Figure 1.
Study Design and DNA vaccines.
(A) DNA vaccines. The unadjuvanted DNA vaccine consisted of a co-delivery of 2 plasmids, one encoding a fusion of SIV RT/Nef/Gag (RNG) and a second encoding SIV gp120 (SIV17E-Fr). The DNA+LT vaccine is a co-delivery of the RNG and SIVgp120 plasmid and a 3rd plasmid encoding the A and B subunits from the heat-labile enterotoxin of E. Coli (LT) downstream from 2 separate CMV promoters. The DNA group received 32 µg of the DNA vaccine (16 µg per plasmid) per dose. The DNA+LT group received 32 µg of the DNA vaccine plus 3.2 µg of the LT genetic adjuvant. (B) Study Design. Anti-retroviral drug therapy (ART) consisting of lopinavir/ritonavir (Kaletra) and PMPA was started 6 weeks after intravenous infection with SIV/DeltaB670 and continued until week 45 post-infection. Arrows indicate time-points of 6 DNA immunizations, administered one month apart. Inverted open triangles indicate time-points when a gut jejunal resection was performed and lymphocytes and tissues isolated for analysis of viral replication, immune responses, and CD4+ T cell counts in the gut. ART was discontinued 7 weeks after the last DNA immunization to assess the effects of vaccination on protecting against viral rebound and disease. Vaccine efficacy, defined by containment of viral rebound was assessed at 5 (66 weeks post-infection) and 10 (88 weeks post-infection) months after stopping ART.
Figure 2.
Therapeutic DNA vaccination reduces viral load and protects from viral rebound after drug is stopped.
Plasma viral RNA load was determined by real time RT-PCR. Boxed areas (ART) indicate period of antiretroviral drug therapy. Arrows indicate DNA vaccine doses. (A) Response to ART (weeks 0–18). Shown are viral loads in 41 SIV-infected macaques before starting ART (weeks 0–6) and during the first 12 weeks of ART only (weeks 6–18). ART responders (black) had declining viral loads during ART only. ART low responders (red) had little or no decline in viral loads during ART only and maintained ≥5×104 viral RNA copies/ml (dotted line). (B) Log-fold reduction in viral load during ART only. The change in viral load during ART in each animal was determined by dividing the mean viral load prior to initiating ART (weeks 4–6) by the mean viral load during the first 12 weeks of ART but prior to initiating the therapeutic vaccinations (weeks 8–18). Shown is the log-fold reduction in mean viral load during ART for each animal. ART low responders had <1 log-fold reduction. ART responders exhibited 1.6–4.9 log-fold reductions in viral load during ART. (C) Mean plasma viral loads in each group prior to initiating ART (week 6, Pre-ART) and after 12 weeks on ART but before starting therapeutic vaccinations (week 18, Pre-vaccine). (D) Mean plasma viral loads during vaccinations and after withdrawing ART. Differences in mean viral loads after withdrawing ART are significant starting at weeks 68 and 48 in the DNA (P = 0.019) and DNA+LT (P = 0.002) groups, respectively, and remained significant thereafter when compared to controls (Wilcoxon test). (E) Viral loads in each animal. Protection from viral rebound was defined as containment of viral load ≤100 viral RNA copies/ml (hashed line) for a period of at least 5 months after drug was withdrawn (weeks 45–66).
Figure 3.
Therapeutic DNA vaccination protects from AIDS.
Maintenance of CD4+ T cell counts as a marker of clinical disease progression was measured by flow cytometry. Shown is % CD4+ T cell count relative to baseline. Animals that maintained their CD4+ T cell counts above 50% of their baseline level (hashed line) level remained clinically healthy throughout the study.
Table 1.
Protection from viral rebound and CD4 decline.
Table 2.
Relationship between TRIM5 haplotype and outcome.
Table 3.
Statistical Analysis of Trim5 haplotype versus outcome.
Figure 4.
Therapeutic DNA vaccines increase SIV-specific IFN-γ T cell responses, but not antibody, in the blood.
(A) IFN-γ T cell responses were measured throughout the study by ELISPOT. Shown is the mean (± SEM) SIV-specific IFN-γ T cell response at each time-point minus background. Background levels were <25 SFC/106 PBMC. Differences between the DNA or DNA+LT vaccine groups and controls were significant (P≤0.0063, repeated measures ANOVA). (B) SIV-gp120 specific binding antibody was measured throughout the study by ELISA. Shown are mean (± SEM) endpoint titers. Mean antibody titers in the DNA+LT group were significantly lower (P = 0.0422) than the controls (C) Neutralizing antibody responses were measured against the vaccine strain (SIV/17E-Fr). Shown are mean (± SEM) neutralization titers after the final DNA vaccine dose but before stopping ART (week 40) and 9 weeks after stopping ART (week 54). *P = 0.0292 (D) Neutralizing antibody titers in each animal before (week 40) and after (week 54) stopping ART against the challenge strain (SIV/DeltaB670).
Figure 5.
Therapeutic DNA vaccination increases CD4+ and CD8+ T cell responses in the blood after stopping ART.
SIV-specific CD4+ and CD8+ T cells with cytokine (IFN-γ, TNF-α, IL-2) or cytolytic (CD107a) effector functions were measured in PBMC at 3 time-points just before (week 44) and after (weeks 50, 58) ART was withdrawn by flow cytometry following in vitro stimulation with overlapping peptide pools (RT, Nef, Gag, and Env) derived from the SIV genes included in the DNA vaccine. Shown is the mean frequency and functional nature of the SIV-specific effector T cell response in the CD4+ (A) and CD8+ (B) T cell subsets. *indicates P<0.05 in only the DNA+LT group.
Figure 6.
LT-adjuvanted DNA vaccine increases T cells with multiple effector functions in the blood.
SIV-specific T cells with 1–4 cytokine (IFN-γ, TNF-α, IL-2) or cytolytic (CD107a) effector functions were measured in PBMC 14 weeks after ART was withdrawn (week 58) by flow cytometry following in vitro stimulation with overlapping peptide pools derived from genes included in the DNA vaccine (RT, Nef, Gag, Env). Boolean gating was performed to identify the total frequency of CD4+ and CD8+ T cells in the blood of ART responders with any one of 1–4 effector functions. (A) Cumulative mean (±SEM) frequency of SIV-specific CD4+ and CD8+ T cells expressing IFN-γ, TNF-α, IL-2 or CD107a. Cumulative mean frequency (± SEM) of SIV-specific (B) CD4+ and (C) CD8+ T cells co-expressing any combination of 2–4 effector functions. Mean (± SEM) frequency of SIV-specific (D) CD4+ and (E) CD8+ T cells expressing the indicated combination of dual effector functions.
Figure 7.
Therapeutic DNA vaccination reduces virus production in the lymph nodes and gut mucosa.
(A) Viral load in gut lamina propria (LP), mesenteric lymph nodes (MLN), and axillary lymph nodes (ALN) in each animal 3 weeks before (week 42, solid circles) and 3 weeks after ART was withdrawn (week 48, open circles). Solid lines indicate the median viral load for the group. Hashed line indicates the lower limit of detection for this analysis (3 viral RNA copies). (B) Correlations between mean virus production in the gut lamina propria (left panel), mesenteric lymph nodes (middle panel) and axillary lymph nodes (right panel) vs. mean plasma viral load in each animal post-ART. Each symbol corresponds to mean virus load in the plasma of each animal after stopping ART (weeks 47–88) on the X axis vs. mean virus production measured in tissues at weeks 42 and 48 on the Y axis. Spearman's rank correlation coefficients are shown.
Figure 8.
Therapeutic DNA vaccination increases mucosal SIV-specific T cell responses in the gut.
(A) The magnitude of the SIV-specific IFN-γ T cell response in the gut was determined by ELISPOT analysis of lamina propria lymphocytes (LPL) isolated from jejunal resections. Results represent the mean number of IFN-γ spot forming cells (± SEM) for each group from 2 separate experiments performed at 2 time-points post-infection (weeks 42 and 48). (B) Breadth of the T cell response in each macaque in the blood (top panels, PBMC) and gut (lower panels, GALT). SIV-specific T cell responses in PBMC and LPL isolated from the jejunum were measured by IFN-γ ELISPOT assay against 11 separate pools of overlapping peptides (15-mers overlapping by 11 amino acids) comprising the indicated amino acid sequences from SIV Gag, Env, Pol, and Nef. The percent contribution of each peptide-pool specific response to the total response was determined by dividing the mean number of IFN-γ spot forming cells (SFC) measured against each individual peptide pool by the sum of the response against all peptide pools. Results represent the average of 2 time-points tested at weeks 42 and 48. (C) Mean absolute GALT CD4+ T cell counts measured by flow cytometric analysis of mononuclear cells isolated from the jejunal lamina propria at weeks 12, 42, and 48 post-infection were similar in animals that controlled vs. failed to control viral rebound after ART was stopped. Controllers are animals that contained virus at ≤100 copies for at least 5 months after stopping ART. Non-controllers are animals that exhibited viral rebound within the same timeframe.
Figure 9.
Viral control correlates to CD8+ T cell responses with broad specificity in the gut.
Correlations between the breadth of the T cell response in the gut (top panels A and B) or the blood (lower panels C and D) vs. viral loads in blood (left panels A and C) and gut (right panels B and D). Each symbol corresponds to mean virus load in the plasma of each animal after stopping ART (weeks 47–88) or gut (weeks 42, 48) on the X axis vs. mean number of peptide pools recognized in the IFN-γ ELISPOT assay at weeks 42 and 48 in the blood or gut lamina propria on the Y axis. Spearman's rank correlation coefficients are shown.