RESUMO
BACKGROUND AND OBJECTIVES: Influenza A viruses cause highly contagious diseases in a variety of hosts, including humans and pigs. To develop a vaccine that can be broadly effective against genetically divergent strains of the virus, in this study we employed molecular breeding (DNA shuffling) technology to create a panel of chimeric HA genes. METHODS AND RESULTS: Each chimeric HA gene contained genetic elements from parental swine influenza A viruses that had a history of zoonotic transmission, and also from a 2009 pandemic virus. Each parental virus represents a major phylogenetic clade of influenza A H1N1 viruses. Nine shuffled HA constructs were initially screened for immunogenicity in mice by DNA immunization, and one chimeric HA (HA-129) was expressed on both a A/Puerto Rico/8/34 backbone with mutations associated with a live, attenuated phenotype (PR8LAIV-129) and a A/swine/Texas/4199-2/98 backbone (TX98-129). When delivered to mice, the PR8LAIV-129 induced antibodies against all four parental viruses, which was similar to the breadth of immunity observed when HA-129 was delivered as a DNA vaccine. This chimeric HA was then tested as a candidate vaccine in a nursery pig model, using inactivated TX98-129 virus as the backbone. The results demonstrate that pigs immunized with HA-129 developed antibodies against all four parental viruses, as well as additional primary swine H1N1 influenza virus field isolates. CONCLUSION: This study established a platform for creating novel genes of influenza viruses using a molecular breeding approach, which will have important applications toward future development of broadly protective influenza virus vaccines.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H1N1/genética , Vacinas contra Influenza/imunologia , Animais , Anticorpos Antivirais/sangue , Embaralhamento de DNA , Feminino , Fusão Gênica , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Imunização , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/genética , Camundongos , Sus scrofa/imunologia , Sus scrofa/virologiaRESUMO
Influenza A virus causes a highly contagious respiratory disease in a variety of avian and mammalian hosts, including humans and pigs. The primary means for preventing influenza epidemics is vaccination. Epitope-based vaccine represents a new approach to achieve protective immunity. The objective of this study was to construct and evaluate the immunogenicity of an epitope-based antigen for its potential application in future influenza vaccine development. The antigen, comprised of a set of consensus influenza A virus epitopes (IAVe), was genetically linked to a subunit of the bacterial heat-labile enterotoxin (LTB) as an adjuvant. Immunogenicity of this LTB-IAVe antigen was evaluated in a pig model. Despite an inability to detect neutralizing antibodies directed toward the whole virus, humoral immunity against the IAVe was demonstrated in both serum (IgA and IgG) and mucosal secretions (IgG) of immunized pigs. Specific cellular immunity was also induced after LTB-IAVe immunization, as evidenced by up-regulating of IL-1ß, IL-8, and IL-4 expression in peripheral blood mononuclear cells (PBMCs) of vaccinated pigs. In comparison to the non-immunized pigs, pigs immunized with the LTB-IAVe showed improved protection against a pathogenic H1N1 swine influenza virus challenge, with about 50% decrease of pneumonic lesions and 10-fold reduction of the viral load in lung and nasal secretion at five days post challenge. This study establishes a platform for future construction of epitope-based vaccines against influenza A virus infection.
Assuntos
Toxinas Bacterianas/farmacologia , Enterotoxinas/farmacologia , Epitopos/imunologia , Proteínas de Escherichia coli/farmacologia , Fenômenos Imunogenéticos/efeitos dos fármacos , Vacinas contra Influenza/imunologia , Leucócitos Mononucleares , Infecções por Orthomyxoviridae/veterinária , Doenças dos Suínos/prevenção & controle , Adjuvantes Imunológicos/farmacologia , Animais , Anticorpos Neutralizantes/sangue , Toxinas Bacterianas/genética , Enterotoxinas/genética , Proteínas de Escherichia coli/genética , Humanos , Imunização , Imunoglobulina A/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Interleucina-4/imunologia , Interleucina-8/imunologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Suínos , Doenças dos Suínos/imunologiaRESUMO
We have developed a porcine intestine epithelial cell line, designated SD-PJEC for the propagation of influenza viruses. The SD-PJEC cell line is a subclone of the IPEC-J2 cell line, which was originally derived from newborn piglet jejunum. Our results demonstrate that SD-PJEC is a cell line of epithelial origin that preferentially expresses receptors of oligosaccharides with Sia2-6Gal modification. This cell line is permissive to infection with human and swine influenza A viruses and some avian influenza viruses, but poorly support the growth of human-origin influenza B viruses. Propagation of swine-origin influenza viruses in these cells results in a rapid growth rate within the first 24 h post-infection and the titres ranged from 4 to 8 log(10) TCID(50) ml(-1). The SD-PJEC cell line was further tested as a potential alternative cell line to Madin-Darby canine kidney (MDCK) cells in conjunction with 293T cells for rescue of swine-origin influenza viruses using the reverse genetics system. The recombinant viruses A/swine/North Carolina/18161/02 (H1N1) and A/swine/Texas/4199-2/98 (H3N2) were rescued with virus titres of 7 and 8.25 log(10) TCID(50) ml(-1), respectively. The availability of this swine-specific cell line represents a more relevant substrate for studies and growth of swine-origin influenza viruses.
