Elastase-Dependent Live Attenuated Swine Influenza A Viruses Are Immunogenic and Confer Protection against Swine Influenza A Virus Infection in Pigs

Influenza A viruses cause significant morbidity in swine, resulting in a substantial economic burden. Swine influenza virus (SIV) infection also poses important human public health concerns. Vaccination is the primary method for the prevention of influenza virus infection. Previously, we generated two elastase-dependent mutant SIVs derived from A/Sw/Saskatchewan/18789/02(H1N1): A/Sw/Sk-R345V (R345V) and A/Sw/Sk-R345A (R345A). These two viruses are highly attenuated in pigs, making them good candidates for a live-virus vaccine. In this study, the immunogenicity and the ability of these candidates to protect against SIV infection were evaluated in pigs. We report that intratracheally administrated R345V and R345A induced antigen-specific humoral and cell-mediated immunity characterized by increased production of immunoglobulin G (IgG) and IgA antibodies in the serum and in bronchoalveolar lavage fluid, high hemagglutination inhibition titers in serum, an enhanced level of lymphocyte proliferation, and higher numbers of gamma interferon-secreting cells at the site of infection. Based on the immunogenicity results, the R345V virus was further tested in a protection trial in which pigs were vaccinated twice with R345V and then challenged with homologous A/Sw/Saskatchewan/18789/02, H1N1 antigenic variant A/Sw/Indiana/1726/88 or heterologous subtypic H3N2 A/Sw/Texas/4199-2/9/98. Our data showed that two vaccinations with R345V provided pigs with complete protection from homologous H1N1 SIV infection and partial protection from heterologous subtypic H3N2 SIV infection. This protection was characterized by significantly reduced macroscopic and microscopic lung lesions, lower virus titers from the respiratory tract, and lower levels of proinflammatory cytokines. Thus, elastase-dependent SIV mutants can be used as live-virus vaccines against swine influenza in pigs.Swine influenza virus (SIV) is the causative pathogen of swine influenza, a highly contagious, acute respiratory viral disease of swine. The mortality of SIV-infected pigs is usually low, although morbidity may approach 100%. Swine influenza is characterized by sudden onset, coughing, respiratory distress, weight loss, fever, nasal discharge, and rapid recovery (38). SIV is a member of the influenza virus A genus in the Orthomyxoviridae family, and the virus has a genome consisting of eight segments of negative-sense single-stranded RNA (29). Epithelial cells in the swine respiratory tract have receptors for both avian and mammalian influenza viruses (13); thus, pigs could potentially serve as “mixing vessels” for the generation of new reassortant strains of influenza A virus that have pandemic capacity. There are a number of reports in which the direct transmission of influenza viruses from pigs to humans has been documented (6, 12, 52), and several of these cases have resulted in human fatalities (19, 35, 40, 53). Consequently, effective control of SIV would be beneficial to both humans and animals.Until 1998, classical H1N1 SIVs were the predominant isolates from pigs in the United States and Canada (5, 28). In 1997 to 1998, a dramatic change in the epidemiologic pattern of SIV began. Serological studies conducted by Olsen and colleagues in 1997 to 1998 detected a significant increase in H3-seropositive individuals, and H3N2 SIVs were isolated from pigs in both the United States and Canada (17, 54). Furthermore, reassortment between H3N2 viruses and classical H1N1 SIV resulted in the appearance of H1N2 reassortant viruses (14, 15). In addition to the isolation of H4N6 viruses, which are of duck origin, in pigs in Canada (16), wholly avian viruses of the H3N3 and H1N1 subtypes have also been isolated from Canadian pigs (18). In general, three major SIV subtypes exist, i.e., H1N1, H1N2, and H3N2, each of which has multiple genetic and antigenic variants circulating in North American swine populations (18, 28). The increased incidence of avian-like or human-like SIV reassortants raises concerns for public health and requires research devoted to the development of cross-protective SIV vaccines.Currently available swine influenza vaccines are based on inactivated whole virus of the H1N1 and H3N2 subtypes. Application of these vaccines reduces the severity of disease but does not provide consistent protection from infection (3, 22). In contrast to killed vaccines that are administered intramuscularly, intranasally administered live attenuated influenza vaccines (LAIV) induce an immune response at the site of natural infection. Therefore, an LAIV has the potential to induce broad humoral and cellular immune responses that could provide protection against antigenically different influenza viruses. LAIV based on attenuation of the virus by cold adaptation are available for humans (2) and horses (41). However, to date, no SIV LAIV are commercially available for use in swine in North America. Recent studies by Solorzano et al. showed that a mutant SIV with a truncated NS1 protein was highly attenuated in pigs (36). In addition, this SIV/NS1 LAIV was capable of stimulating a protective immune response against homologous SIVs and a partial protection against heterologous subtypic wild-type (WT) SIVs (31, 50). Stech and colleagues demonstrated that the conversion of a conserved cleavage site in the influenza virus hemagglutinin (HA) protein from a trypsin-sensitive site to an elastase-sensitive site results in in vivo attenuation of the influenza virus in mouse models (9, 37). Furthermore, these elastase-dependent LAIV were able to induce protective systemic and mucosal immune responses. Recently, we showed that two elastase-dependent SIVs derived from A/Sw/Saskatchewan/18789/02 (SIV/Sk02), R345V and R345A, are attenuated in their natural host, pigs (23). In the current study, we addressed the immunogenic and cross-protective abilities of these mutants.