Swine influenza virus (SwIV) is one of the important zoonotic pathogens. Current flu vaccines have failed to provide cross-protection against evolving viruses in the field. Poly(lactic-co-glycolic acid) (PLGA) is a biodegradable FDA approved polymer and widely used in drug and vaccine delivery. In this study, inactivated SwIV H1N2 antigens (KAg) encapsulated in PLGA nanoparticles (PLGA-KAg) were prepared, which were spherical in shape with 200 to 300nm diameter, and induced maturation of antigen presenting cells in vitro. Pigs vaccinated twice with PLGA-KAg via intranasal route showed increased antigen specific lymphocyte proliferation and enhanced the frequency of T-helper/memory and cytotoxic T cells (CTLs) in peripheral blood mononuclear cells (PBMCs). In PLGA-KAg vaccinated and heterologous SwIV H1N1 challenged pigs, clinical flu symptoms were absent, while the control pigs had fever for four days. Grossly and microscopically, reduced lung pathology and viral antigenic mass in the lung sections with clearance of infectious challenge virus in most of the PLGA-KAg vaccinated pig lung airways were observed. Immunologically, PLGA-KAg vaccine irrespective of not significantly boosting the mucosal antibody response, it augmented the frequency of IFN-γ secreting total T cells, T-helper and CTLs against both H1N2 and H1N1 SwIV. In summary, inactivated influenza virus delivered through PLGA-NPs reduced the clinical disease and induced cross-protective cell-mediated immune response in a pig model. Our data confirmed the utility of a pig model for intranasal particulate flu vaccine delivery platform to control flu in humans.

Pigs are believed to be one of the important sources of emerging human and swine influenza viruses (SwIV). Influenza virus conserved peptides have the potential to elicit cross-protective immune response, but without the help of potent adjuvant and delivery system they are poorly immunogenic. Biodegradable polylactic-co-glycolic acid (PLGA) nanoparticle (PLGA-NP) based vaccine delivery system enhances cross-presentation of antigens by the professional antigen presenting cells. In this study, Norovirus P particle containing SwIV M2e (extracellular domain of the matrix protein 2) chimera and highly conserved two each of H1N1 peptides of pandemic 2009 and classical human influenza viruses were entrapped in PLGA-NPs. Influenza antibody-free pigs were vaccinated with PLGA-NPs peptides cocktail vaccine twice with or without an adjuvant, Mycobacterium vaccae whole cell lysate, intranasally as mist. Vaccinated pigs were challenged with a virulent heterologous zoonotic SwIV H1N1, and one week later euthanized and the lung samples were analyzed for the specific immune response and viral load. Clinically, pigs vaccinated with PLGA-NP peptides vaccine had no fever and flu symptoms, and the replicating challenged SwIV was undetectable in the bronchoalveolar lavage fluid. Immunologically, PLGA-NP peptides vaccination (without adjuvant) significantly increased the frequency of antigen-specific IFNγ secreting CD4 and CD8 T cells response in the lung lymphocytes, despite not boosting the antibody response both at pre- and post-challenge. In summary, our data indicated that nanoparticle-mediated delivery of conserved H1N1 influenza peptides induced the virus specific T cell response in the lungs and reduced the challenged heterologous virus load in the airways of pigs.

We have recently demonstrated the effectiveness of an influenza A virus (IAV) subunit vaccine based on biodegradable polyanhydride nanoparticles delivery in mice. In the present study, we evaluated the efficacy of ∼200nm polyanhydride nanoparticles encapsulating inactivated swine influenza A virus (SwIAV) as a vaccine to induce protective immunity against a heterologous IAV challenge in pigs. Nursery pigs were vaccinated intranasally twice with inactivated SwIAV H1N2 (KAg) or polyanhydride nanoparticle-encapsulated KAg (KAg nanovaccine), and efficacy was evaluated against a heterologous zoonotic virulent SwIAV H1N1 challenge. Pigs were monitored for fever daily. Local and systemic antibody responses, antigen-specific proliferation of peripheral blood mononuclear cells, gross and microscopic lung lesions, and virus load in the respiratory tract were compared among the groups of animals. Our pre-challenge results indicated that KAg nanovaccine induced virus-specific lymphocyte proliferation and increased the frequency of CD4CD8αα T helper and CD8 cytotoxic T cells in peripheral blood mononuclear cells. KAg nanovaccine-immunized pigs were protected from fever following SwIAV challenge. In addition, pigs immunized with the KAg nanovaccine presented with lower viral antigens in lung sections and had 6 to 8-fold reduction in nasal shedding of SwIAV four days post-challenge compared to control animals. Immunologically, increased IFN-γ secreting T lymphocyte populations against both the vaccine and challenge viruses were detected in KAg nanovaccine-immunized pigs compared to the animals immunized with KAg alone. However, in the KAg nanovaccine-immunized pigs, hemagglutination inhibition, IgG and IgA antibody responses, and virus neutralization titers were comparable to that in the animals immunized with KAg alone. Overall, our data indicated that intranasal delivery of polyanhydride-based SwIAV nanovaccine augmented antigen-specific cellular immune response in pigs, with promise to induce cross-protective immunity.

Porcine epidemic diarrhea virus (PEDV) is an economically devastating enteric disease in the swine industry. The virus infects pigs of all ages, but it cause severe clinical disease in neonatal suckling pigs with up to 100% mortality. Currently, available vaccines are not completely effective and feedback methods utilizing PEDV infected material has variable success in preventing reinfection. Comprehensive information on the levels and duration of effector/memory IgA and IgG antibody secreting B cell response in the intestines and lymphoid organs of PEDV-infected sows, and their association with specific antibody levels in clinical samples such as plasma, oral fluid, and feces is important. Therefore, our goal in this study was to quantify PEDV specific IgA and IgG B cell responses in sows at approximately 1 and 6 months post-infection in commercial swine herds, including parity one and higher sows. Our data indicated that evaluation of both PEDV specific IgA and IgG antibody levels in the plasma and oral fluid (but not feces) samples is beneficial in disease diagnosis. PEDV specific B cell response in the intestine and spleen of infected sows decline by 6 months, and this associates with specific antibody levels in the plasma and oral fluid samples; but the virus neutralization titers in plasma remains high beyond 6 months post-infection. In conclusion, in sows infected with PEDV the presence of effector/memory B cell response and strong virus neutralization titers in plasma up to 6 months post-infection, suggests their potential to protect sows from reinfection and provide maternal immunity to neonates, but challenge studies are required to confirm such responses.

Porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural protein 1␤ (nsp1␤) is a multifunctional viral protein, which is involved in suppressing the host innate immune response and activating a unique ؊2/؊1 programmed ribosomal frameshifting (PRF) signal for the expression of frameshifting products. In this study, site-directed mutagenesis analysis showed that the R128A or R129A mutation introduced into a highly conserved motif ( 123 GKYLQRRLQ 131 ) reduced the ability of nsp1␤ to suppress interferon beta (IFN-␤) activation and also impaired nsp1␤'s function as a PRF transactivator. Three recombinant viruses, vR128A, vR129A, and vRR129AA, carrying single or double mutations in the GKYLQRRLQ motif were characterized. In comparison to the wild-type (WT) virus, vR128A and vR129A showed slightly reduced growth abilities, while the vRR129AA mutant had a significantly reduced growth ability in infected cells. Consistent with the attenuated growth phenotype in vitro, pigs infected with nsp1␤ mutants had lower levels of viremia than did WT virus-infected pigs. Compared to the WT virus in infected cells, all three mutated viruses stimulated high levels of IFN-␣ expression and exhibited a reduced ability to suppress the mRNA expression of selected interferon-stimulated genes (ISGs). In pigs infected with nsp1␤ mutants, IFN-␣ production was increased in the lungs at early time points postinfection, which was correlated with increased innate NK cell function. Furthermore, the augmented innate response was consistent with the increased production of IFN-␥ in pigs infected with mutated viruses. These data demonstrate that residues R128 and R129 are critical for nsp1␤ function and that modifying these key residues in the GKYL QRRLQ motif attenuates virus growth ability and improves the innate and adaptive immune responses in infected animals. IMPORTANCEPRRSV infection induces poor antiviral innate IFN and cytokine responses, which results in weak adaptive immunity. One of the strategies in next-generation vaccine construction is to manipulate viral proteins/genetic elements involved in antagonizing the host immune response. PRRSV nsp1␤ was identified to be a strong innate immune antagonist. In this study, two basic amino acids, R128 and R129, in a highly conserved GKYLQRRLQ motif were determined to be critical for nsp1␤ function. Mutations introduced into these two residues attenuated virus growth and improved the innate and adaptive immune responses of infected animals. Technologies developed in this study could be broadly applied to current commercial PRRSV modified live-virus (MLV) vaccines and other candidate vaccines. P orcine reproductive and respiratory syndrome (PRRS), a disease described in the United States in 1987 (1) and in Europe in 1990 (2), has caused tremendous economic losses to the swine industry since its appearance. Hallmark symptoms of PRRS are mild to severe respiratory disease in infected newborn and growing pigs and reproductive failure in pregnant sows. The etiologic agent, PRR...