FLUAV RAM-IGIP: A modified live influenza virus vaccine that enhances humoral and mucosal responses against influenza.

Current influenza A vaccines fall short, leaving both humans and animals vulnerable. To address this issue, we have developed attenuated modified live virus (MLV) vaccines against influenza using genome rearrangement techniques targeting the internal gene segments of FLUAV. The rearranged M2 (RAM) strategy involves cloning the M2 ORF downstream of the PB1 ORF in segment 2 and incorporating multiple early stop codons within the M2 ORF in segment 7. Additionally, the IgA-inducing protein (IGIP) coding region was inserted into the HA segment to further attenuate the virus and enhance protective mucosal responses. RAM-IGIP viruses exhibit similar growth rates to wild type (WT) viruses in vitro and remain stable during multiple passages in cells and embryonated eggs. The safety, immunogenicity, and protective efficacy of the RAM-IGIP MLV vaccine against the prototypical 2009 pandemic H1N1 strain A/California/04/2009 (H1N1) (Ca/04) were evaluated in Balb/c mice and compared to a prototypic cold-adapted live attenuated virus vaccine. The results demonstrate that the RAM-IGIP virus exhibits attenuated virulence in vivo. Mice vaccinated with RAM-IGIP and subsequently challenged with an aggressive lethal dose of the Ca/04 strain exhibited complete protection. Analysis of the humoral immune response revealed that the inclusion of IGIP enhanced the production of neutralizing antibodies and augmented the antibody-dependent cellular cytotoxicity response. Similarly, the RAM-IGIP potentiated the mucosal immune response against various FLUAV subtypes. Moreover, increased antibodies against NP and NA responses were observed. These findings support the development of MLVs utilizing genome rearrangement strategies in conjunction with the incorporation of immunomodulators. IMPORTANCE: Current influenza vaccines offer suboptimal protection, leaving both humans and animals vulnerable. Our novel attenuated MLV vaccine, built by rearranging FLUAV genome segments and incorporating the IgA-inducing protein, shows promising results. This RAM-IGIP vaccine exhibits safe attenuation, robust immune responses, and complete protection against lethal viral challenge in mice. Its ability to stimulate broad-spectrum humoral and mucosal immunity against diverse FLUAV subtypes makes it a highly promising candidate for improved influenza vaccines.

Influenza A virus reassortment in mammals gives rise to genetically distinct within-host subpopulations.

Influenza A virus (IAV) genetic exchange through reassortment has the potential to accelerate viral evolution and has played a critical role in the generation of multiple pandemic strains. For reassortment to occur, distinct viruses must co-infect the same cell. The spatio-temporal dynamics of viral dissemination within an infected host therefore define opportunity for reassortment. Here, we used wild type and synonymously barcoded variant viruses of a pandemic H1N1 strain to examine the within-host viral dynamics that govern reassortment in guinea pigs, ferrets and swine. The first two species are well-established models of human influenza, while swine are a natural host and a frequent conduit for cross-species transmission and reassortment. Our results show reassortment to be pervasive in all three hosts but less frequent in swine than in ferrets and guinea pigs. In ferrets, tissue-specific differences in the opportunity for reassortment are also evident, with more reassortants detected in the nasal tract than the lower respiratory tract. While temporal trends in viral diversity are limited, spatial patterns are clear, with heterogeneity in the viral genotypes detected at distinct anatomical sites revealing extensive compartmentalization of reassortment and replication. Our data indicate that the dynamics of viral replication in mammals allow diversification through reassortment but that the spatial compartmentalization of variants likely shapes their evolution and onward transmission.

Pathobiology and dysbiosis of the respiratory and intestinal microbiota in 14 months old Golden Syrian hamsters infected with SARS-CoV-2

The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS2) affected the geriatric population. Among research models, Golden Syrian hamsters (GSH) are one of the most representative to study SARS2 pathogenesis and host responses. However, animal studies that recapitulate the effects of SARS2 in the human geriatric population are lacking. To address this gap, we inoculated 14 months old GSH (resembling people over 60 years old) with a prototypic ancestral strain of SARS2 and studied the effects on virus pathogenesis, virus shedding, and respiratory and gastrointestinal microbiome changes. SARS2 infection led to high vRNA loads in the nasal turbinates (NT), lungs, and trachea as well as higher pulmonary lesions scores later in infection. Dysbiosis throughout SARS2 disease progression was observed in the pulmonary microbial dynamics with the enrichment of opportunistic pathogens (Haemophilus, Fusobacterium, Streptococcus, Campylobacter, and Johnsonella) and microbes associated with inflammation (Prevotella). Changes in the gut microbial community also reflected an increase in multiple genera previously associated with intestinal inflammation and disease (Helicobacter, Mucispirillum, Streptococcus, unclassified Erysipelotrichaceae, and Spirochaetaceae). Influenza A virus (FLUAV) pre-exposure resulted in slightly more pronounced pathology in the NT and lungs early on (3 dpc), and more notable changes in lungs compared to the gut microbiome dynamics. Similarities among aged GSH and the microbiome in critically ill COVID-19 patients, particularly in the lower respiratory tract, suggest that GSHs are a representative model to investigate microbial changes during SARS2 infection. The relationship between the residential microbiome and other confounding factors, such as SARS2 infection, in a widely used animal model, contributes to a better understanding of the complexities associated with the host responses during viral infections. Author SummaryThe SARS-CoV-2 pandemic led to millions of human losses, notably affecting the geriatric population, who are at greater risk of developing acute respiratory distress infection leading to prolonged hospitalization and death. However, the mechanism of age-related pathogenicity is not fully understood. Here, we utilized an aged Syrian hamster model resembling ~60-year-old humans to analyze the pathobiology, host response, and effects of SARS2 on the respiratory and intestinal microbiome. We identified specific microbial markers observed in severe COVID-19 patients within the lungs of aged hamsters infected with SARS-CoV-2. Prior influenza A virus (H1N1) exposure amplified these changes. Similarities among aged GSH and critically ill COVID-19 patients suggest that GSHs are a valuable model for investigating microbial changes during SARS2 infection. The relationship between the age, residential microbiome and viral pathogens contributes to a better understanding of the complexities associated with the host responses during viral infection while limiting potential environmental factors that may contribute to inter-individual variation.

Surveillance of seasonal respiratory viruses among Chilean patients during the COVID-19 pandemic

The StudySARS-CoV-2 has generated over 122 million cases worldwide. Non-pharmaceuticals interventions such as confinements and lockdowns started in Chile on March 18th 2020. In Europe, confinements and lockdowns have been accompanied by a decrease in the circulation of other respiratory viruses such as Influenza A virus(IAV), Influenza B virus(IBV) or respiratory syncytial virus(RSV) (1). Although changes in circulation patterns of respiratory viruses have been reported, limited information regarding the southern hemisphere is available where the SARS-CoV-2 pandemic merged with the winter season. We conducted viral surveillance of respiratory viruses and we evaluated their presence and establishing whether they were co-circulating with SARS-CoV-2.

Mild and severe SARS-CoV-2 infection induces respiratory and intestinal microbiome changes in the K18-hACE2 transgenic mouse model

Transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in millions of deaths and declining economies around the world. K18-hACE2 mice develop disease resembling severe SARS-CoV-2 infection in a virus dose-dependent manner. The relationship between SARS-CoV-2 and the intestinal or respiratory microbiome is not fully understood. In this context, we characterized the cecal and lung microbiome of SARS-CoV-2 challenged K18-hACE2 transgenic mice in the presence or absence of treatment with the Mpro inhibitor GC376. Cecum microbiome showed decreased Shannon and Inv Simpson diversity index correlating with SARS-CoV-2 infection dosage and a difference of Bray-Curtis dissimilarity distances among control and infected mice. Bacterial phyla such as Firmicutes, particularly Lachnospiraceae and Oscillospiraceae, were significantly less abundant while Verrucomicrobiota, particularly the family Akkermansiaceae, were increasingly more prevalent during peak infection in mice challenged with a high virus dose. In contrast to the cecal microbiome, the lung microbiome showed similar microbial diversity among the control, low and high challenge virus groups, independent of antiviral treatment. Bacterial phyla in the lungs such as Bacteroidota decreased while Firmicutes and Proteobacteria were significantly enriched in mice challenged with a high dose of SARS-CoV-2. In summary, we identified changes in the cecal and lung microbiome of K18-hACE2 mice with severe clinical signs of SARS-CoV-2 infection. IMPORTANCEThe COVID-19 pandemic has resulted in millions of deaths. The hosts respiratory and intestinal microbiome can affect directly or indirectly the immune system during viral infections. We characterized the cecal and lung microbiome in a relevant mouse model challenged with a low and high dose of SARS-CoV-2 in the presence or absence of an antiviral Mpro inhibitor, GC376. Decreased microbial diversity and taxonomic abundances of the phyla Firmicutes, particularly Lachnospiraceae, correlating with infection dosage was observed in the cecum. In addition, microbes within the family Akkermansiaceae were increasingly more prevalent during peak infection, which is observed in other viral infections. The lung microbiome showed similar microbial diversity to the control, independent of antiviral treatment. Decreased Bacteroidota and increased Firmicutes and Proteobacteria were observed in the lungs in a virus dose-dependent manner. These studies add to a better understanding of the complexities associated with the intestinal microbiome during respiratory infections.

Efficacy of GC-376 against SARS-CoV-2 virus infection in the K18 hACE2 transgenic mouse model.

The COVID-19 pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the defining global health emergency of this century. GC-376 is a M pro inhibitor with antiviral activity against SARS-CoV-2 in vitro . Using the K18-hACE2 mouse model, the in vivo antiviral efficacy of GC-376 against SARS-CoV-2 was evaluated. GC-376 treatment was not toxic in K18-hACE2 mice and produced milder tissue lesions, reduced viral loads, fewer presence of viral antigen, and reduced inflammation in comparison to vehicle-treated controls, most notably in the brain in mice challenged with a low virus dose. Although GC-376 was not sufficient to improve neither clinical symptoms nor survival, it did show a positive effect against SARS-CoV-2 in vivo . This study supports the notion that the K18-hACE2 mouse model is suitable to study antiviral therapies against SARS-CoV-2, and GC-376 represents a promising lead candidate for further development to treat SARS-CoV-2 infection.

Efficacy of GC-376 against SARS-CoV-2 virus infection in the K18 hACE2 transgenic mouse model

The COVID-19 pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the defining global health emergency of this century. GC-376 is a Mpro inhibitor with antiviral activity against SARS-CoV-2 in vitro. Using the K18-hACE2 mouse model, the in vivo antiviral efficacy of GC-376 against SARS-CoV-2 was evaluated. GC-376 treatment was not toxic in K18-hACE2 mice and produced milder tissue lesions, reduced viral loads, fewer presence of viral antigen, and reduced inflammation in comparison to vehicle-treated controls, most notably in the brain in mice challenged with a low virus dose. Although GC-376 was not sufficient to improve neither clinical symptoms nor survival, it did show a positive effect against SARS-CoV-2 in vivo. This study supports the notion that the K18-hACE2 mouse model is suitable to study antiviral therapies against SARS-CoV-2, and GC-376 represents a promising lead candidate for further development to treat SARS-CoV-2 infection.