Development and Evaluation of PLGA-Nanoparticle Entrapped Influenza Virus Peptides Vaccine and Effect on Molecular Phenotype of Alveolar Macrophages with reference to DAP12 Signaling Pathway in Pigs

Present vaccines for swine influenza are strain specific and less effective against changing viral strains. A novel approach to develop a vaccine that would protect against varying strains is essential to control the disease. Hence in the present study we developed and evaluated a PLGA NP based influenza conserved viral peptide vaccine and evaluated its efficacy in pig. In this study, norovirus P particle M2e [extracellular domain of the matrix protein 2 (M2e) of influenza virus] chimera and conserved four influenza peptides were entrapped in PLGA-NPs. Influenza antibody free 4-5 weeks old caesarean section delivered pigs were intranasally vaccinated twice at two weeks interval with PLGA-NP entrapped or un-entrapped peptide antigen with or without Mycobacterium vaccae whole cell lysates adjuvant. Two weeks post-booster, pigs were challenged with a heterologous SwIV via intratracheal and intranasal routes; and 7 days later pigs were euthanized and samples were analyzed to determine the protective efficacy of the vaccine. Post challenge, pigs vaccinated with PLGA-NP entrapped peptides had no fever despite having similar gross lung lesions as that of pigs vaccinated with un-entrapped peptides. Further, the replicating infective virus was undetectable in these pigs. Immunologically, PLGA-NP vaccinated pigs had significantly increased frequencies of IFN gamma secreting CD3+CD4 +CD8-, CD3+CD4-CD8+ and CD3+ CD4+CD8+ cells in response to stimulation of the lung mononuclear cells by peptides and virus. Our data indicate that the PLGA-NP entrapped influenza peptides vaccine can induce the viral peptide specific cell-mediated immune response in intranasally vaccinated pigs. Though the PLGA based conserved peptide vaccine showed protection by significantly reducing the infective virus load in bronchoalveolar lavage fluid but vaccine did not protect against development of lung lesions. Antigen presenting cells through DAP12 signaling regulates immunopathogenic T cell response to conserved peptide antigen. Thus, second objective was to understand the effects of SwIV infection or vaccination/challenge on expression of DAP12 and its associated receptors, MDL-1, TREM-1 and TREM-2, in pig alveolar monocyte/macrophages (AMM) and their association with phenotype of AMM. In vitro stimulation of healthy specific pathogen free pig AMM cells with the IFN gamma or IL-4 led to activation of AMM into classically activated (M1) or alternatively activated (M2) state, respectively. Activational states were defined based on the expression profile of M1 (TNF alpha, iNOS) and M2 (ARG1, MMR) markers. The M1 markers TNFa & iNOS expression was significantly increased on IFN gamma stimulation, but surprisingly increased expression of MMR marker was also observed with IFN gamma stimulation. The M2 marker ARG1 expression was increased on IL-4 stimulation and TREM2 expression is associated with M2 phenotype. The AMM from mock infected pigs on post infection day 7 showed increased expression of TREM2 without defined phenotype whereas AMM from PLGA-NP peptide vaccinated/challenged pigs were showing increased expression of M1 (TNF alpha, IL1beta) and M2 (ARG1) markers along with receptors MDL1 and TREM1. In conclusion the results indicate that SwIV infection causes differential expression of DAP12 associated receptors and is altered by the vaccination. The overall conclusion of this study was the PLGA-NP entrapped SwIV peptides vaccine can induce the viral peptide specific cell-mediated immune response in intranasally vaccinated pigs which also alters the phenotype of AM and expression of DAP12 associated receptors.