CD40L-adjuvanted DNA/modified vaccinia virus Ankara simian immunodeficiency virus (SIV) vaccine enhances protection against neutralization-resistant mucosal SIV infection

Abstract

Here, we show that a CD40L-adjuvanted DNA/modified vaccinia virus Ankara (MVA) simian immunodeficiency virus (SIV) vaccine enhances protection against a pathogenic neutralization-resistant mucosal SIV infection, improves long-term viral control, and prevents AIDS. Analyses of serum IgG antibodies to linear peptides of SIV Env revealed a strong response to V2, with targeting of fewer epitopes in the immunodominant region of gp41 (gp41-ID) and the V1 region as a correlate for enhanced protection. Greater expansion of antiviral CD8 T cells in the gut correlated with long-term viral control.

FIG 1

CD40L-adjuvanted DNA/MVA SIVmac239 vaccine enhances protection against SIVmac251 infection. (A) Kaplan-Meier plots showing the number of SIV challenges required for acquisition of SIVmac251 infection. The P value reflects a significantly lower hazard ratio than that for controls when the log rank (Mantel-Cox) method was used. (B) Geometric mean values for viral RNA in plasma. Scatter plots show number of copies of viral RNA for individual animals postinfection. (C) Change in percentage of rectal CD4 T cells from preinfection baseline levels in SIV-infected animals. The CD4 T cell level was measured as a percentage of total CD3 cells (3). Data reflect means ± the standard error of the mean (SEM). Scatter plots show changes in rectal CD4 T cell levels for individual animals postinfection. Preinfection data were not available for 4 control animals and thus were not included in the analysis. However, we observed a profound depletion of CD4 T cells in the rectums of these 4 animals as early as 2 weeks postinfection. P values show differences between the indicated groups. (D) Survival of animals post-SIVmac251 infection. The P values indicate a higher survival rate in adjuvanted animals than in nonadjuvanted and unvaccinated animals as determined by the log rank (Mantel-Cox) method. Five additional unvaccinated controls that were challenged simultaneously for a parallel study were included. The P value is 0.052 without these additional controls.

FIG 2

D_40LM_40L vaccine alters binding-antibody specificity and promotes a V2-focused IgG response. (A) Geometric mean anti-Env IgG binding antibodies in serum after MVA boosting. Concentrations of Env-specific IgG were measured by enzyme-linked immunosorbent assay (ELISA) (1) using rgp140mac239 (Immune Technologies). Error bars represent SEM. M, MVA vaccine; dashed lines, day of MVA vaccination. (B) Serum binding IgG responses in the nonadjuvanted (DM) and adjuvanted (D_40LM_40L) animals against 15-mer peptides (overlapping by 12 amino acids) specific to the V1 and V2 variable regions of gp120 and the immunodominant region of gp41 (gp41-ID) of SIVmac239. Each line represents an individual animal. Linear epitope mapping analyses were performed as previously described (6) with modifications, including background correction and inclusion of a positivity cutoff of ≥500 signal intensity units. (C) Breadth of IgG response specific to individual regions of gp160. Tukey box plots and individual animals in each vaccination group are represented. (D) Correlations between the binding intensity of antibody to V2 peptides and the binding intensity of antibody to gp41-ID or V1 peptides at 2 weeks after the second MVA boost. (E) Correlation between the breadth of reactivity to total Env, gp41-ID, or V1 peptides and the number of challenges required for productive infection. For correlation tests, uninfected animals were assigned the number 8.

FIG 3

D_40LM_40L vaccine enhances T-FH and germinal center B cell responses. (A) Immunofluorescence imaging of a nondraining lymph node from a CD40L-adjuvanted animal at 2 weeks after the final MVA boost shows germinal center follicles with T-FH cells. Sections were stained with CD4 (red), CD20 (blue), and PD-1 (green) as described previously (7, 8). The merged image shows PD-1^high CD4 T-FH cells abundantly present within germinal centers of lymphoid follicles. F, follicle; GC, germinal center; TZ, T cell zone. Scale bars = 50 microns. (B) Sections of lymph node germinal center follicles stained with CD3 (red), CD20 (blue), and Ki67 (green) show proliferating (Ki67⁺) B cells in germinal center follicles. (C) Numbers of follicles with germinal centers in nonadjuvanted (DM) and adjuvanted (D_40LM_40L) animals. The P value reflects significance as determined by the Mann-Whitney t test. (D) Total number of T-FH cells and density of Ki67⁺ B cells/mm² of section. The total number of T-FH cells was calculated as a product of the density of T-FH cells/mm² and the number of follicles in the section. (E) Correlation of the density of T-FH cells and Ki67⁺ B cells with the avidity index of anti-SIVmac239 Env IgG at the time of challenge. P values reflect significance as determined by Spearman's rank correlation test. These analyses were performed on seven DM and eight D_40LM_40L animals.

FIG 4

Enhanced control of SIVmac251 is associated with rapid expansion of SIV Gag-specific CD8 T cells in the rectum. (A) Temporal viral RNA levels in controllers (<10⁴ copies/ml at set point) and noncontrollers (>10⁴ copies/ml at set point). Results for nonadjuvanted animals are shown in pink and for CD40L-adjuvanted animals in blue. Results for A*01 animals are indicated by filled symbols with a black border and for non-A*01 animals by open symbols. Viral RNA levels were determined using qPCR (9). Dashed lines indicate the viral load threshold used to define controllers and noncontrollers. (B) Temporal frequencies of postinfection Gag-specific CD8 IFN-γ⁺ T cells in the rectum determined using an intracellular-cytokine-staining (ICS) assay (1). Dashed lines indicate the day of productive SIVmac251 infection. (C) Temporal frequencies of postinfection Gag-specific CD8 IFN-γ⁺ T cells in the blood determined using an ICS assay. Dashed lines indicate the day of productive SIVmac251 infection. (D) Temporal viral RNA levels in blood (top panel) and expansion of Gag-specific CD8 T cells in the rectum (middle panel) and blood (bottom panel) of atypical controllers postinfection. The vertical dashed lines indicate the day of productive SIVmac251 infection. (E) Correlation between Gag-specific CD8 IFN-γ T cells in rectum at 2 weeks postinfection and viral set point at week 12 postinfection. (F) Correlation between post-MVA vaccination memory IFN-γ⁺ CD8 T cells in blood and postinfection IFN-γ⁺ CD8 T cells in rectum. (G) Correlation between post-MVA vaccination peak IFN-γ⁺ CD8 T cells in blood and viral RNA copies at 3 weeks postinfection. P values for panels E through G were obtained using the two-tailed Spearman rank correlation test.