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Entrapment of H1N1 Influenza Virus Derived Conserved Peptides in PLGA Nanoparticles Enhances T Cell Response and Vaccine Efficacy in Pigs.

Hiremath J, Kang KI, Xia M, Elaish M, Binjawadagi B, Ouyang K, Dhakal S, Arcos J, Torrelles JB, Jiang X, Lee CW, Renukaradhya GJ - PLoS ONE (2016)

Bottom Line: Biodegradable polylactic-co-glycolic acid (PLGA) nanoparticle (PLGA-NP) based vaccine delivery system enhances cross-presentation of antigens by the professional antigen presenting cells.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.

View Article: PubMed Central - PubMed

Affiliation: Food Animal Health Research Program, Ohio Agricultural Research and Development Center, 1680 Madison Avenue, Wooster, Ohio, 44691, United States of America, and Department of Veterinary Preventive Medicine, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio, 43210, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Immunophenotyping of activated lymphocyte subsets in LMNCs of vaccinated and SwIV challenged pigs.(A) A representative flow cytometry plots depicting porcine IFNγ positive T lymphocytes subpopulations. Gating pattern of lymphocytes of a pig infected with SwIV H1N1 and restimulated ex vivo with the virus for 3 days and immunostained using fluorochrome conjugated pig specific CD3έ, CD4α and CD8α markers and intracellular IFNγ to identify the frequency of CD3+CD4+CD8α+IFNγ+, CD3+CD4-CD8α+IFNγ+ and CD3+CD4+CD8α-IFNγ+ cells is shown. Lung MNCs (LMNCs) isolated on the day of necropsy from all the experimental pigs were either unstimulated (Control, C) or stimulated with the SwIV (V), conserved peptides (A1, A2, A7 and A8 peptides) or M2e (M) for 3 days and immunostained cells were gated for T cell subsets as described above for: (B) CD3+CD4+CD8α+IFNγ+; (C) CD3+CD4-CD8α+IFNγ+; and (D) CD3+CD4+CD8α-IFNγ+ cells. (E) Delineating the SwIV specific T cell response in NPP vaccinated pigs. Three activated (IFNγ+) T lymphocyte subsets in the LMNCs of only NPP vaccinated pigs either unstimulated (Control, C) or stimulated with the SwIV or indicated peptides are shown. A white dotted line drawn across each T cell subset separates the Ags specific recalled T cell response over the control unstimulated cells of NPP vaccinated pigs. (F) Secreted IFNγ in the culture supernatant of virus restimulated LMNCs of vaccinated and SwIV challenged pigs was analyzed by ELISA. Background cytokine levels from each pig groups were subtracted from the specific stimulation. Each bar indicates the average frequency of indicated T cell subset or cytokine from 6 or 7 pigs ± SEM. Asterisk denotes statistically significant difference at P<0.05 (*), P<0.01 (**) and P<0.001 (***) between the indicated two pig groups determined by One way ANOVA followed by Tukey Post-hoc test for the significance between the indicated pig groups.
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pone.0151922.g002: Immunophenotyping of activated lymphocyte subsets in LMNCs of vaccinated and SwIV challenged pigs.(A) A representative flow cytometry plots depicting porcine IFNγ positive T lymphocytes subpopulations. Gating pattern of lymphocytes of a pig infected with SwIV H1N1 and restimulated ex vivo with the virus for 3 days and immunostained using fluorochrome conjugated pig specific CD3έ, CD4α and CD8α markers and intracellular IFNγ to identify the frequency of CD3+CD4+CD8α+IFNγ+, CD3+CD4-CD8α+IFNγ+ and CD3+CD4+CD8α-IFNγ+ cells is shown. Lung MNCs (LMNCs) isolated on the day of necropsy from all the experimental pigs were either unstimulated (Control, C) or stimulated with the SwIV (V), conserved peptides (A1, A2, A7 and A8 peptides) or M2e (M) for 3 days and immunostained cells were gated for T cell subsets as described above for: (B) CD3+CD4+CD8α+IFNγ+; (C) CD3+CD4-CD8α+IFNγ+; and (D) CD3+CD4+CD8α-IFNγ+ cells. (E) Delineating the SwIV specific T cell response in NPP vaccinated pigs. Three activated (IFNγ+) T lymphocyte subsets in the LMNCs of only NPP vaccinated pigs either unstimulated (Control, C) or stimulated with the SwIV or indicated peptides are shown. A white dotted line drawn across each T cell subset separates the Ags specific recalled T cell response over the control unstimulated cells of NPP vaccinated pigs. (F) Secreted IFNγ in the culture supernatant of virus restimulated LMNCs of vaccinated and SwIV challenged pigs was analyzed by ELISA. Background cytokine levels from each pig groups were subtracted from the specific stimulation. Each bar indicates the average frequency of indicated T cell subset or cytokine from 6 or 7 pigs ± SEM. Asterisk denotes statistically significant difference at P<0.05 (*), P<0.01 (**) and P<0.001 (***) between the indicated two pig groups determined by One way ANOVA followed by Tukey Post-hoc test for the significance between the indicated pig groups.

Mentions: Since pigs were vaccinated and challenged via nasal route, we quantified influenza Ag specific T cell response in the lungs of pigs using lung mononuclear cells (LMNCs). On the day of necropsy, isolated LMNCs were either unstimulated or stimulated with the SwIV or individual peptides and analyzed for the frequency of activated (IFNγ+) T lymphocyte subsets. A representative graph depicting the gating pattern of restimulated porcine lymphocyte subsets secreting IFNγ belongs to SwIV infected pigs is shown (Fig 2A). Porcine immune system has a unique abundant population of CD4 and CD8 double positive T cell subset, which have combined T-helper, memory and cytotoxic T cell properties [35,36]. Our results detected both the virus and peptides (A1, A2, A7 and A8) specific increased frequency of CD4+CD8α+IFNγ+ T cells in LMNCs of NPP vaccinated pigs compared to all the other vaccine formulations received animals (Fig 2B). This data indicated influenza Ags specific activation of CD4 and CD8 double positive T cell subset in the lungs of NPP vaccinated pigs.


Entrapment of H1N1 Influenza Virus Derived Conserved Peptides in PLGA Nanoparticles Enhances T Cell Response and Vaccine Efficacy in Pigs.

Hiremath J, Kang KI, Xia M, Elaish M, Binjawadagi B, Ouyang K, Dhakal S, Arcos J, Torrelles JB, Jiang X, Lee CW, Renukaradhya GJ - PLoS ONE (2016)

Immunophenotyping of activated lymphocyte subsets in LMNCs of vaccinated and SwIV challenged pigs.(A) A representative flow cytometry plots depicting porcine IFNγ positive T lymphocytes subpopulations. Gating pattern of lymphocytes of a pig infected with SwIV H1N1 and restimulated ex vivo with the virus for 3 days and immunostained using fluorochrome conjugated pig specific CD3έ, CD4α and CD8α markers and intracellular IFNγ to identify the frequency of CD3+CD4+CD8α+IFNγ+, CD3+CD4-CD8α+IFNγ+ and CD3+CD4+CD8α-IFNγ+ cells is shown. Lung MNCs (LMNCs) isolated on the day of necropsy from all the experimental pigs were either unstimulated (Control, C) or stimulated with the SwIV (V), conserved peptides (A1, A2, A7 and A8 peptides) or M2e (M) for 3 days and immunostained cells were gated for T cell subsets as described above for: (B) CD3+CD4+CD8α+IFNγ+; (C) CD3+CD4-CD8α+IFNγ+; and (D) CD3+CD4+CD8α-IFNγ+ cells. (E) Delineating the SwIV specific T cell response in NPP vaccinated pigs. Three activated (IFNγ+) T lymphocyte subsets in the LMNCs of only NPP vaccinated pigs either unstimulated (Control, C) or stimulated with the SwIV or indicated peptides are shown. A white dotted line drawn across each T cell subset separates the Ags specific recalled T cell response over the control unstimulated cells of NPP vaccinated pigs. (F) Secreted IFNγ in the culture supernatant of virus restimulated LMNCs of vaccinated and SwIV challenged pigs was analyzed by ELISA. Background cytokine levels from each pig groups were subtracted from the specific stimulation. Each bar indicates the average frequency of indicated T cell subset or cytokine from 6 or 7 pigs ± SEM. Asterisk denotes statistically significant difference at P<0.05 (*), P<0.01 (**) and P<0.001 (***) between the indicated two pig groups determined by One way ANOVA followed by Tukey Post-hoc test for the significance between the indicated pig groups.
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Related In: Results  -  Collection

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pone.0151922.g002: Immunophenotyping of activated lymphocyte subsets in LMNCs of vaccinated and SwIV challenged pigs.(A) A representative flow cytometry plots depicting porcine IFNγ positive T lymphocytes subpopulations. Gating pattern of lymphocytes of a pig infected with SwIV H1N1 and restimulated ex vivo with the virus for 3 days and immunostained using fluorochrome conjugated pig specific CD3έ, CD4α and CD8α markers and intracellular IFNγ to identify the frequency of CD3+CD4+CD8α+IFNγ+, CD3+CD4-CD8α+IFNγ+ and CD3+CD4+CD8α-IFNγ+ cells is shown. Lung MNCs (LMNCs) isolated on the day of necropsy from all the experimental pigs were either unstimulated (Control, C) or stimulated with the SwIV (V), conserved peptides (A1, A2, A7 and A8 peptides) or M2e (M) for 3 days and immunostained cells were gated for T cell subsets as described above for: (B) CD3+CD4+CD8α+IFNγ+; (C) CD3+CD4-CD8α+IFNγ+; and (D) CD3+CD4+CD8α-IFNγ+ cells. (E) Delineating the SwIV specific T cell response in NPP vaccinated pigs. Three activated (IFNγ+) T lymphocyte subsets in the LMNCs of only NPP vaccinated pigs either unstimulated (Control, C) or stimulated with the SwIV or indicated peptides are shown. A white dotted line drawn across each T cell subset separates the Ags specific recalled T cell response over the control unstimulated cells of NPP vaccinated pigs. (F) Secreted IFNγ in the culture supernatant of virus restimulated LMNCs of vaccinated and SwIV challenged pigs was analyzed by ELISA. Background cytokine levels from each pig groups were subtracted from the specific stimulation. Each bar indicates the average frequency of indicated T cell subset or cytokine from 6 or 7 pigs ± SEM. Asterisk denotes statistically significant difference at P<0.05 (*), P<0.01 (**) and P<0.001 (***) between the indicated two pig groups determined by One way ANOVA followed by Tukey Post-hoc test for the significance between the indicated pig groups.
Mentions: Since pigs were vaccinated and challenged via nasal route, we quantified influenza Ag specific T cell response in the lungs of pigs using lung mononuclear cells (LMNCs). On the day of necropsy, isolated LMNCs were either unstimulated or stimulated with the SwIV or individual peptides and analyzed for the frequency of activated (IFNγ+) T lymphocyte subsets. A representative graph depicting the gating pattern of restimulated porcine lymphocyte subsets secreting IFNγ belongs to SwIV infected pigs is shown (Fig 2A). Porcine immune system has a unique abundant population of CD4 and CD8 double positive T cell subset, which have combined T-helper, memory and cytotoxic T cell properties [35,36]. Our results detected both the virus and peptides (A1, A2, A7 and A8) specific increased frequency of CD4+CD8α+IFNγ+ T cells in LMNCs of NPP vaccinated pigs compared to all the other vaccine formulations received animals (Fig 2B). This data indicated influenza Ags specific activation of CD4 and CD8 double positive T cell subset in the lungs of NPP vaccinated pigs.

Bottom Line: Biodegradable polylactic-co-glycolic acid (PLGA) nanoparticle (PLGA-NP) based vaccine delivery system enhances cross-presentation of antigens by the professional antigen presenting cells.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.

View Article: PubMed Central - PubMed

Affiliation: Food Animal Health Research Program, Ohio Agricultural Research and Development Center, 1680 Madison Avenue, Wooster, Ohio, 44691, United States of America, and Department of Veterinary Preventive Medicine, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio, 43210, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus