Increased mucosal CD4+ T cell activation in rhesus macaques following vaccination with an adenoviral vector

Abstract

The possibility that vaccination with adenovirus (AdV) vectors increased mucosal T cell activation remains a central hypothesis to explain the potential enhancement of HIV acquisition within the Step trial. Modeling this within rhesus macaques is complicated because human adenoviruses, including human adenovirus type 5 (HAdV-5), are not endogenous to macaques. Here, we tested whether vaccination with a rhesus macaque-derived adenoviral vector (simian adenovirus 7 [SAdV-7]) enhances mucosal T cell activation within rhesus macaques. Following intramuscular SAdV-7 vaccination, we observed a pronounced increase in SAdV-7-specific CD4(+) T cell responses in peripheral blood and, more dramatically, in rectal mucosa tissue. Vaccination also induced a significant increase in the frequency of activated memory CD4(+) T cells in SAdV-7- and HAdV-5-vaccinated animals in the rectal mucosa but not in peripheral blood. These fluctuations within the rectal mucosa were also associated with a pronounced decrease in the relative frequency of naive resting CD4(+) T cells. Together, these results indicate that peripheral vaccination with an AdV vector can increase the activation of mucosal CD4(+) T cells, potentially providing an experimental model to further evaluate the role of host-vector interactions in increased HIV acquisition after AdV vector vaccination.

Importance: The possibility that vaccination with a human adenovirus 5 vector increased mucosal T cell activation remains a central hypothesis to explain the potential enhancement of human immunodeficiency virus (HIV) acquisition within the Step trial. In this study, we tested whether vaccination with a rhesus macaque-derived adenoviral vector in rhesus macaques enhances mucosal CD4(+) T cell activation, the main cell target of simian immunodeficiency virus (SIV)/HIV. The results showed that vaccination with an adenoviral vector indeed increases activation of mucosal CD4(+) T cells and potentially increases susceptibility to SIV infection.

FIG 1

Phylogenetic relationships between hexon proteins from human and monkey adenoviruses. (The amino acid sequences were aligned by using ClustalW version 2.0.3 and refined by using Gblocks version 0.91b. The alignment was used to construct the phylogenetic tree by using PhyML version 3.0 aLRT and rendered by using Treedyn 198.3. Branch support values are indicated as percentages. The scale bar indicates the number of substitutions per site.) Representatives of human adenoviruses of species A through G (serotypes in parentheses and including HAdV-5, in boldface type) and several monkey adenoviruses are shown. Monkey adenoviruses (including SAdV-7, which was used to construct vectors used in this study [highlighted in red]) are seen to belong to a phylogenetically distinct group (gray box).

FIG 2

Vaccination and sample collection timeline. Shown is the macaque trial design with 10 tissue (PBMCs and rLPLs) collection time points and either SAdV-7 or HAdV-5 vector vaccinations interspersed at weeks 0, 17, and 31.

FIG 3

Gating strategy for identification of CD4⁺ memory T cells. First, to ensure that only live single cells were collected from either PBMCs or rectal lamina propria lymphocytes (rLPLs), forward scatter height (FSC-H)-versus-forward scatter area (FSC-A) and side scatter area (SSC-A)-versus-FSC-A plots were used to exclude doublets and focus on singlet lymphocytes. Dead cells were excluded by gating on cells negative for the viability marker Aqua Blue. Monocytes and B cells were excluded via the CD14/CD20 dump gate. To select for CD4⁺ T cells specifically, we included CD4⁺ and CD8⁻ T cells. To determine the memory phenotype, we used CD28 versus CD95 cells and excluded naive cells (CD28⁺/CD95⁻) from the analysis. From here, activation markers (CD25, CD69, HLA-DR, and Ki67) were gated as shown on the bottom plots on unstimulated memory CD4⁺ T cells, and cytokines (IL-2, TNF-α, and IFN-γ) were analyzed. All gating is shown for rectal lamina propria lymphocytes, unless indicated otherwise.

FIG 4

AdV vector vaccination increases memory AdV-specific CD4⁺ T cell percentages. (A) Representative memory CD4⁺ T cell IL-2/IFN-γ/TNF-α response after stimulation with 1 × 10¹⁰ particles/ml of whole SAdV-7 vector. The number at the bottom right of the plot represents the proportion of cells that express IL-2/IFN-γ/TNF-α. (B) Percent CD4⁺ memory T cell IL-2/IFN-γ/TNF-α expression in response to whole SAdV-7 vector stimulation (SAdV-7 stimulated) overnight after background (no stimulation) subtraction. Levels of IL-2, IFN-γ, and TNF-α production were measured on the same fluorochrome. Peripheral blood and rectal lamina propria T cells from 12 SAdV-7-vaccinated and 5 HAdV-5-vaccinated rhesus macaques (symbols for each macaque are listed in the key) were collected at 10 time points, including baseline (week −2), interspersed with three AdV vector immunizations. Significance at any time point was calculated as the percent increase relative to the baseline value and was determined as described in Materials and Methods. Statistically significant increases relative to the baseline value are indicated above the red bars (week 19 approached statistical significance). +, animals with no rectal biopsy data at the baseline time point.

FIG 5

Increased activation marker expression on total unstimulated memory CD4⁺ T cells in rectal mucosa after AdV vector vaccination. Shown are percentages of total unstimulated memory CD4⁺ T cells expressing at least one of four activation markers (HLA-DR, CD25, Ki67, and CD69). Peripheral blood and rectal lamina propria T cells from 12 SAdV-7-vaccinated and 5 HAdV-5-vaccinated rhesus macaques (symbols for each macaque are listed in the key) were collected at 10 time points, including baseline (week −2), interspersed with three AdV vector immunizations. Significance at any time point was calculated as the percent change relative to the baseline value, shown by red lines above each plot, and was determined as described in Materials and Methods. +, animals with no rectal biopsy data at the baseline time point.

FIG 6

Increased activation marker expression on total SAdV-7-stimulated memory CD4⁺ T cells in rectal mucosa after AdV vector vaccination. Shown are percentages of total memory CD4⁺ T cells stimulated with whole SAdV-7 vector overnight and expressing at least one of four activation markers (HLA-DR, CD25, Ki67, and CD69). Cells are from the same rhesus macaques and time points as those in Fig. 5, with the same statistical significance. +, animals with no rectal biopsy data at the baseline time point.

FIG 7

Naive CD4⁺ T cell percentages at baseline and after AdV vector immunization. (A) Representative example of a flow cytometric plot gating on CD4⁺ T cells stimulated with SAdV-7 overnight using the memory markers CD28 and CD95, where naive indicates CD28⁺ CD95⁻ cells, central memory indicates CD28⁺ CD95⁺ cells, effector memory indicates CD28⁻ CD95⁺ cells, and effector indicates CD28⁻ CD95⁻ cells. The number at the top right of the plot represents the percentage of naive CD4⁺ T cells. (B) Percentage of naive CD4⁺ T cells stimulated overnight with whole SAdV-7 vector. Samples were collected from macaques, and immunizations were administered as described in the legend of Fig. 5. Significance at any time point was calculated as a percent change relative to the baseline value, shown by red lines above each plot, and was determined as described in Materials and Methods. +, animals with no rectal biopsy data at the baseline time point.