Resistance to infection, early and persistent suppression of simian immunodeficiency virus SIVmac251 viremia, and significant reduction of tissue viral burden after mucosal vaccination in female rhesus macaques

Abstract

The efficacy of oral, intestinal, nasal, and vaginal vaccinations with DNA simian immunodeficiency virus (SIV)/interleukin-2 (IL-2)/IL-15, SIV Gag/Pol/Env recombinant modified vaccinia virus Ankara (rMVA), and AT-2 SIVmac239 inactivated particles was compared in rhesus macaques after low-dose vaginal challenge with SIVmac251. Intestinal immunization provided better protection from infection, as a significantly greater median number of challenges was necessary in this group than in the others. Oral and nasal vaccinations provided the most significant control of disease progression. Fifty percent of the orally and nasally vaccinated animals suppressed viremia to undetectable levels, while this occurred to a significantly lower degree in intestinally and vaginally vaccinated animals and in controls. Viremia remained undetectable after CD8(+) T-cell depletion in seven vaccinated animals that had suppressed viremia after infection, and tissue analysis for SIV DNA and RNA was negative, a result consistent with a significant reduction of viral activity. Regardless of the route of vaccination, mucosal vaccinations prevented loss of CD4(+) central memory and CD4(+)/α4β7(+) T-cell populations and reduced immune activation to different degrees. None of the orally vaccinated animals and only one of the nasally vaccinated animals developed AIDS after 72 to 84 weeks of infection, when the trial was closed. The levels of anti-SIV gamma interferon-positive, CD4(+), and CD8(+) T cells at the time of first challenge inversely correlated with viremia and directly correlated with protection from infection and longer survival.

FIG 1

Protection from mucosal infection. (a) Kaplan-Meier graph of the number of nontraumatic challenges received for each vaccinated group until a positive infection was detected in plasma. The nontraumatic challenges were stopped after 32 challenges, and the animals that were still uninfected received traumatic challenges. The dotted line intersects the point in each curve corresponding to 50% of the animals being infected. (b) Median number of challenges for each group and total number of challenges received by each animal, whether nontraumatic or traumatic. Three animals (249, 246, and 148) remained noninfected after 32 nontraumatic and 2 traumatic challenges, and they are shown as open symbols. Wilcoxon signed-rank test (P < 0.05) was used for the median comparison. (c) IFN-γ⁺ CD8⁺ T-cell responses in quartiles, detected 3 weeks before challenge in vaginal MNC. The quartile partition is based on the number of resisted challenges. Animals in the 1st quartile resisted up to 7 challenges, in the 2nd quartile 8 to 13 challenges, in the 3rd quartile 14 to 23 challenges, and in the 4th quartile more than 23 challenges. Prechallenge vaginal MNC were not available for the control animals that were not included in the calculation. Mann-Whitney t test was used.

FIG 2

Postinfection viremia and neutralizing antibodies. (a) Dynamics of viral loads during infection: comparison between the mean viremia of the vaccinated animals and of the control macaques during the course of infection (top left graph). The values of viral loads for each individual animal and the time points for individual group averages (black line in each graph) are represented for all groups as logarithmic values. Mann-Whitney t test was used to compare mean viremia of controls and vaccinated animals (P = 0.01). Error bars represent standard errors of the means (SEM). (b) Plasma viral loads at the peak of viremia (left graph) and area under the curve (AUC) from week 16 to 60 (right graph) for each animal, calculated using the logarithmic values of the viral loads. One-way ANOVA and a Bonferroni post hoc test were used to compare groups (P = 0.05 and 0.03, respectively). (c) Logarithmic geometric means and SEM of serum neutralizing antibody levels, measured against SIV_mac251, are reported for each group (top left graph) and for each animal of all groups. Open symbols and dotted lines are used for animals that control viral replication to undetectable levels. Two-way ANOVA was used for multiple comparisons. No significant differences were detected.

FIG 3

Protection from disease progression. (a) Kaplan-Meier curves of survival during infection, plotted for each group. The dotted line intersects the point in each curve corresponding to 50% of the animals still surviving. A log-rank test (Mantel-Cox) was used for comparison (P = 0.05). (b) Correlation between the levels of viral loads measured early in the chronic phase (16 weeks postinfection) and during survival (P = 0.001 and r = −0.66 by Spearman test). (c) Correlation between anti-Gag⁺ Env CD4⁺/IFN-γ and CD8⁺/IFN-γ T-cell responses detected in PBMCs on the day of first challenge and control of early chronic viremia (week 16 postinfection viral loads). The Spearman test was used to determine statistical significance (P = 0.0034 and r = −0.56 for anti-Gag⁺ Env CD4⁺/IFN-γ and P = 0.0045 and r = −0.54 for CD8⁺/IFN-γ).

FIG 4

Immune responses after infection. (a) PBMC, (b) rectal, and (c) vaginal SIV Gag⁺ Env-specific T-cell immune responses early in chronic infection (12 weeks postinfection, the only postinfection time point available for rectal and vaginal MNC). The pie plots illustrate the diversity and the amount (group average percentages are given under the pie) of the CD4⁺ or CD8⁺ T-cell responses, detected as the ability to produce IL-2, IFN-γ, or TNF-α or multiple cytokines. Sampling in the vaginal and rectal mucosa was carried out 3 weeks before vaginal challenge to allow for the healing of the vaginal mucosa. Comparisons of the responses within each group were done using a Mann-Whitney t test, and for each cytokine, single-, double-, and triple-positive cells that express that cytokine are included in the numbers used for statistical analysis. The arched black line marks populations that are significantly larger than the same population at another time point. The red numbers indicate values for total populations that are significantly higher before challenge than the corresponding values at week 12 (oral and nasal groups) and are higher than those for the same population at the same time point in the other groups (intestinal group).

FIG 5

Immunological parameters associated with vaccine efficacy. (a) Circulating C_M (CD95⁺/CD28⁺) CD4⁺ T cells are given as percentages of total CD4⁺ T cells in vaccinated (black) and control (green) animals (left; P = 0.005) and for each animal in the groups (right; P = 0.02). Values observed on week 16 after infection in PBMC are reported for C_M CD4⁺ T cells. (b) PBMC CD4⁺ T-cell levels reported for each group as average percentages (left) or average absolute counts/ml (right) during the course of the infection. (c) Percentages of circulating α4β7^high+/CD4⁺ T cells in vaccinated (black) and control (green) groups (left; P = 0.0001 by Mann-Whitney t test) and in each group (right; P = 0.0001 by one-way ANOVA followed by a Bonferroni posttest, which reveals which between-group comparisons contribute to the significant ANOVA result) early during chronic infection (16 weeks postinfection). (d) Levels of immune activation in CD4⁺ and CD8⁺ T-cell populations during infection. Geometric means of the percentages of CD38^high+/HLA-DR⁺ CD4⁺ (left; P = 0.0001) or CD8⁺ (middle; P = 0.005) C_M T cells in PBMC in each group are reported. Shown are comparisons of the geometric mean of CD38^high+/HLA-DR⁺ CD4⁺ or CD8⁺ C_M T-cell percentages in each group at the peak of immune activation 4 weeks after infection (right; P = 0.03). Mann-Whitney t test was used for the pair average comparisons. Two-way ANOVA and a Bonferroni post hoc test were used for multiple comparisons. Error bars represent SEM. (e) Correlation between levels of immune activation detected as percentages of CD38^high+/HLA-DR⁺ CD8⁺ C_M in PBMC 20 weeks postinfection and early control of the viral replication 16 weeks postinfection (left; P = 0.0001 and r = 0.79), preservation of the CD4⁺ C_M T-cell population 16 weeks after infection (middle; P = 0.03 and r = −0.38), and long-term survival (right; P = 0.0006 and r = −0.55). The Spearman test was used to test the statistical significance of the correlation.

FIG 6

CD8⁺ T-cell depletion and SIV detection in serum and tissues. (a) Percentage of CD8⁺ T cells during and after CD8⁺ T cell depletion in PBMC of three animals (numbers 148, 246, and 249) that resisted 32 nontraumatic and two traumatic challenges and nine animals that had controlled viremia to undetectable levels between 4 and 28 weeks postinfection (numbers 255, 253, 153, 155, 150, 250, 147, 175, and 301). The x axis represents days since first anti-CD8 antibody injection (day 1). Positive RT-PCR time points are indicated for the two animals (numbers 301 and 175) that became viremic after CD8⁺ T-cell depletion. SIV copies/ml were 48,000, 56,000, and 45,000 for animal 175 and 12,000, 7,700, and 280,000 for animal 301. All of the other animals were consistently negative during the time course. (b) Detection of SIV gag DNA in mesenteric lymph nodes, tonsils, and spleen and in jejunum, ileum, and colon of 4 nonviremic animals, two vaccinated nasally, one orally, and one intestinally, and two viremic animals, one orally and one intestinally vaccinated. The color for each animal is according to its vaccine group, as coded before. DNA copy numbers were normalized according to the detection of IL-15 copies in the same amount of DNA. Each tissue symbol represents averages from multiple fragments of tissue or lymph nodes, varying from 3 to 5 for each. (c) Detection of SIV Gag RNA in mesenteric lymph nodes (LN) and PBMC of nonviremic and viremic animals. RNA copy numbers were normalized according to the detection of 18S RNA in the same amount of RNA for each sample. Only samples from one viremic animal were tested.

FIG 7

Virus-specific immune responses stimulated during multiple low-dose challenges. (a) Total anti-SIV CD4⁺ and CD8⁺ Gag or Env responses detected before challenge (open bars) or after 32 nontraumatic vaginal exposures to SIV_mac251 (closed bars) in vaginal MNC of animals that remained RT-PCR negative after 32 nontraumatic challenges. (b) Anti-SIV gp140 IgA-specific activity (ng gp140-specific IgA per μg total IgA) detected before challenge (open symbols) or after 32 nontraumatic vaginal exposures to SIV_mac251 (closed symbols) in rectal and vaginal secretions of animals that remained RT-PCR negative after 32 nontraumatic challenges. The SIV antibody concentration in secretions is expressed as specific activity (ng SIV-specific IgA per microgram of total IgA). Asterisks indicate animals that resisted two traumatic challenges that were administered after the sampling reported here.