Two novel recombinant strains of modified vaccinia Ankara (rMVA) for the vaccination of horses against equine influenza virus were developed, and preliminary evidence of their immunogenicity in ponies was demonstrated [Breathnach CC, Rudersdorf R, Lunn DP. Use of recombinant modified vaccinia Ankara vectors for equine influenza vaccination. Vet Immunol Immunopathol 2004:98;127-36]. The present study assessed the protective efficacy of these rMVA strains in ponies, examined the advantage of combining rMVA vaccination with a DNA priming dose, and investigated the protection resulting from equine influenza nucleoprotein (NP) versus haemagglutinin (HA) vaccination. Twenty yearling ponies, seronegative for equine influenza virus, were divided into four groups of five. Group 1 and Group 2 ponies were vaccinated using a DNA prime-rMVA boost vaccination regimen, with HA- or NP-expressing vectors, respectively. Group 3 ponies were vaccinated with rMVA-HA only. Group 4 ponies served as unvaccinated controls. Vaccines were administered on days 0, 42 and 70, and all ponies were challenge infected with influenza virus on day 100. Antigen-specific antibody and cellular immune responses to each vaccination regimen were monitored throughout the experiment. Both groups of HA-vaccinated ponies were significantly protected from clinical disease following challenge infection, demonstrating the efficacy of rMVA vaccination with or without a DNA prime. NP-vaccination provided more limited protection from clinical disease. The protective post-vaccinal immune responses were characterized by antigen-specific IgGa, IgGb and IgA antibodies which were induced both in serum and in nasal secretions. Virus-specific lymphoproliferative and IFN-gamma mRNA responses were also elicited by each vaccination regimen. These data demonstrate that vaccination of horses with rMVA alone, or as part of a prime-boost regimen, is an effective means of inducing protective immunity to influenza virus infection, and also indicate that NP-specific immune responses can contribute to protection of horses.