HIV-1 Tat immunization restores immune homeostasis and attacks the HAART-resistant blood HIV DNA: results of a randomized phase II exploratory clinical trial

Abstract

Background: The phase II multicenter, randomized, open label, therapeutic trial (ISS T-002, Clinicaltrials.gov NCT00751595) was aimed at evaluating the immunogenicity and the safety of the biologically active HIV-1 Tat protein administered at 7.5 or 30 μg, given 3 or 5 times monthly, and at exploring immunological and virological disease biomarkers. The study duration was 48 weeks, however, vaccinees were followed until the last enrolled subject reached the 48 weeks. Reported are final data up to 144 weeks of follow-up. The ISS T-002 trial was conducted in 11 clinical centers in Italy on 168 HIV positive subjects under Highly Active Antiretroviral Therapy (HAART), anti-Tat Antibody (Ab) negative at baseline, with plasma viremia <50 copies/mL in the last 6 months prior to enrollment, and CD4(+) T-cell number ≥200 cells/μL. Subjects from a parallel observational study (ISS OBS T-002, Clinicaltrials.gov NCT0102455) enrolled at the same clinical sites with the same criteria constituted an external reference group to explore biomarkers of disease.

Results: The vaccine was safe and well tolerated and induced anti-Tat Abs in most patients (79%), with the highest frequency and durability in the Tat 30 μg groups (89%) particularly when given 3 times (92%). Vaccination promoted a durable and significant restoration of T, B, natural killer (NK) cells, and CD4(+) and CD8(+) central memory subsets. Moreover, a significant reduction of blood proviral DNA was seen after week 72, particularly under PI-based regimens and with Tat 30 μg given 3 times (30 μg, 3x), reaching a predicted 70% decay after 3 years from vaccination with a half-life of 88 weeks. This decay was significantly associated with anti-Tat IgM and IgG Abs and neutralization of Tat-mediated entry of oligomeric Env in dendritic cells, which predicted HIV-1 DNA decay. Finally, the 30 μg, 3x group was the only one showing significant increases of NK cells and CD38(+)HLA-DR(+)/CD8(+) T cells, a phenotype associated with increased killing activity in elite controllers.

Conclusions: Anti-Tat immune responses are needed to restore immune homeostasis and effective anti-viral responses capable of attacking the virus reservoir. Thus, Tat immunization represents a promising pathogenesis-driven intervention to intensify HAART efficacy.

Trial registration: ClinicalTrials.gov NCT01024556.

Figures

Figure 1

Study flow chart. Two hundred seventy-two HAART-treated patients were assessed for eligibility. One hundred three patients were excluded from the study (n = 94 for either not meeting the inclusion criteria or for meeting exclusion criteria; n = 8 declined to participate, and 1 was lost to follow-up). Thus, 169 subjects were randomized to one of the 4 treatment groups. One subject was excluded after randomization for concomitant treatment with a contraindicated medication, therefore 168 volunteers were allocated to intervention and analyzed for safety (safety population). Since 11 subjects (from the different treatment groups) received less than 3 immunizations and 2 subjects were non compliant with antiretroviral therapy, 155 subjects were considered for the analyses of immunogenicity (immunogenicity population).

Figure 2

Anti-Tat humoral immune response in vaccinees. (A) Percentage of subjects producing Anti-Tat Abs (responders) after Tat immunization (7.5 μg, 3x n = 40; 7.5 μg, 5x n = 40; 30 μg, 3x n = 38; 30 μg, 5x n = 37). (B) Kaplan-Meier estimates showing the cumulative probability of anti-Tat Ab durability in responders stratified according to treatment groups and up to week 144 of follow-up (n = 123, median follow-up of 96 weeks). (C) Percentage of subjects in each treatment group producing anti-Tat IgM (light blue), IgG (red), or IgA (white) Abs. (D) Percentage of subjects in each treatment group producing two (IgM and IgG, in blue) or three (IgM, IgG and IgA, in red) anti-Tat Ab classes. (E) Anti-Tat IgM (light blue) and IgA (white) peak titers between 4 and 24 weeks since the first immunization in subjects positive for IgM (7.5 μg, 3x n = 16; 7.5 μg, 5x n = 20; 30 μg, 3x n = 24; 30 μg, 5x n = 23) or IgA anti-Tat Abs (7.5 μg, 3x n = 7; 7.5 μg, 5x n = 9; 30 μg, 3x n = 19; 30 μg, 5x n = 22). (F) Anti-Tat IgG Ab peak titers between 4 and 24 weeks since the first immunization in subjects positive for IgG anti-Tat Abs (7.5 μg, 3x n = 24; 7.5 μg, 5x n = 24; 30 μg, 3x n = 32; 30 μg, 5x n = 31). Box plots represent the median, 25th and 75th percentile, with the minimum and maximum values; the outliers are not represented in the graphs.

Figure 3

Anti-Tat cellular immune response. IFN-γ, IL-2 or IL-4 production and CD4+ or CD8+ T cell proliferation to Tat in (A) vaccinees or (C) OBS subjects. The percentages of responders at baseline (white bars) and up to 48 weeks from baseline (blue bars) (vaccinees: IFN-γ, IL-2, or IL-4 n = 151, proliferation n = 140; OBS: IFN-γ, IL-2, or IL-4 n = 60, proliferation n = 54) are shown. Peak intensity of anti-Tat cellular responses in (B) vaccinees or (D) OBS subjects at baseline (white bars) and up to 48 weeks (blue bars) in responders (subjects with at least one positive response after baseline) (vaccinees: IFN-γ n = 39, IL-2 n = 38, IL-4 n = 12, CD4+ proliferation n = 86, CD8+ proliferation n = 77; OBS: IFN-γ n = 12, IL-2 n = 11, IL-4 n = 6, CD4+ proliferation n = 33, CD8+ proliferation n = 34). Box plots represent the median, 25th and 75th percentile, with the minimum and maximum values; the outliers are not represented in the graphs. The McNemar’s and Wilcoxon signed-rank tests were applied. P-values assess the values of frequencies and intensity, respectively, up to 48 weeks after immunization versus baseline values.

Figure 4

CD4+, CD8+, B and NK cell numbers. Changes from baseline of (A, B) CD4+, (C, D) CD8+, (E, F) B and (G, H) NK cells at years 1, 2 and 3 in vaccinees (left panels) (n = 152 at year 1 ; n = 114 at year 2 ; n = 69 at year 3) and in OBS subjects (right panels) (n = 79 at year 1 ; n = 42 at year 2 ; n = 30 at year 3), respectively. Data are presented as mean values with standard error. A longitudinal analysis for repeated measurements was applied. P-values assess the values at year 1, 2 or 3 after immunization versus baseline values.

Figure 5

CD4+, CD8+, B and NK cell numbers stratified by antiretroviral regimens. Changes from baseline of (A, B) CD4+, (C, D) CD8+, (E, F) B and (G, H) NK cells at years 1, 2 and 3 according to antiretroviral regimens in vaccinees (left panels) (NNRTI- or NRTI-based n = 104 at year 1 ; n = 80 at year 2 ; n = 49 at year 3; PI-based n = 48 at year 1 ; n = 34 at year 2 ; n = 20 at year 3) and OBS subjects (right panels) (NNRTI- or NRTI-based n = 49 at year 1 ; n = 21 at year 2 ; n = 14 at year 3; PI-based n = 30 at year 1 ; n = 20 at year 2 ; n = 16 at year 3). Data are presented as mean values with standard error. A longitudinal analysis for repeated measurements was applied. P-values assess the values at year 1, 2 or 3 after immunization versus baseline values, both overall and stratified by drug regimens (NNRTI/NRTI- or PI-based).

Figure 6

CD4+T cells stratified by CD4+nadir and CD4+/CD8+T cell ratio. Changes from baseline of (A) CD4+ by CD4+ nadir and (B) CD4+/CD8+ T cell ratio at years 1, 2 and 3 in vaccinees (left panels) (n = 152 at year 1 ; n = 114 at year 2 ; n = 69 at year 3) and in OBS subjects (right panels) (n = 79 at year 1 ; n = 42 at year 2 ; n = 30 at year 3), respectively. Vaccinees with CD4+ T cell nadir ≤250 cells/μl: n = 32, >250 cells/μl n = 120; OBS subjects with CD4+ T cell nadir ≤250 cells/μl: n = 45 ; >250 cells/μl n = 34. Data are presented as mean values with standard error. A longitudinal analysis for repeated measurements was applied. P-values assess the values at year 1, 2 or 3 after immunization versus baseline values.

Figure 7

Naïve, central and effector memory CD4+and CD8+T cells. Changes from baseline of naïve, Tem and Tcm (A, B) CD4+ and (C, D) CD8+ T cell percentage at years 1, 2 and 3 for vaccinees (left panels) (n = 94 at year 1 ; n = 75 at year 2 ; n = 32 at year 3) and OBS subjects (right panels) (n = 32 at year 1 ; n = 20 at year 2). Data are presented as mean values with standard error. A longitudinal analysis for repeated measurements was applied. P-values assess the values at year 1, 2 or 3 after immunization versus baseline values.

Figure 8

Naïve, central and effector memory CD4+and CD8+T cells stratified by antiretroviral regimens. Changes from baseline of naïve, Tem and Tcm (A, B) CD4+ and (C, D) CD8+ T-cell percentage at years 1, 2 and 3 according to antiretroviral regimens in vaccinees (left panels) (NNRTI- or NRTI-based n = 59 at year 1 ; n = 49 at year 2 ; n = 21 at year 3; PI-based n = 35 at year 1 ; n = 26 at year 2 ; n = 11 at year 3) and OBS subjects (right panels) (NNRTI- or NRTI-based n = 19 at year 1 ; n = 9 at year 2; PI-based n = 13 at year 1 ; n = 11 at year 2). Data are presented as mean values with standard error. A longitudinal analysis for repeated measurements was applied. P-values assess the values at year 1, 2 or 3 after immunization versus baseline values, both overall and stratified by drug regimens (NNRTI/NRTI- or PI-based).

Figure 9

Blood HIV-1 DNA load (expressed as log10copies/μg DNA) stratified by antiretroviral regimens. (A) Changes of HIV-1 DNA (expressed as log10 copies/μg DNA) versus baseline according to antiretroviral regimens in vaccinees (NNRTI- or NRTI-based n = 102; PI-based n = 45) or OBS subjects (NNRTI- or NRTI-based n = 36; PI-based n = 26). (B) Changes of HIV-1 DNA versus baseline in vaccinees of each treatment group stratified according to ARV regimens (7.5 μg, 3x n = 38; 7.5 μg, 5x n = 37; 30 μg, 3x n = 37; 30 μg, 5x n = 35). The dotted lines represent the 99% confidence interval of HIV-1 DNA mean change from baseline (−0.21, 0.04 log10 copies/μg) in OBS subjects (all time points). In all panels data are presented as mean values with standard error. Median time of follow-up was 96 weeks for vaccinees and 120 weeks for OBS subjects. A longitudinal analysis for repeated measurements was applied. P-values assess the values at each week after immunization versus baseline values stratified by drug regimens (NNRTI/NRTI- or PI-based).

Figure 10

Blood HIV-1 DNA load (expressed as log10copies/106CD4+T cells) stratified by antiretroviral regimens. (A) Changes of HIV-1 DNA (expressed as log10 copies/106 CD4+ T cells) in vaccinees (NNRTI- or NRTI-based n = 97; PI-based n = 45) or OBS subjects (NNRTI- or NRTI-based n = 30; PI-based n = 24). (B) Changes of HIV-1 DNA in vaccinees of each treatment group stratified according to ARV regimens (7.5 μg, 3x n = 36; 7.5 μg, 5x n = 36; 30 μg, 3x n = 36; 30 μg, 5x n = 34). The dotted lines represent the 99% confidence interval of HIV-1 DNA mean change from baseline (−0.24, 0.02 log10 copies/106 CD4+ T cells) in OBS subjects (all time points). In all panels data are presented as mean values with standard error. Median time of follow-up was 96 weeks for vaccinees and 120 weeks for OBS subjects. A longitudinal analysis for repeated measurements was applied. P-values assess the values at each week after immunization versus baseline values stratified by drug regimens (NNRTI/NRTI- or PI-based).

Figure 11

HIV-1 DNA decay in individuals immunized with Tat at 30 μg, 3x or OBS subjects. (A) Longitudinal regression analysis of HIV-1 DNA (log10 copies/106 CD4+ T cells) using a random-effect regression model up to 144 weeks since the first immunization in vaccinees (Tat 30 μg, 3x, n = 37) or OBS subjects (n = 62). All longitudinal data were included in the analysis with a median of 108 weeks of follow-up in vaccinees and 120 weeks in OBS subjects, respectively. (B) Estimates of HIV-1 DNA decay based on the regression model after 1, 2 and 3 years since the first immunization in vaccinees (Tat 30 μg, 3x in red) and in OBS subjects (in blue). (C) Estimates of HIV-1 DNA annual decay in vaccinees immunized with Tat 30 μg, 3x stratified according to NNRTI- or NRTI-based (in blue, n = 25) or PI-based (in red, n = 12) regimens. Results in panels B and C are expressed as the percentage of HIV-1 DNA decay with 95% confidence interval (CI).

Figure 12

Neutralization of Tat-mediated Env entry in DC and HIV-1 DNA in vaccinees. (A) Relationship between Tat-mediated entry of trimeric Env in DC and anti-Tat IgM (blue), IgG (red) and IgA (violet) Ab binding titers in subjects immunized with Tat 30 μg, 3x (generalized estimating equations with adjustment for repeated measures in the same patient; longitudinal samples from 31 individuals). (B) Percentage of Env entry in DC in the presence or absence of Tat in subjects immunized with Tat 30 μg, 3x at 48 weeks since the first immunization (n = 32) (Student’s t-test). Data are expressed as mean changes from baseline with standard error. (C) Inhibition of Tat-mediated Env entry in DC by anti-Tat Ab positive (n = 20) and anti-Tat Ab negative (n = 12) sera at week 48 from subjects immunized with Tat 30 μg, 3x. Of the 32 subjects evaluated, 30 were positive for anti-Tat Abs before week 48. Data are expressed as the percentage of subjects showing neutralization of Tat-mediated Env entry at 48 weeks as compared to baseline. (Fisher's Exact test) (D) Pearson correlation between anti-Tat IgM and IgG Ab titers at 48 weeks and Tat-mediated Env entry in DC at 48 weeks versus baseline in subjects immunized with Tat 30 μg, 3x (n = 32). (E) Reduction from baseline of HIV-1 DNA (log10 copies/106 CD4+ T cells) over time in sera from vaccinees (Tat 30 μg, 3x) positive for neutralization (≥50%) of Env entry in DC at week 48 since vaccination (n = 20). Data in panels are presented as mean values with standard error. Changes from baseline of HIV-1 DNA in subjects with anti-Tat Abs and neutralizing activity were evaluated with a longitudinal analysis for repeated measurements. P-values assess the values at each week after immunization versus values at baseline.

Figure 13

CD38+HLA-DR+/CD8+T cells in individuals immunized with Tat at the 30 μg doses or in OBS subjects. Changes from baseline of CD38+HLA-DR+/CD8+ at years 1, 2 and 3 in (A) vaccinees of the Tat 30 μg, 3x group (year 1: n = 28; year 2: n = 20; year 3: n = 10) and the Tat 30 μg, 5x group (year 1: n = 28; year: 2 n = 19; year 3: n = 9); (B) OBS subjects (year 1: n = 38; year 2: n = 25). (C) Changes from baseline of CD38+HLA-DR+/CD8+ at week 8, 12, 20 and 48 in vaccinees of the Tat 30 μg, 3x group (n = 28); and the Tat 30 μg, 5x group (n = 28). Data are presented as mean values with standard error. A longitudinal analysis for repeated measurements was applied. P-values assess the values at year 1, 2, or 3 or week 8, 12, 20 or 48 after immunization versus baseline values.

Figure 14

NK cells in immunized with Tat at the 30 μg doses or in OBS subjects. Changes from baseline of NK cells/μl at years 1, 2 and 3 in (A) vaccinees of the Tat 30 μg, 3x (year 1 n = 37; year 2 n = 27; year 3: n = 18); and the Tat 30 μg, 5x group (year 1: n = 37; year 2: n = 26; year 3: n = 16); and (B) OBS subjects (year 1: n = 79; year 2: n = 42; year 3: n = 30). Data are presented as mean values with standard error. A longitudinal analysis for repeated measurements was applied. P-values assess the values at year 1, 2 or 3 after immunization versus baseline values.