Intranasal vaccination with replication-defective adenovirus type 5 encoding influenza virus hemagglutinin elicits protective immunity to homologous challenge and partial protection to heterologous challenge in pigs

Abstract

Influenza A virus (IAV) is widely circulating in the swine population and causes significant economic losses. To combat IAV infection, the swine industry utilizes adjuvanted whole inactivated virus (WIV) vaccines, using a prime-boost strategy. These vaccines can provide sterilizing immunity toward homologous virus but often have limited efficacy against a heterologous infection. There is a need for vaccine platforms that induce mucosal and cell-mediated immunity that is cross-reactive to heterologous viruses and can be produced in a short time frame. Nonreplicating adenovirus 5 vector (Ad5) vaccines are one option, as they can be produced rapidly and given intranasally to induce local immunity. Thus, we compared the immunogenicity and efficacy of a single intranasal dose of an Ad5-vectored hemagglutinin (Ad5-HA) vaccine to those of a traditional intramuscular administration of WIV vaccine. Ad5-HA vaccination induced a mucosal IgA response toward homologous IAV and primed an antigen-specific gamma interferon (IFN-γ) response against both challenge viruses. The Ad5-HA vaccine provided protective immunity to homologous challenge and partial protection against heterologous challenge, unlike the WIV vaccine. Nasal shedding was significantly reduced and virus was cleared from the lung by day 5 postinfection following heterologous challenge of Ad5-HA-vaccinated pigs. However, the WIV-vaccinated pigs displayed vaccine-associated enhanced respiratory disease (VAERD) following heterologous challenge, characterized by enhanced macroscopic lung lesions. This study demonstrates that a single intranasal vaccination with an Ad5-HA construct can provide complete protection from homologous challenge and partial protection from heterologous challenge, as opposed to VAERD, which can occur with adjuvanted WIV vaccines.

Figures

Fig 1

Ad5-HA vaccination elicits IFN-γ responses to both homologous and heterologous viruses. Pigs were vaccinated intranasally with Ad5-empty or Ad5-HA (CA09) on day 0. PBMC were isolated on day 21 or 42 postvaccination from pigs vaccinated with Ad5-empty or Ad5-HA, and an ELISpot assay was used to determine the number of IFN-γ SC in 5 × 105 PBMC following stimulation in vitro for 18 h with live IAV strain A/CA/04/09 (A) or A/SW/MN/2011/08 (B). (C) PBMC were also collected from pigs in the kaCA group, and the number of IFN-γ SC was determined by ELISpot assay. Results are reported as means ± standard errors of the means (SEM), and statistical differences between nonvaccinated and vaccinated groups challenged with the same virus are indicated with connecting bars and asterisks (P < 0.05).

Fig 2

Macroscopic lung lesions on day 5 postinfection were reduced by Ad5-HA vaccination and enhanced in kaCA-vaccinated pigs. Pigs were vaccinated intranasally with Ad5-empty or Ad5-HA 42 days prior to challenge or intramuscularly with kaCA 42 and 21 days prior to challenge. Pigs were challenged intranasally with A/CA/04/09 (CA09), A/SW/MN/2011/08 (MN08), or PBS (NC). The percentage of macroscopic lung lesions in the Ad5-HA- or Ad5-empty-vaccinated pigs (A) and kaCA-vaccinated pigs (B) were evaluated 5 days after infection with the indicated virus. Results are reported as means ± SEM, and statistical differences between nonvaccinated and vaccinated groups challenged with the same virus are indicated with connecting bars and asterisks (P ≤ 0.05).

Fig 3

Microscopic pneumonia scores and IAV antigen scores at 5 days postinfection. Tissues were collected from pigs vaccinated intranasally with Ad5-empty (white bars) or Ad5-HA (black bars) 42 days prior to challenge or intramuscularly with kaCA (gray bars) 42 and 21 days prior to challenge. Pigs were challenged intranasally with A/CA/04/09 (CA09), A/SW/MN/2011/08 (MN08), or PBS (NC). Trachea (A) and lung (B) histopathology scores are shown for hematoxylin-and-eosin-stained formalin-fixed tissues collected 5 days following challenge with CA09 or MN08. (C) Lung IAV antigen scores identified using an anti-NP (HB65) antibody on formalin-fixed tissue 5 days after infection with CA09 or MN08 as described in Materials and Methods. Results are reported as means ± SEM, and statistical differences between nonvaccinated and vaccinated groups challenged with the same virus are indicated with connecting bars and asterisks (P ≤ 0.05).

Fig 4

Ad5-HA vaccination elicits IAV-specific IgG and IgA in the lung. Pigs were vaccinated with Ad5-empty or Ad5-HA (CA09) intranasally 42 days prior to infection with A/CA/04/09 (CA09) or A/SW/MN/2011/08 (MN08). ELISA plates were coated with CA09 or MN08 as the antigen, and levels of IgA (A and C) and IgG (B and D) antibodies in BALF samples (diluted as described in Materials and Methods) collected 5 days after infection with the indicated challenge virus are shown. Results are reported as the mean OD ± SEM for each group. Statistical differences between nonvaccinated and vaccinated groups challenged with the same virus are indicated with connecting bars and asterisks (P ≤ 0.05).