Therapeutic immunization with HIV-1 Tat reduces immune activation and loss of regulatory T-cells and improves immune function in subjects on HAART

Abstract

Although HAART suppresses HIV replication, it is often unable to restore immune homeostasis. Consequently, non-AIDS-defining diseases are increasingly seen in treated individuals. This is attributed to persistent virus expression in reservoirs and to cell activation. Of note, in CD4(+) T cells and monocyte-macrophages of virologically-suppressed individuals, there is continued expression of multi-spliced transcripts encoding HIV regulatory proteins. Among them, Tat is essential for virus gene expression and replication, either in primary infection or for virus reactivation during HAART, when Tat is expressed, released extracellularly and exerts, on both the virus and the immune system, effects that contribute to disease maintenance. Here we report results of an ad hoc exploratory interim analysis (up to 48 weeks) on 87 virologically-suppressed HAART-treated individuals enrolled in a phase II randomized open-label multicentric clinical trial of therapeutic immunization with Tat (ISS T-002). Eighty-eight virologically-suppressed HAART-treated individuals, enrolled in a parallel prospective observational study at the same sites (ISS OBS T-002), served for intergroup comparison. Immunization with Tat was safe, induced durable immune responses, and modified the pattern of CD4(+) and CD8(+) cellular activation (CD38 and HLA-DR) together with reduction of biochemical activation markers and persistent increases of regulatory T cells. This was accompanied by a progressive increment of CD4(+) T cells and B cells with reduction of CD8(+) T cells and NK cells, which were independent from the type of antiretroviral regimen. Increase in central and effector memory and reduction in terminally-differentiated effector memory CD4(+) and CD8(+) T cells were accompanied by increases of CD4(+) and CD8(+) T cell responses against Env and recall antigens. Of note, more immune-compromised individuals experienced greater therapeutic effects. In contrast, these changes were opposite, absent or partial in the OBS population. These findings support the use of Tat immunization to intensify HAART efficacy and to restore immune homeostasis.

Trial registration: ClinicalTrials.gov NCT00751595.

Trial registration: ClinicalTrials.gov NCT00751595 NCT01024556.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Flow diagram of the study participants.

One hundred and forty-four HAART-treated patients were screened for enrollment. Of them, 87 met the inclusion criteria and were randomized on schedule and dose of immunization. This represents all the study population prior to the protocol amendment. All recruited individuals were included in the safety analysis (n = 87). Six subjects discontinued the immunization schedule. Of them, 5 were evaluated only for safety, since received at least one immunization, and 1 was evaluated also for immunogenicity since received 3 immunizations out of 5 (as indicated in the Protocol S1). A total of 82 individuals completed the 20-weeks period of the study and 68 have completed the 48-weeks period after the first immunizations.

Figure 2. Anti-Tat humoral immune responses.

(A) Percentage of subjects developing anti-Tat IgM (blue bar), IgG (purple bar), IgA (violet bar), or total anti-Tat Ab (green bar), stratified by Tat dose and treatment groups. Responder frequency up to week 20 among groups was analyzed by the Cochran-Armitage Trend test (p = 0.0117 and p = 0.0051 for IgM and IgA, respectively). Tat 7.5 µg, 3x, n = 21; Tat 7.5 µg, 5x, n = 18; Tat 30 µg, 3x, n = 21; Tat 30 µg, 5x, n = 22 subjects, respectively. (B) Percentage of subjects positive for anti-Tat Ab at week 48 (Tat 7.5 µg, 3x, n = 20; Tat 7.5 µg, 5x, n = 15; Tat 30 µg, 3x, n = 17; Tat 30 µg, 5x, n = 16 subjects, respectively). Responder frequency among groups was analyzed as described above (p = 0.0024 and p = 0.0048 for IgM and IgG, respectively, p = 0.0007 for total Ig). (C) Peak of anti-Tat IgM, IgG or IgA titers (geometric mean and range) up to 20 weeks after the first immunization from subjects positive for anti-Tat Ab (blue diamond Tat 7.5 µg and red square Tat 30 µg). Anti-Tat IgM: Tat 7.5 µg, n = 14; Tat 30 µg, n = 28. Anti-Tat IgG: Tat 7.5 µg, n = 26; Tat 30 µg, n = 33. Anti-Tat IgA: Tat 7.5 µg, n = 7; Tat 30 µg, n = 24 subjects, respectively. (D) Anti-Tat IgM, IgG or IgA titers at 48 weeks after the first immunization from subjects positive for anti-Tat Ab. Anti-Tat IgM: Tat 7.5 µg, n = 6; Tat 30 µg, n = 20. Anti-Tat IgG: Tat 7.5 µg, n = 8; Tat 30 µg, n = 18. Anti-Tat IgA: Tat 7.5 µg, n = 5; Tat 30 µg, n = 10 subjects, respectively.

Figure 3. Anti-Tat cellular immune responses.

(A) Percentage of subjects showing anti-Tat specific production of IFN-γ, IL-2 or IL-4, respectively, measured at baseline (blue bar) and up to week 48 after the first immunization (red bar) and stratified by Tat dose (Tat 7.5 µg, n = 35; Tat 30 µg, n = 33 subjects, respectively). (B) Percentage of subjects showing anti-Tat CD4+ or CD8+ lymphoproliferative responses measured at baseline and up to week 48 and stratified by Tat dose (Tat 7.5 µg, n = 31; Tat 30 µg, n = 31). The analysis was performed using the McNemar's test: *p<0.05, **p<0.01.

Figure 4. Expression of activation markers on CD8+ T cells after Tat immunization.

Changes from baseline of CD8+ T cells (gating on CD8+ T cells) expressing (A) CD38, (B) HLA-DR, or (C) both CD38 and HLA-DR. Results are shown according to Tat dose and time after the first immunization. Data are presented as the mean % changes (± standard error) at week 8, 12, 20 and 48. Blue bars: Tat 7.5 µg, n = 17 up to week 20 and n = 12 at week 48; red bars: Tat 30 µg, n = 21 up to week 20, n = 16 at week 48, respectively. The t-Test for paired data was used for the analyses: *p<0.05, **p<0.01. (D) Correlation between CD38+/CD8+ T cells (%) and anti-Tat IgA antibody titers (Multivariate regression model for repeated measures). Blue diamond: Tat 7.5 µg, n = 39; red square: Tat 30 µg, n = 43, respectively.

Figure 5. Expression of activation markers on CD4+ T cells after Tat immunization.

Changes from baseline of CD4+ T cells (gating on CD4+ T cells) expressing (A) CD38, (B) HLA-DR, or (C) both CD38 and HLA-DR. Results are shown according to Tat dose and time after the first immunization. Data are presented as the mean % changes (± standard error) at week 8, 12, 20 and 48. Blue bars: Tat 7.5 µg, n = 17 up to week 20 and n = 12 at week 48; red bars: Tat 30 µg, n = 20 up to week 20, n = 15 at week 48, respectively; The t-Test for paired data was used for the analyses: *p<0.05, **p<0.01.

Figure 6. Production of β2-microglobulin and neopterin after Tat immunization.

Changes from baseline, according to Tat dose, of (A) β2-microglobulin serum levels (mg/L) and (B) Neopterin (nmol/L). Blue bars: Tat 7.5 µg; red bars: Tat 30 µg. Data are presented as the mean changes (± standard error) at 4, 16 and 24 weeks (Tat 7.5 µg, n = 37, Tat 30 µg, n = 40 subjects, respectively). The t-Test for paired data was used for the analyses: **p<0.01.

Figure 7. Production of total Ig after Tat immunization.

Total IgM (A), IgG (B) and IgA (C) serum levels (mg/dL) are shown. Blue bars: Tat 7.5 µg, n = 37; red bars: Tat 30 µg, n = 40. Data are presented as the mean changes (± standard error). The t-Test for paired data was used for the analyses: *p<0.05, **p<0.01.

Figure 8. CD25 and FOXP3 expression on CD4+ T cells after Tat immunization.

(A) Changes from baseline of CD4+ lymphocytes expressing CD25 are shown according to Tat dose and time after the first immunization (Tat 7.5 µg, n = 32 up to week 20 and n = 24 at week 48; Tat 30 µg, n = 34 up to week 20 and n = 28 at week 48, respectively). (B) Changes from baseline of the percentage of CD4+CD25+ lymphocytes expressing FOXP3+ (Tat 7.5 µg, n = 31 up to week 20 and n = 23 at week 48; Tat 30 µg, n = 29 up to week 20 and n = 28 at week 48, respectively). (C) Changes from baseline of the percentage of CD4+ T cells expressing CD25+FOXP3+ (Tat 7.5 µg, n = 31 up to week 20 and n = 23 at week 48; Tat 30 µg, n = 29 up to week 20 and n = 24 at week 48). (D) Changes from baseline of the absolute number of CD4+ lymphocytes expressing CD25+ FOXP3+ (Tat 7.5 µg, n = 30 up to week 20 and n = 20 at week 48; Tat 30 µg, n = 29 up to week 20 and n = 22 at week 48, respectively). Blue bars: Tat 7.5 µg; red bars: Tat 30 µg. Data are presented as the mean changes (± standard error) evaluated at 8, 12, 20 and 48 weeks after the first immunization. The t-Test for paired data was used for the analyses: *p<0.05, **p<0.01.

Figure 9. Evaluation of PBMC viability, CD4+ T cell and B cell counts after Tat immunization.

(A) Changes from baseline of in vitro PBMC viability, stratified by Tat dose. Blue bars: Tat 7.5 µg, n = 35 up to week 20 and n = 32 at week 48; red bars: Tat 30 µg, n = 40 up to week 20 and n = 33 at week 48, respectively. (B) Changes from baseline of CD4+ T cells/µL (data from clinical sites), stratified by Tat dose. Blue bars: Tat 7.5 µg, n = 39 up to week 24 and n = 30 at week 48; red bars Tat 30 µg, n = 43 up to week 24 and n = 34 at week 48. (C) Changes from baseline of B cells/µL, stratified by Tat dose. Blue bars: Tat 7.5 µg, n = 38 up to week 20 and n = 30 at week 48; red bars Tat 30 µg, n = 40 up to week 20 and n = 30 at week 48, respectively. The t-Test for paired data was used for the analyses: *p<0.05, **p<0.01.

Figure 10. Evaluation of the percentage of CD4+, CD8+, NK and B cells in all Tat-immunized subjects and after stratification by Tat dose.

(A) Changes from baseline of CD4+, CD8+, NK and B cells (percentage) for all immunized subjects (n = 78 up to week 20, n = 60 at week 48). (B, C) Changes from baseline of CD4+, CD8+, NK and B cells (percentage), stratified by Tat doses. (B) Tat 7.5 µg, n = 38 up to week 20 and n = 30 at week 48; (C) Tat 30 µg, n = 40 up to week 20 and n = 30 at week 48, respectively. The t-Test for paired data was used for the analyses: *p<0.05, **p<0.01.

Figure 11. Evaluation of the percentage of CD4+, CD8+, NK and B cells in all Tat-immunized subjects after stratification by HAART regimens.

Changes from baseline of CD4+, CD8+, NK and B cells (percentage) from all immunized patients are shown for NNRTI-based (A) and PI-based (B) treatments. NNRTI-based: n = 51 up to week 20 and n = 41 at week 48; PI-based: n = 19 up to week 20 and n = 12 at week 48, respectively. The t-Test for paired data was used for the analyses: *p<0.05, **p<0.01.

Figure 12. Effect of Tat immunization on naïve, central and effector memory CD4+ and CD8+ T cells.

Percentage of naïve (CD45RA+/CD62L+), effector RA+ (CD45RA+/CD62L−, Temra) or RA- (CD45RA−/CD62L−, Temro) and central memory (CD45RA−/CD62L+, Tcm) CD4+ (A) or CD8+ (B) T cells at baseline and at week 8, 12, 20 and 48 after the first immunization for subjects immunized with both Tat doses (n = 18 up to week 20 and n = 10 at week 48). Asterisk indicates significant changes from baseline. The t-Test for paired data was used for the analyses: *p<0.05, **p<0.01.

Figure 13. Cellular immune responses against Env or recall antigens after Tat immunization.

Percentage of responders at baseline (blue bar) and up to week 48 (red bar) are stratified by Tat dose. Percentage of subjects showing (A) anti-Env production of IFN-γ, IL-2 and IL-4 (Tat 7.5 µg, n = 31; Tat 30 µg, n = 29) and (B) CD4+ or CD8+ lymphoproliferative responses (Tat 7.5 µg, n = 31; Tat 30 µg, n = 30); (C) anti-Candida cytokines production (Tat 7.5 µg, n = 32; Tat 30 µg, n = 29), and (D) CD4+ or CD8+ lymphoproliferative responses (Tat 7.5 µg, n = 31; Tat 30 µg, n = 29); (E) anti-CEF production of IFN-γ, IL-2 and IL-4 (Tat 7.5 µg, n = 34; Tat 30 µg, n = 32), and (F) CD4+ or CD8+ lymphoproliferative responses (Tat 7.5 µg, n = 31; Tat 30 µg, n = 29). The analysis was performed using the McNemar's test: *p<0.05, **p<0.01.

Figure 14. Correlation of the reduction of the percentage of CD38+/CD8+T cells with the increase of anti-Tat IgA antibody titers, CD8+ T central memory and anti-Tat specific IL-2 production after Tat immunization.

Multivariate regression model for repeated measures was applied to CD38+/CD8+ T cells (%), including as explicative factors anti-Tat antibody titers (IgM, IgG, IgA), CD8+ central memory (%) and anti-Tat IFN-γ, IL-2 and IL-4 cytokines, for immunized subjects (n = 70).