A bivalent live-attenuated influenza vaccine for the control and prevention of H3N8 and H3N2 canine influenza viruses

Canine influenza viruses (CIVs) cause a contagious respiratory disease in dogs. CIV subtypes include H3N8, which originated from the transfer of H3N8 equine influenza virus (EIV) to dogs; and the H3N2, which is an avian-origin virus adapted to infect dogs. Only inactivated influenza vaccines (IIVs) are currently available against the different CIV subtypes. However, the efficacy of these CIV IIVs is not optimal and improved vaccines are necessary for the efficient prevention of disease caused by CIVs in dogs. Since live-attenuated influenza vaccines (LAIVs) induce better immunogenicity and protection efficacy than IIVs, we have combined our previously described H3N8 and H3N2 CIV LAIVs to create a bivalent vaccine against both CIV subtypes. Our findings show that, in a mouse model of infection, the bivalent CIV LAIV is safe and able to induce, upon a single intranasal immunization, better protection than that induced by a bivalent CIV IIV against subsequent challenge with H3N8 or H3N2 CIVs. These protection results also correlated with the ability of the bivalent CIV LAIV to induce better humoral immune responses. This is the first description of a bivalent LAIV for the control and prevention of H3N8 and H3N2 CIV infections in dogs

Development of a novel equine influenza virus live-attenuated vaccine

H3N8 equine influenza virus (EIV) is an important and significant respiratory pathogen of horses. EIV is enzootic in Europe and North America, mainly due to the suboptimal efficacy of current vaccines. We describe, for the first time, the generation of a temperature sensitive (ts) H3N8 EIV live-attenuated influenza vaccine (LAIV) using reverse-genetics approaches. Our EIV LAIV was attenuated (att) in vivo and able to induce, upon a single intranasal administration, protection against H3N8 EIV wild-type (WT) challenge in both a mouse model and the natural host, the horse. Notably, since our EIV LAIV was generated using reverse genetics, the vaccine can be easily updated against drifting or emerging strains of EIV using the safety backbone of our EIV LAIV as master donor virus (MDV). These results demonstrate the feasibility of implementing a novel EIV LAIV approach for the prevention and control of currently circulating H3N8 EIVs in horse populations

Inhibition of the Type I Interferon Antiviral Response During Arenavirus Infection

Arenaviruses merit interest both as tractable experimental model systems to study acute and persistent viral infections, and as clinically-important human pathogens. Several arenaviruses cause hemorrhagic fever (HF) disease in humans. In addition, evidence indicates that the globally-distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a human pathogen of clinical significance in congenital infections, and also poses a great danger to immunosuppressed individuals. Arenavirus persistence and pathogenesis are facilitated by their ability to overcome the host innate immune response. Mammalian hosts have developed both membrane toll-like receptors (TLR) and cytoplasmic pattern recognition receptors (PRRs) that recognize specific pathogen-associated molecular patterns (PAMPs), resulting in activation of the transcription factors IRF3 or IRF7, or both, which together with NF-κB and ATF-2/c-JUN induce production of type I interferon (IFN-I). IFN-I plays a key role in host anti-microbial defense by mediating direct antiviral effects via up-regulation of IFN-I stimulated genes (ISGs), activating dendritic cells (DCs) and natural killer (NK) cells, and promoting the induction of adaptive responses. Accordingly, viruses have developed a plethora of strategies to disrupt the IFN-I mediated antiviral defenses of the host, and the viral gene products responsible for these disruptions are often major virulence determinants. IRF3- and IRF7-dependent induction of host innate immune responses is frequently targeted by viruses. Thus, the arenavirus nucleoprotein (NP) was shown to inhibit the IFN-I response by interfering with the activation of IRF3. This NP anti-IFN activity, together with alterations in the number and function of DCs observed in mice chronically infected with LCMV, likely play an important role in LCMV persistence in its murine host. In this review we will discuss current knowledge about the cellular and molecular mechanisms by which arenaviruses can subvert the host innate immune response and their implications for understanding HF arenaviral disease as well as arenavirus persistence in their natural hosts

The K186E amino acid substitution in the canine influenza virus H3N8 NS1 protein restores its ability to inhibit host gene expression

Canine influenza viruses (CIVs) are the causative agents of canine influenza, a contagious respiratory disease in dogs, and include the equine-origin H3N8 and the avian-origin H3N2. Influenza A virus (IAV) non-structural protein 1 (NS1) is a virulence factor essential for counteracting the innate immune response. Here, we evaluated the ability of H3N8 CIV NS1 to inhibit host innate immune responses. We found that H3N8 CIV NS1 was able to efficiently counteract interferon (IFN) responses but was unable to block general gene expression in human or canine cells. Such ability was restored by a single amino acid substitution in position 186 (K186E) that resulted in NS1 binding to the 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), a cellular protein involved in pre-mRNA processing. We also examined the frequency distribution of K186 and E186 among H3N8 CIVs and equine influenza viruses (EIVs), the ancestors of H3N8 CIV, and experimentally determined the impact of amino acid 186 in the ability of different CIV and EIV NS1s to inhibit general gene expression. In all cases, the presence of E186 was responsible for the control of host gene expression. Contrastingly, the NS1 protein of H3N2 CIV harbors E186 and blocks general gene expression in canine cells. Altogether, our results confirm previous studies on the strain-dependent ability of NS1 to block general gene expression. Moreover, the observed polymorphism on amino acid 186 between H3N8 and H3N2 CIVs might be the result of adaptive changes acquired during long-term circulation of avian-origin IAVs in mammals. IMPORTANCE: Canine influenza is a respiratory disease of dogs caused by two CIV subtypes, the H3N8 and H3N2 viruses of equine and avian origin, respectively. Influenza NS1 is the main viral factor responsible for the control of host innate immune responses and changes in NS1 can play an important role in host adaptation. Here we assessed the ability of H3N8 CIV NS1 to inhibit host innate immune responses and gene expression. The H3N8 CIV NS1 did not block host gene expression but this activity was restored by a single amino acid substitution (K186E), which was responsible for NS1 binding to the host factor CPSF30. In contrast, the H3N2 CIV NS1, that contains E186, blocks general gene expression. Our results suggest that the ability to block host gene expression is not required for influenza replication in mammals but might be important in the long-term adaptation of avian-origin influenza viruses to mammals

Live attenuated influenza A virus vaccines with modified NS1 proteins for veterinary use

Influenza A viruses (IAV) spread rapidly and can infect a broad range of avian or mammalian species, having a tremendous impact in human and animal health and the global economy. IAV have evolved to develop efficient mechanisms to counteract innate immune responses, the first host mechanism that restricts IAV infection and replication. One key player in this fight against host-induced innate immune responses is the IAV non-structural 1 (NS1) protein that modulates antiviral responses and virus pathogenicity during infection. In the last decades, the implementation of reverse genetics approaches has allowed to modify the viral genome to design recombinant IAV, providing researchers a powerful platform to develop effective vaccine strategies. Among them, different levels of truncation or deletion of the NS1 protein of multiple IAV strains has resulted in attenuated viruses able to induce robust innate and adaptive immune responses, and high levels of protection against wild-type (WT) forms of IAV in multiple animal species and humans. Moreover, this strategy allows the development of novel assays to distinguish between vaccinated and/or infected animals, also known as Differentiating Infected from Vaccinated Animals (DIVA) strategy. In this review, we briefly discuss the potential of NS1 deficient or truncated IAV as safe, immunogenic and protective live-attenuated influenza vaccines (LAIV) to prevent disease caused by this important animal and human pathogen

Host-Range Restriction of Vaccinia Virus E3L Deletion Mutant Can Be Overcome In Vitro, but Not In Vivo, by Expression of the Influenza Virus NS1 Protein

During the last decades, research focused on vaccinia virus (VACV) pathogenesis has been intensified prompted by its potential beneficial application as a vector for vaccine development and anti-cancer therapies, but also due to the fear of its potential use as a bio-terrorism threat. Recombinant viruses lacking a type I interferon (IFN) antagonist are attenuated and hence good vaccine candidates. However, vaccine virus growth requires production in IFN-deficient systems, and thus viral IFN antagonists that are active in vitro, yet not in vivo, are of great value. The VACV E3 and influenza virus NS1 proteins are distinct double-stranded RNA-binding proteins that play an important role in pathogenesis by inhibiting the mammalian IFN-regulated innate antiviral response. Based on the functional similarities between E3 and NS1, we investigated the ability of NS1 to replace the biological functions of E3 of VACV in both in vitro and in vivo systems. For this, we generated a VACV recombinant virus lacking the E3L gene, yet expressing NS1 (VVΔE3L/NS1). Our study revealed that NS1 can functionally replace E3 in cultured cells, rescuing the protein synthesis blockade, and preventing apoptosis and RNA breakdown. In contrast, in vivo the VVΔE3L/NS1 virus was highly attenuated after intranasal inoculation, as it was unable to spread to the lungs and other organs. These results indicate that there are commonalities but also functional differences in the roles of NS1 and E3 as inhibitors of the innate antiviral response, which could potentially be utilized for vaccine production purposes in the future

A temperature sensitive live-attenuated canine influenza virus H3N8 vaccine

Canine influenza is a respiratory disease of dogs caused by canine influenza virus (CIV). CIV subtypes responsible for influenza in dogs include H3N8, which originated from the transfer of H3N8 equine influenza virus to dogs; and the H3N2 CIV, which is an avian-origin virus that adapted to infect dogs. Influenza infections are most effectively prevented through vaccination to reduce transmission and future infection. Currently, only inactivated influenza vaccines (IIVs) are available for the prevention of CIV in dogs. However, the efficacy of IIVs is suboptimal, and novel approaches are necessary for the prevention of disease caused by this canine respiratory pathogen. Using reverse genetics techniques, we have developed a live-attenuated CIV vaccine (LACIV) for the prevention of H3N8 CIV. The H3N8 LACIV replicates efficiently in canine cells at 33°C but is impaired at temperatures of 37 to 39°C and was attenuated compared to wild-type H3N8 CIV in vivo and ex vivo. The LACIV was able to induce protection against H3N8 CIV challenge with a single intranasal inoculation in mice. Immunogenicity and protection efficacy were better than that observed with a commercial CIV H3N8 IIV but provided limited cross-reactive immunity and heterologous protection against H3N2 CIV. These results demonstrate the feasibility of implementing a LAIV approach for the prevention and control of H3N8 CIV in dogs and suggest the need for a new LAIV for the control of H3N2 CIV. Importance: Two influenza A virus subtypes has been reported in dogs in the last 16 years: the canine influenza viruses (CIV) H3N8 and H3N2 of equine and avian origins, respectively. To date, only inactivated influenza vaccines (IIVs) are available to prevent CIV infections. Here, we report the generation of a recombinant, temperature-sensitive H3N8 CIV as a live-attenuated influenza vaccine (LAIV), which was attenuated in mice and dog tracheal, explants compared to CIV H3N8 wild type. A single dose of H3N8 LACIV showed immunogenicity and protection against a homologous challenge that was better than that conferred with an H3N8 IIV, demonstrating the feasibility of implementing a LAIV approach for the improved control of H3N8 CIV infections in dogs

The K186E Amino Acid Substitution in the Canine Influenza Virus H3N8 NS1 Protein Restores Its Ability To Inhibit Host Gene Expression

Canine influenza viruses (CIVs) are the causative agents of canine influenza, a contagious respiratory disease in dogs, and include the equine-origin H3N8 and the avian-origin H3N2. Influenza A virus (IAV) non-structural protein 1 (NS1) is a virulence factor essential for counteracting the innate immune response. Here, we evaluated the ability of H3N8 CIV NS1 to inhibit host innate immune responses. We found that H3N8 CIV NS1 was able to efficiently counteract interferon (IFN) responses but was unable to block general gene expression in human or canine cells. Such ability was restored by a single amino acid substitution in position 186 (K186E) that resulted in NS1 binding to the 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), a cellular protein involved in pre-mRNA processing. We also examined the frequency distribution of K186 and E186 among H3N8 CIVs and equine influenza viruses (EIVs), the ancestors of H3N8 CIV, and experimentally determined the impact of amino acid 186 in the ability of different CIV and EIV NS1s to inhibit general gene expression. In all cases, the presence of E186 was responsible for the control of host gene expression. Contrastingly, the NS1 protein of H3N2 CIV harbors E186 and blocks general gene expression in canine cells. Altogether, our results confirm previous studies on the strain-dependent ability of NS1 to block general gene expression. Moreover, the observed polymorphism on amino acid 186 between H3N8 and H3N2 CIVs might be the result of adaptive changes acquired during long-term circulation of avian-origin IAVs in mammals. IMPORTANCE: Canine influenza is a respiratory disease of dogs caused by two CIV subtypes, the H3N8 and H3N2 viruses of equine and avian origin, respectively. Influenza NS1 is the main viral factor responsible for the control of host innate immune responses and changes in NS1 can play an important role in host adaptation. Here we assessed the ability of H3N8 CIV NS1 to inhibit host innate immune responses and gene expression. The H3N8 CIV NS1 did not block host gene expression but this activity was restored by a single amino acid substitution (K186E), which was responsible for NS1 binding to the host factor CPSF30. In contrast, the H3N2 CIV NS1, that contains E186, blocks general gene expression. Our results suggest that the ability to block host gene expression is not required for influenza replication in mammals but might be important in the long-term adaptation of avian-origin influenza viruses to mammals

Transcriptional role of p53 in interferon-mediated antiviral immunity

Tumor suppressor p53 is activated by several stimuli, including DNA damage and oncogenic stress. Previous studies (Takaoka, A., S. Hayakawa, H. Yanai, D. Stoiber, H. Negishi, H. Kikuchi, S. Sasaki, K. Imai, T. Shibue, K. Honda, and T. Taniguchi. 2003. Nature. 424:516–523) have shown that p53 is also induced in response to viral infections as a downstream transcriptional target of type I interferon (IFN) signaling. Moreover, many viruses, including SV40, human papillomavirus, Kaposi's sarcoma herpesvirus, adenoviruses, and even RNA viruses such as polioviruses, have evolved mechanisms designated to abrogate p53 responses. We describe a novel p53 function in the activation of the IFN pathway. We observed that infected mouse and human cells with functional p53 exhibited markedly decreased viral replication early after infection. This early inhibition of viral replication was mediated both in vitro and in vivo by a p53-dependent enhancement of IFN signaling, specifically the induction of genes containing IFN-stimulated response elements. Of note, p53 also contributed to an increase in IFN release from infected cells. We established that this p53-dependent enhancement of IFN signaling is dependent to a great extent on the ability of p53 to activate the transcription of IFN regulatory factor 9, a central component of the IFN-stimulated gene factor 3 complex. Our results demonstrate that p53 contributes to innate immunity by enhancing IFN-dependent antiviral activity independent of its functions as a proapoptotic and tumor suppressor gene