Oral Fluids as a Live-Animal Sample Source for Evaluating Cross-Reactivity and Cross-Protection following Intranasal Influenza A Virus Vaccination in Pigs.

In North American swine, there are numerous antigenically distinct H1 influenza A virus (IAV) variants currently circulating, making vaccine development difficult due to the inability to formulate a vaccine that provides broad cross-protection. Experimentally, live-attenuated influenza virus (LAIV) vaccines demonstrate increased cross-protection compared to inactivated vaccines. However, there is no standardized assay to predict cross-protection following LAIV vaccination. Hemagglutination-inhibiting (HI) antibody in serum is the gold standard correlate of protection following IAV vaccination. LAIV vaccination does not induce a robust serum HI antibody titer; however, a local mucosal antibody response is elicited. Thus, a live-animal sample source that could be used to evaluate LAIV immunogenicity and cross-protection is needed. Here, we evaluated the use of oral fluids (OF) and nasal wash (NW) collected after IAV inoculation as a live-animal sample source in an enzyme-linked immunosorbent assay (ELISA) to predict cross-protection in comparison to traditional serology. Both live-virus exposure and LAIV vaccination provided heterologous protection, though protection was greatest against more closely phylogenetically related viruses. IAV-specific IgA was detected in NW and OF samples and was cross-reactive to representative IAV from each H1 cluster. Endpoint titers of cross-reactive IgA in OF from pigs exposed to live virus was associated with heterologous protection. While LAIV vaccination provided significant protection, LAIV immunogenicity was reduced compared to live-virus exposure. These data suggest that OF from pigs inoculated with wild-type IAV, with surface genes that match the LAIV seed strain, could be used in an ELISA to assess cross-protection and the antigenic relatedness of circulating and emerging IAV in swine.

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.

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.

Absence of Tlr2 protects against high-fat diet-induced inflammation and results in greater insulin-stimulated glucose transport in cultured adipocytes.

We have previously shown that toll-like receptor-4 (Tlr4) is involved in obesity-induced inflammation in adipose tissue (AT). However, less is known about the role of Tlr2 in this process. To determine the involvement of this receptor in obesity-induced inflammation, we utilized male Tlr2(-/-) mice that were backcrossed onto a mouse model of diet-induced obesity (DIO). Mice were fed either low-fat control (LFD) or high-fat diet (HFD) ad libitum for 16 weeks. Despite negligible differences in body weight or energy intake, Tlr2(-/-) mice were protected from HFD-induced adiposity as was evident by reduced epididymal fat pad weight and carcass lipid content. Corresponding with these effects was a blunted accumulation of F4/80-positive macrophages in AT of Tlr2(-/-) mice. Furthermore, transcript abundance of proinflammatory mediators, including monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-α (TNFα) and nitric oxide synthase-2 (NOS2) in AT of Tlr2(-/-) mice, was lower or less responsive to DIO. There were no significant differences in serum markers of insulin sensitivity (data not shown). However, adipocytes derived from stromal vascular cells (SVCs) isolated from AT of Tlr2(-/-) mice had considerably greater basal and insulin-stimulated glucose uptake as compared with those obtained from Tlr2(+/+) mice. Furthermore, the absence of Tlr2(-/-) precluded the induction of insulin resistance by zymosan A (ZymA) but not by palmitate. These data indicate that Tlr2 may be directly involved in HFD-induced inflammation and may also regulate basal and insulin-stimulated glucose uptake in adipocytes.