Systems analysis of MVA-C induced immune response reveals its significance as a vaccine candidate against HIV/AIDS of clade C

Based on the partial efficacy of the HIV/AIDS Thai trial (RV144) with a canarypox vector prime and protein boost, attenuated poxvirus recombinants expressing HIV-1 antigens are increasingly sought as vaccine candidates against HIV/AIDS. Here we describe using systems analysis the biological and immunological characteristics of the attenuated vaccinia virus Ankara strain expressing the HIV-1 antigens Env/Gag-Pol-Nef of HIV-1 of clade C (referred as MVA-C). MVA-C infection of human monocyte derived dendritic cells (moDCs) induced the expression of HIV-1 antigens at high levels from 2 to 8 hpi and triggered moDCs maturation as revealed by enhanced expression of HLA-DR, CD86, CD40, HLA-A2, and CD80 molecules. Infection ex vivo of purified mDC and pDC with MVA-C induced the expression of immunoregulatory pathways associated with antiviral responses, antigen presentation, T cell and B cell responses. Similarly, human whole blood or primary macrophages infected with MVA-C express high levels of proinflammatory cytokines and chemokines involved with T cell activation. The vector MVA-C has the ability to cross-present antigens to HIV-specific CD8 T cells in vitro and to increase CD8 T cell proliferation in a dose-dependent manner. The immunogenic profiling in mice after DNA-C prime/MVA-C boost combination revealed activation of HIV-1-specific CD4 and CD8 T cell memory responses that are polyfunctional and with effector memory phenotype. Env-specific IgG binding antibodies were also produced in animals receiving DNA-C prime/MVA-C boost. Our systems analysis of profiling immune response to MVA-C infection highlights the potential benefit of MVA-C as vaccine candidate against HIV/AIDS for clade C, the prevalent subtype virus in the most affected areas of the world

Systems Analysis of MVA-C Induced Immune Response Reveals Its Significance as a Vaccine Candidate against HIV/AIDS of Clade C

Based on the partial efficacy of the HIV/AIDS Thai trial (RV144) with a canarypox vector prime and protein boost, attenuated poxvirus recombinants expressing HIV-1 antigens are increasingly sought as vaccine candidates against HIV/AIDS. Here we describe using systems analysis the biological and immunological characteristics of the attenuated vaccinia virus Ankara strain expressing the HIV-1 antigens Env/Gag-Pol-Nef of HIV-1 of clade C (referred as MVA-C). MVA-C infection of human monocyte derived dendritic cells (moDCs) induced the expression of HIV-1 antigens at high levels from 2 to 8 hpi and triggered moDCs maturation as revealed by enhanced expression of HLA-DR, CD86, CD40, HLA-A2, and CD80 molecules. Infection ex vivo of purified mDC and pDC with MVA-C induced the expression of immunoregulatory pathways associated with antiviral responses, antigen presentation, T cell and B cell responses. Similarly, human whole blood or primary macrophages infected with MVA-C express high levels of proinflammatory cytokines and chemokines involved with T cell activation. The vector MVA-C has the ability to cross-present antigens to HIV-specific CD8 T cells in vitro and to increase CD8 T cell proliferation in a dose-dependent manner. The immunogenic profiling in mice after DNA-C prime/MVA-C boost combination revealed activation of HIV-1-specific CD4 and CD8 T cell memory responses that are polyfunctional and with effector memory phenotype. Env-specific IgG binding antibodies were also produced in animals receiving DNA-C prime/MVA-C boost. Our systems analysis of profiling immune response to MVA-C infection highlights the potential benefit of MVA-C as vaccine candidate against HIV/AIDS for clade C, the prevalent subtype virus in the most affected areas of the world

Signaling pathways induced upon MVA-C infection of mDCs and pDCs.

<p>Selected significant top 10 pathways and their genes members resulting from comparing myeloid DCs infected with MVA-C (MDC_MVC) and mock-infected (MDC_Mock) groups (A) or plasmacytoid DCs infected with MVA-C (PDC_MVC) and mock-infected (PDC_Mock) groups (B). Each row is an upregulated canonical pathway for innate immunity (Ingenuity software); each column represents an up- (red) or down- (blue) regulated gene (p<0.05 and |FC|>2) included in 1 or more regulated pathway(s). The over-representation test was performed using Fisher Exact Test and the significance, displayed on the right side, is achieved for p<0.05 (-log(p)>1.3).</p

Humoral immune responses elicited by MVA-C against HIV-1 gp140 protein.

<p>Levels of Env-specific IgG binding antibodies (A) or IgG subtypes (B) were measured in serum from naïve and immunized mice at 53 days after the boost. The values represent the mean antibodies titer for each group. ** p<0.005.</p

MVA-C infection induces antigen cross-presentation to CD8 T cells and T cell proliferation.

<p>A) Human moDCs were incubated with apoptotic infected-HeLa cells before a CD8 T cell clone was added. After overnight incubation, cells were harvested and among the lymphocyte population, CD8 cells were gated and analyzed for IFN-γ, TNF-α, IL-2 and MIP-1β production. Cytokine production by HIV-specific CD8 T cells was determined. Percentages of CD8 T cells producing any cytokine are indicated at the various virus multiplicities. B) MVA-C and parental MVA-WT were evaluated <i>in vitro</i> using cryopreserved PBMCs from an HIV-1-infected subject. Cell proliferation using the CFSE dilution assay was measured 6 days after stimulation. At the end of the stimulation period, cells were stained for CD3, CD4, CD8 and a viability marker and analyzed by flow cytometry. Mean values and standard deviation of at least three experiments are shown in panels A and B.</p

MVA-C induces cytokine and chemokine production in human primary macrophages.

<p>A) Human primary macrophages were infected at 5 pfu/cell with MVA-WT or MVA-C. At 3 h.p.i., cells were collected, RNA extracted and levels of different mRNAs were defined by RT-PCR. B) Human primary macrophages were infected (1 or 5 pfu/cell) with MVA-WT or MVA-C. Supernatants were collected at 24 h and used to quantify IL-1β, IL-8, IFNβ, MIP-1α, RANTES, IP-10 and IFNα by ELISA. Experiments were performed in triplicate samples from two human donors. Data are means and standard deviation from one experiment representative of at least two experiments.</p

Long-lived memory immune response to HIV antigens elicited by MVA-C.

<p>A) Magnitude of vaccine-specific CD4⁺ and CD8⁺ T cells. The HIV-1-specific CD4 and CD8 T cells were measured 53 days after the last immunization by ICS assay following stimulation with the different HIV-1 peptide pools. The values represent the sum of the percentages of T cells secreting IFN-γ and/or TNF-α and/or IL-2 against Env+Gag+GPN peptide pools. All data are background subtracted. B) Functional profile of memory HIV-1-specific CD4 and CD8 T cell responses. All the possible combinations of the responses are shown on the <i>x</i> axis, whereas the percentage of the functionally distinct cell populations within the total CD4 or CD8 T cell populations are shown on the <i>y</i> axis. Responses are grouped and colour-coded on the basis of the number of functions. The non-specific responses obtained in the control group DNA-Φ/MVA-WT were subtracted in all populations. C) Phenotypic profile of memory HIV-1-specific CD4 or CD8 T cells. Representative FACS plots showing the percentage of Env-specific CD4 or CD8 T cells with central memory (TCM, CD44^highCD62L⁺) or effector memory (TEM, CD44^highCD62L⁻) phenotype. * p<0.05; ** p<0.005. p-values indicate significantly higher responses compared to DNA-Φ/MVA-WT immunization group.</p

Genes induced by MVA-C in human mDCs and pDCs.

<p>A) Gene expression heatmap using the top 50 differentially expressed genes resulting from comparing myeloid DCs infected with MVA-C (MDC_MVC) and mock-infected (MDC_Mock) groups. B) Gene expression heatmap using the top 50 differentially expressed genes resulting from comparing plasmacytoid DCs infected with MVA-C (PDC_MVC) and mock-infected (PDC_Mock) groups. Genes selected as differentially expressed based on the following criteria: adjusted p-value<0.05 and |FC|>1.3. The scale shows the level of gene expression where Red and Blue correspond to up- and down-regulation respectively.</p

Significant genes associated with specific immune function in MVA-C-infected mDCs and pDCs.

<p>A) Gene expression heatmap using selected set of immune related genes resulting from comparing myeloid DCs infected with MVA-C (MDC_MVC) and mock-infected (MDC_Mock) groups. B) Gene expression heatmap using selected set of IFN-induced genes resulting from comparing myeloid DCs infected with MVA-C (MDC_MVC) and mock-infected (MDC_Mock) groups. C) Gene expression heatmap using selected set of IFN signaling genes resulting from comparing plasmacytoid DCs infected with MVA-C (PDC_MVC) and mock-infected (PDC_Mock) groups. D) Gene expression heatmap using selected set of inflammation associated genes resulting from comparing myeloid DCs infected with MVA-C (MDC_MVC) and mock-infected (MDC_Mock) groups. E) Gene expression heatmap using selected set of inflammation associated genes resulting from comparing plasmacytoid DCs infected with MVA-C (PDC_MVC) and mock-infected (PDC_Mock) groups. The genes selected are differentially expressed (FC>1.3 and adjusted p-value<0.05). Red and Blue correspond to up- and down-regulation respectively.</p