Temperature-Sensitive Live-Attenuated Canine Influenza Virus H3N8 Vaccine

Abstract

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.

Interplay of PA-X and NS1 Proteins in Replication and Pathogenesis of a Temperature-Sensitive 2009 Pandemic H1N1 Influenza A Virus

“…1B). Both combinations of plasmids (WT and LAIV) resulted in similar Gaussia luciferase (Gluc) expression levels at 33°C; however, Gluc expression was significantly reduced at higher temperatures (37°C and 39°C) in cells transfected with the pH1N1 LAIV plasmids, consistent with previous results shown for other IAV LAIV strains (9)(10)(11). Importantly, Western blot analysis shows similar levels of NP expression at each temperature for WT-and LAIV-transfected cells (Fig.…”

“…In order to generate a pH1N1 LAIV virus, we introduced four ts mutations identified in previous studies into the PB2 (N265S) and PB1 (K391E, D581G, and A661T) ORFs of pH1N1 (Fig. 1A) (7,9,10,39). No mutation was introduced into the viral NP, since pH1N1 NP already contains a G at position 34.…”

“…First, because a recombinant pH1N1 harboring inhibitory PA-X and NS1 proteins could lead to a more virulent virus (35)(36)(37)(38), we selected, for safety reasons, to use the ca, ts, and att pH1N1 LAIV virus. Second, we and others have previously shown that LAIVs provide a more sensitive approach to study mutations affecting overall viral replication and pathogenicity (5,7,(9)(10)(11)39). Finally, the results obtained from our studies could be used to identify and develop more efficient LAIV candidates with acceptable safety profiles by modulating the virus' ability to control the host immune responses (7,11).…”

“…The attenuation mechanisms are not totally understood but could involve several steps in the replication cycle of the virus (4). Importantly, when the ts signature of A/AA/6/60 was introduced into three different viruses (influenza virus A/Puerto Rico/8/34 H1N1 [PR8] [7,8], canine influenza virus H3N8 [9,10], or pandemic influenza virus A/New York/1682/2009 H1N1 [11]), a similar ts viral phenotype was observed in tissue culture cells and in mouse models of infection. Moreover, ca, ts, and att viruses have been previously utilized to evaluate additional mutations that can increase the safety profile of a vaccine candidate (7,11).…”

A Live Attenuated Influenza Vaccine Elicits Enhanced Heterologous Protection When the Internal Genes of the Vaccine Are Matched to Those of the Challenge Virus

“…The amino acid residues conferring these ts, ca, and att phenotypes have been mapped for both viruses and share no residues in common, although they fall almost exclusively into the genes encoding viral RNAdependent RNA polymerase (28,29). Moreover, our group and others have introduced the mutations responsible for the ts and att phenotype of AA/60 H2N2 into different IAV subtype and strain backgrounds and have shown that they confer a ts phenotype in cell culture and an att phenotype in animal models (30)(31)(32)(33)(34)(35)(36)(37).…”