)pdm09 (pdmCa09) with 4'-FlU alleviated pneumonia. Three doses mediated complete survival when treatment was initiated up to 60 hours after infection, indicating a broad time window for effective intervention. Therapeutic oral 4'-FlU ensured survival of animals infected with HPAI A/VN/12/2003 (H5N1) and of immunocompromised mice infected with pdmCa09. Recoverees were protected against homologous reinfection. This study defines the mechanistic foundation for high sensitivity of influenza viruses to 4'-FlU and supports 4'-FlU as developmental candidate for the treatment of seasonal and pandemic influenza.

Ferrets as a model for tuberculosis transmission.

Even with the COVID-19 pandemic, tuberculosis remains a leading cause of human death due to a single infectious agent. Until successfully treated, infected individuals may continue to transmit Mycobacterium tuberculosis bacilli to contacts. As with other respiratory pathogens, such as SARS-CoV-2, modeling the process of person-to-person transmission will inform efforts to develop vaccines and therapies that specifically impede disease transmission. The ferret (Mustela furo), a relatively inexpensive, small animal has been successfully employed to model transmissibility, pathogenicity, and tropism of influenza and other respiratory disease agents. Ferrets can become naturally infected with Mycobacterium bovis and are closely related to badgers, well known in Great Britain and elsewhere as a natural transmission vehicle for bovine tuberculosis. Herein, we report results of a study demonstrating that within 7 weeks of intratracheal infection with a high dose (>5 x 103 CFU) of M. tuberculosis bacilli, ferrets develop clinical signs and pathological features similar to acute disease reported in larger animals, and ferrets infected with very-high doses (>5 x 104 CFU) develop severe signs within two to four weeks, with loss of body weight as high as 30%. Natural transmission of this pathogen was also examined. Acutely-infected ferrets transmitted M. tuberculosis bacilli to co-housed naïve sentinels; most of the sentinels tested positive for M. tuberculosis in nasal washes, while several developed variable disease symptomologies similar to those reported for humans exposed to an active tuberculosis patient in a closed setting. Transmission was more efficient when the transmitting animal had a well-established acute infection. The findings support further assessment of this model system for tuberculosis transmission including the testing of prevention measures and vaccine efficacy.

SARS-CoV-2 VOC type and biological sex affect molnupiravir efficacy in severe COVID-19 dwarf hamster model.

SARS-CoV-2 variants of concern (VOC) have triggered infection waves. Oral antivirals such as molnupiravir promise to improve disease management, but efficacy against VOC delta was questioned and potency against omicron is unknown. This study evaluates molnupiravir against VOC in human airway epithelium organoids, ferrets, and a lethal Roborovski dwarf hamster model of severe COVID-19-like lung injury. VOC were equally inhibited by molnupiravir in cells and organoids. Treatment reduced shedding in ferrets and prevented transmission. Pathogenicity in dwarf hamsters was VOC-dependent and highest for delta, gamma, and omicron. All molnupiravir-treated dwarf hamsters survived, showing reduction in lung virus load from one (delta) to four (gamma) orders of magnitude. Treatment effect size varied in individual dwarf hamsters infected with omicron and was significant in males, but not females. The dwarf hamster model recapitulates mixed efficacy of molnupiravir in human trials and alerts that benefit must be reassessed in vivo as VOC evolve.

Influenza-induced Tpl2 expression within alveolar epithelial cells is dispensable for host viral control and anti-viral immunity.

Tumor progression locus 2 (Tpl2) is a serine/threonine kinase that regulates the expression of inflammatory mediators in response to Toll-like receptors (TLR) and cytokine receptors. Global ablation of Tpl2 leads to severe disease in response to influenza A virus (IAV) infection, characterized by respiratory distress, and studies in bone marrow chimeric mice implicated Tpl2 in non-hematopoietic cells. Lung epithelial cells are primary targets and replicative niches of influenza viruses; however, the specific regulation of antiviral responses by Tpl2 within lung epithelial cells has not been investigated. Herein, we show that Tpl2 is basally expressed in primary airway epithelial cells and that its expression increases in both type I and type II airway epithelial cells (AECI and AECII) in response to influenza infection. We used Nkx2.1-cre to drive Tpl2 deletion within pulmonary epithelial cells to delineate epithelial cell-specific functions of Tpl2 during influenza infection in mice. Although modest increases in morbidity and mortality were attributed to cre-dependent deletion in lung epithelial cells, no alterations in host cytokine production or lung pathology were observed. In vitro, Tpl2 inhibition within the type I airway epithelial cell line, LET1, as well as genetic ablation in primary airway epithelial cells did not alter cytokine production. Overall, these findings establish that Tpl2-dependent defects in cells other than AECs are primarily responsible for the morbidity and mortality seen in influenza-infected mice with global Tpl2 ablation.

Immunisation of ferrets and mice with recombinant SARS-CoV-2 spike protein formulated with Advax-SM adjuvant protects against COVID-19 infection.

The development of a safe and effective vaccine is a key requirement to overcoming the COVID-19 pandemic. Recombinant proteins represent the most reliable and safe vaccine approach but generally require a suitable adjuvant for robust and durable immunity. We used the SARS-CoV-2 genomic sequence and in silico structural modelling to design a recombinant spike protein vaccine (Covax-19™). A synthetic gene encoding the spike extracellular domain (ECD) was inserted into a baculovirus backbone to express the protein in insect cell cultures. The spike ECD was formulated with Advax-SM adjuvant and first tested for immunogenicity in C57BL/6 and BALB/c mice. Covax-19 vaccine induced high spike protein binding antibody levels that neutralised the original lineage B.1.319 virus from which the vaccine spike protein was derived, as well as the variant B.1.1.7 lineage virus. Covax-19 vaccine also induced a high frequency of spike-specific CD4 + and CD8 + memory T-cells with a dominant Th1 phenotype associated with the ability to kill spike-labelled target cells in vivo. Ferrets immunised with Covax-19 vaccine intramuscularly twice 2 weeks apart made spike receptor binding domain (RBD) IgG and were protected against an intranasal challenge with SARS-CoV-2 virus given two weeks after the last immunisation. Notably, ferrets that received the two higher doses of Covax-19 vaccine had no detectable virus in their lungs or in nasal washes at day 3 post-challenge, suggesting that in addition to lung protection, Covax-19 vaccine may have the potential to reduce virus transmission. This data supports advancement of Covax-19 vaccine into human clinical trials.

Protection of K18-hACE2 mice and ferrets against SARS-CoV-2 challenge by a single-dose mucosal immunization with a parainfluenza virus 5-based COVID-19 vaccine.

Transmission-blocking vaccines are urgently needed to reduce transmission of SARS-CoV 2, the cause of the COVID-19 pandemic. The upper respiratory tract is an initial site of SARS-CoV-2 infection and, for many individuals, remains the primary site of virus replication. An ideal COVID-19 vaccine should reduce upper respiratory tract virus replication and block transmission as well as protect against severe disease. Here, we optimized a vaccine candidate, parainfluenza virus 5 (PIV5) expressing the SARS-CoV-2 S protein (CVXGA1), and then demonstrated that a single-dose intranasal immunization with CVXGA1 protects against lethal infection of K18-hACE2 mice, a severe disease model. CVXGA1 immunization also prevented virus infection of ferrets and blocked contact transmission. This mucosal vaccine strategy inhibited SARS-CoV-2 replication in the upper respiratory tract, thus preventing disease progression to the lower respiratory tract. A PIV5-based mucosal vaccine provides a strategy to induce protective innate and cellular immune responses and reduce SARS-CoV-2 infection and transmission in populations.