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. 2017 Jun 9;91(13):e00575-17.
doi: 10.1128/JVI.00575-17. Print 2017 Jul 1.

Distinct Roles of Vaccinia Virus NF-κB Inhibitor Proteins A52, B15, and K7 in the Immune Response

Mauro Di Pilato  1 , Ernesto Mejías-Pérez  2 , Carlos Oscar S Sorzano  3 , Mariano Esteban  1
Affiliations

Distinct Roles of Vaccinia Virus NF-κB Inhibitor Proteins A52, B15, and K7 in the Immune Response

Mauro Di Pilato et al. J Virol. .

Abstract

Poxviruses use a complex strategy to escape immune control, by expressing immunomodulatory proteins that could limit their use as vaccine vectors. To test the role of poxvirus NF-κB pathway inhibitors A52, B15, and K7 in immunity, we deleted their genes in an NYVAC (New York vaccinia virus) strain that expresses HIV-1 clade C antigens. After infection of mice, ablation of the A52R, B15R, and K7R genes increased dendritic cell, natural killer cell, and neutrophil migration as well as chemokine/cytokine expression. Revertant viruses with these genes confirmed their role in inhibiting the innate immune system. To different extents, enhanced innate immune responses correlated with increased HIV Pol- and Gag-specific polyfunctional CD8 T cell and HIV Env-specific IgG responses induced by single-, double-, and triple-deletion mutants. These poxvirus proteins thus influence innate and adaptive cell-mediated and humoral immunity, and their ablation offers alternatives for design of vaccine vectors that regulate immune responses distinctly.IMPORTANCE Poxvirus vectors are used in clinical trials as candidate vaccines for several pathogens, yet how these vectors influence the immune system is unknown. We developed distinct poxvirus vectors that express heterologous antigens but lack different inhibitors of the central host-cell signaling pathway. Using mice, we studied the capacity of these viruses to induce innate and adaptive immune responses and showed that these vectors can distinctly regulate the magnitude and quality of these responses. These findings provide important insights into the mechanism of poxvirus-induced immune response and alternative strategies for vaccine vector design.

Keywords: NF-κB; NYVAC; T cell immunity; adaptive immunity; human immunodeficiency virus; immunomodulation; innate immunity; poxvirus; vaccines; vaccinia virus.

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Figures

FIG 1
FIG 1
Lack of transcription of the A52R, B15R, and K7R genes in NYVAC deletion mutant-infected cells. BSC-40 cells were infected with NYVAC-C and NYVAC deletion mutant viruses (1 PFU per cell, 24 h). RT-PCR analysis of infected cells confirms the deletion of the A52R (A), B15R (B), and K7R (C) genes by loss of RNA expression.
FIG 2
FIG 2
Deletion of the A52R, B15R, and K7R genes influences peritoneal cell migration. Shown are the percentages and absolute numbers of CD11c+ MHC-II+ dendritic cells (DCs) (A and B), Ly6G+ CD11b+ neutrophils (N) (C and D), NKp46+ CD3− natural killer cells (NK) (E and F), CD19+ B cells (B) (G), F4/80low CD11blow monocytes (M) (H), CD4+ CD3+ T cells (CD4) (I), and CD8+ CD3+ T cells (CD8) (J) in the peritoneal cavity of BALB/c mice at 6 h postinjection of 107 PFU of NYVAC-C or the NYVAC-C deletion mutants. Graphs show the mean ± standard error of the mean (SEM); each point represents an individual mouse. Data are representative of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG 3
FIG 3
Deletion of the A52R, B15R, and K7R genes affects cytokine/chemokine levels. Shown are concentrations of cytokine IL-6 (A) and chemokines CXCL1 (B), CXCL2 (C), CCL2 (D), CCL3 (E), CXCL10 (F), and CXCL9 (G) at 6 h postinfection in the peritoneal cavity of NYVAC-C- or NYVAC-C deletion mutant-injected mice. Graphs show the mean ± SEM; each point represents an individual mouse. Data are representative of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG 4
FIG 4
Characterization of the NYVAC-C-Δ3 revertant viruses. RT-PCR analysis of RNA from BSC-40 cells infected with the NYVAC-C-Δ3 B15R-rev and NYVAC-C-Δ3 A52R-rev revertants (Rev) (1 PFU per cell, 24 h) confirms the correct B15R (A) and A52R (B) gene reinsertion and transcription.
FIG 5
FIG 5
Revertant viruses reduce cell migration and cytokine/chemokine levels. Shown are the percentages and absolute numbers of Ly6G+ CD11b+ neutrophils (N) (A and B), CD11c+ MHCII+ DCs (C and D), NKp46+ CD3− NK cells (E and F), CD19+ B cells (B) (G), F4/80low CD11blow monocytes (M) (H), CD4+ CD3+ T cells (CD4) (I) and CD8+ CD3+ T cells (CD8) (J) in the peritoneal cavity of BALB/c mice at 6 h postinjection of 107 PFU of NYVAC-C triple-deletion mutants and revertant viruses. (K to N) Concentrations of IL-6 (K), CCL2 (L), CXCL2 (M), and CXCL1 (N) in the peritoneal cavity of infected mice. Data are representative of two independent experiments. Graphs show the mean ± SEM; each point represents an individual mouse. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG 6
FIG 6
Deletion of the A52R, B15R, and K7R genes influences adaptive HIV-specific CD8 T cell immune responses. Shown are the vaccine-induced HIV-specific CD8 T cell response in mice (n = 4/group) immunized following a heterologous DNA prime/virus boost regimen (107 PFU of NYVAC-C or NYVAC-C deletion mutants). (A) Percentages of TNF-α+ CD107a+, IFN-γ+ TNF-α+, IFN-γ+ CD107a+ double-positive and IL-2 single-positive T cells. The total value (magnitude) is the sum of percentages of CD8 T cells that express IFN-γ and/or TNF-α and/or IL-2 and/or CD107a, measured by intracellular cytokine staining (ICS). Nonspecific responses of mice infected with control NYVAC-WT were subtracted from the total magnitude. (B to D) Magnitude of Pol-1 (B)-, Gag-Pool (C)-, or Pol-2 (D)-specific CD8 T cell responses. Graphs show the mean ± confidence interval (CI). (E) Functional profile of adaptive Gag-Pol-specific CD8 T cells. Combinations of responses (x axis) and percentages of functionally distinct cell subsets (y axis) are shown in the bar graph. Responses are grouped and color coded based on the number of functions. Pie chart colors indicate the percentage of cytokine-producing cells based on the number of functions (inside) and the different activation markers (outside). Data are representative of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG 7
FIG 7
Deletion of the A52R, B15R, and K7R genes affects humoral Env-specific IgG immune responses. Shown are vaccine-induced HIV-specific IgG responses in mice (n = 4/group) immunized following a heterologous DNA prime/virus boost regimen (107 PFU of NYVAC-C or NYVAC-C deletion mutants). (A to C) Levels of Env-specific total IgG (A), IgG1 (B), and IgG3 (C) antibodies in serum of mice (n = 4/group) 11 days postinfection with NYVAC-C or NYVAC-C deletion mutants. Data are represented as optical density (OD) in arbitrary units (AU). Nonspecific responses of mice infected with control NYVAC-WT were subtracted from total values. Data are representative of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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References

    1. Garcia-Arriaza J, Esteban M. 2014. Enhancing poxvirus vectors vaccine immunogenicity. Hum Vaccin Immunother 10:2235–2244. doi:10.4161/hv.28974. - DOI - PMC - PubMed
    1. Rerks-Ngarm S, Pitisuttithum P, Nitayaphan S, Kaewkungwal J, Chiu J, Paris R, Premsri N, Namwat C, de Souza M, Adams E, Benenson M, Gurunathan S, Tartaglia J, McNeil JG, Francis DP, Stablein D, Birx DL, Chunsuttiwat S, Khamboonruang C, Thongcharoen P, Robb ML, Michael NL, Kunasol P, Kim JH. 2009. Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. N Engl J Med 361:2209–2220. doi:10.1056/NEJMoa0908492. - DOI - PubMed
    1. Harari A, Rozot V, Cavassini M, Bellutti Enders F, Vigano S, Tapia G, Castro E, Burnet S, Lange J, Moog C, Garin D, Costagliola D, Autran B, Pantaleo G, Bart PA. 2012. NYVAC immunization induces polyfunctional HIV-specific T-cell responses in chronically-infected, ART-treated HIV patients. Eur J Immunol 42:3038–3048. doi:10.1002/eji.201242696. - DOI - PubMed
    1. Bart PA, Huang Y, Karuna ST, Chappuis S, Gaillard J, Kochar N, Shen X, Allen MA, Ding S, Hural J, Liao HX, Haynes BF, Graham BS, Gilbert PB, McElrath MJ, Montefiori DC, Tomaras GD, Pantaleo G, Frahm N. 2014. HIV-specific humoral responses benefit from stronger prime in phase Ib clinical trial. J Clin Invest 124:4843–4856. doi:10.1172/JCI75894. - DOI - PMC - PubMed
    1. Mooij P, Koopman G, Drijfhout JW, Nieuwenhuis IG, Beenhakker N, Koestler J, Bogers WM, Wagner R, Esteban M, Pantaleo G, Heeney JL, Jacobs BL, Melief CJ. 2015. Synthetic long peptide booster immunization in rhesus macaques primed with replication competent NYVAC-C-KC induces a balanced CD4/CD8 T-cell and antibody response against the conserved regions of HIV-1. J Gen Virol 96:1478–1483. doi:10.1099/vir.0.000074. - DOI - PubMed

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