Infection with host-range mutant adenovirus 5 suppresses innate immunity and induces systemic CD4+ T cell activation in rhesus macaques

Abstract

Ad5 is a common cause of respiratory disease and an occasional cause of gastroenteritis and conjunctivitis, and seroconversion before adolescence is common in humans. To gain some insight into how Ad5 infection affects the immune system of rhesus macaques (RM) 18 RM were infected with a host-range mutant Ad5 (Ad5hr) by 3 mucosal inoculations. There was a delay of 2 to 6 weeks after the first inoculation before plasmacytoid dendritic cell (pDC) frequency and function increased in peripheral blood. Primary Ad5hr infection suppressed IFN-γ mRNA expression, but the second Ad5hr exposure induced a rapid increase in IFN-gamma mRNA in peripheral blood mononuclear cells (PBMC). Primary Ad5hr infection suppressed CCL20, TNF and IL-1 mRNA expression in PBMC, and subsequent virus exposures further dampened expression of these pro-inflammatory cytokines. Primary, but not secondary, Ad5hr inoculation increased the frequency of CXCR3+ CD4+ T cells in blood, while secondary, but not primary, Ad5hr infection transiently increased the frequencies of Ki67+, HLADR+ and CD95+/CCR5+ CD4+ T cells in blood. Ad5hr infection induced polyfunctional CD4 and CD8+ T cells specific for the Ad5 hexon protein in all of the animals. Thus, infection with Ad5hr induced a complex pattern of innate and adaptive immunity in RM that included transient systemic CD4+ T cell activation and suppressed innate immunity on re-exposure to the virus. The complex effects of adenovirus infection on the immune system may help to explain the unexpected results of testing Ad5 vector expressing HIV antigens in Ad5 seropositive people.

Figure 1. Effect of Ad5hr infection on the number and function of circulating pDC.

A) number of pDC per ml of blood. B) number of IFN-α secreting pDCs per ml of blood. Arrows on the X axis indicate the time of Ad5hr inoculation. The mean values for all animals are indicated by blue bars. P values ≤0.05 from the ANOVA and Dunnetts's post-hoc tests are shown.

Figure 2. Effect of Ad5hr infection on CD4+ Treg frequency.

A) The frequency of FOXP3⁺/CD4⁺ T cells that express a high level of CD25 in blood. B) The frequency of CTLA4⁺/CD25^hi/ CD4⁺ T cells in blood. C) The frequency of FOXP3⁺/CTLA4+/CD25^hi/ CD4⁺ T cells in blood. Arrows on the X axis indicate the time of Ad5hr inoculation. The mean values for all animals are indicated by blue bars. P values £ 0.05 from the ANOVA and Dunnetts’s post-hoc tests are shown.

Figure 3. Effect of Ad5hr infection on the frequency of circulating CD4+T cells expressing activation markers.

A) The percentage of CD95+/CD3+/CD4+ cells expressing CCR5. B) The percentage of CD3+/CD4+ cells expressing a high level of HLA-DR. C) The percentage of CD3+/CD4+ cells expressing Ki67. Arrows on the X axis indicate the time of Ad5hr inoculation. The mean values for all animals are indicated by blue bars. P values ≤0.05 from the ANOVA and Dunnetts's post-hoc tests are shown.

Figure 4. Effect of Ad5hr infection on proinflammatory cytokine and chemokine gene expression in PBMC.

A) The fold-change of IFN-gammamRNA in PBMCs; B) IFN-α; C) IL-1; D) TNF and E) CCL20. The fold-change in target gene mRNA levels in study animals post-Ad5hr infection relative to the same target gene mRNA level before Ad5hr infection was calculated as described in Materials and Methods. Arrows on the X axis indicate the time of Ad5hr inoculation. The mean values for all animals are indicated by blue bars P values ≤0.05 from the ANOVA and Dunnetts's post-hoc tests are shown.

Figure 5. Ad5 hexon-specific T cell responses in PBMCs after Ad5hr infection as detected by multiparameter flow cytometry.

Pie charts present the average CD4+ or CD8+T cell responses for all positive animals at weeks 2, 10 and 14 from the first Ad5hr inoculation, as described in Materials and Methods. Pie slice colors denote the number of positive functions (expression of IL-2, IFN-γ, TNF and CD107 degranulation). Pie arc colors denote the 4 functions separately. Red numbers in the center of pie charts are the mean ± standard deviation of hexon-specific T cells normalized to 10⁵ CD3+ T cells. Fractional numbers below a pie chart refer to the number of animals in a group of 18 that responded.