Elastase-dependent live attenuated swine influenza A viruses are immunogenic and confer protection against swine influenza A virus infection in pigs

Abstract

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.

FIG. 1.

Cell mediated immune responses induced by mutant viruses. (A) The number of SIV-specific IFN-γ secreting cells in pigs after the first and second vaccinations with R345V and R345A, as measured by ELISPOT assay. The results are reported as the average number of spots observed in the wells with SIV antigen-stimulated LNCs (seeded in triplicates) minus the average number of spots in the negative control wells (LNCs simulated with medium alone). (B) SIV-specific lymphocyte proliferation response of LNCs in pigs after the first and second vaccinations with R345V and R345A, as measured by LPR assay. The LPR was calculated as the mean cpm of triplicate cultures and is expressed as a stimulation index (cpm in the presence of stimulus/cpm in the absence of stimulus). Each data point represents an individual animal, and median values are indicated by horizontal bars. *, P < 0.05; **, P < 0.01.

FIG. 2.

SIV-specific antibodies induced by the mutant viruses. (A to C) SIV/Sk02-specific HI (A), IgG (B), and IgA (C) levels induced by R345V and R345A were detected in the serum after the first and second vaccinations. (D to G) Mucosal IgG (D and G) and IgA (E and F) antibody titers from nasal swabs (D and E) and from BALF (F and G) were also determined. Each data point represents an individual animal in each treatment group, and median values are indicated by horizontal bars. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG. 3.

Cross-reactive antibodies induced by the R345V virus. The levels of serum HI (A) and IgG (B) and mucosal IgA and IgG from BALF (C) that cross-reacted with SIV/Ind88 and SIV/Tx98 were determined after the second immunization. Each data point represents an individual animal in each treatment group, and median values are indicated by horizontal bars.

FIG. 4.

Macroscopic lung lesions. The percentage of the area affected by pneumonia was estimated visually for each lung lobe. The total percentage for the entire lung was calculated based on the volume proportions of each lung lobe with respect to the total lung volume. Results are the mean score of the lung lesions ± standard error of the mean for the seven animals in each group. *, P < 0.05; **, P < 0.01.

FIG. 5.

Microscopic lung lesions. (A) Medium-size bronchioles from the lung of a control pig inoculated with MEM only. (B) Severe necrotizing bronchiolitis with moderate multifocal necrosis and attenuation of surviving bronchiolar epithelium in the lungs of unvaccinated SIV/Sk02 challenged pigs. (C) Normal bronchioles and the surrounding blood vessels from the lungs of pigs vaccinated with R345V and challenged with H1N1 SIV/Sk02. (D) Severe acute necrotizing bronchiolitis, interstitial pneumonia and severe bronchiolar necrosis in unvaccinated H1N1 SIV/Ind88-challenged pigs. (E) Normal bronchioles from the lungs of pigs vaccinated with R345V and challenged with H1N1 SIV/Ind88. (F) Moderate bronchiolitis with focal necrosis and severe neutrophil infiltration in the lumen of the bronchioles and bronchi in unvaccinated H3N2 SIV/Tx98-challenged pigs. (G) Mild to moderate bronchiolitis with rare epithelial necrosis in R345V-vaccinated H3N2 SIV/Tx98-challenged pigs. Magnification, ×20; scale bars, 200 μm.

FIG. 6.

Lung virus titers. Lung tissues from the right apical, cardiac, and diaphragmatic lobes were collected and homogenized, and virus titers were determined on MDCK cells. Titers were calculated according to the Reed-Muench method. Each data point represents an individual animal in each treatment group, and median values are indicated by horizontal bars. **, P < 0.01.

FIG. 7.

Levels of proinflammatory cytokines IFN-α (A), IL-1β (B), and IL-6 (C) in the lungs of unvaccinated and R345V-vaccinated challenged pigs. Each data point represents an individual animal in each treatment group, and median values are indicated by horizontal bars. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG. 8.

Antibody titers after vaccination and challenge. The levels of serum HI (A) and IgG (B) titers and mucosa IgA titers from BALF (C) specific for SIV/Sk02, SIV/Ind88, and SIV/Tx98 were determined after pigs were vaccinated twice with R345V and challenged with WT SIVs. Each data point represents an individual animal in each treatment group, and median values are indicated by horizontal bars.