The ISS T-002 EF-UP (ClinicalTrials.gov NCT01024556) was an observational study to prospectively follow up the patients enrolled in the ISS T-002 clinical trial (ClinicalTrials.gov NCT02118168) (15, 33). Ninety-two volunteers (59% of the total vaccinees evaluated at trial end), previously immunized intradermally with the biologically active Tat at 7.5 or 30 μg doses (in the absence of adjuvants) given 3 × or 5 × 1 month apart, as by the ISS T-002 study protocol (15), were followed up for a total of 132 weeks. The ISS T-002 EF-UP study started on September 2013 and was closed on July 2016, when the participants had a median follow-up of 120 weeks (range 72–132). The primary endpoint of this study was to evaluate the persistence of the anti-Tat humoral immune response in vaccinees who had received at least 3 immunizations. Evaluations of immunological and virological disease biomarkers, including assessment of the immune recovery and virus reservoirs, were the secondary endpoints. Clinical and routine laboratory evaluations performed at each clinical site were also recorded. The immunological and virological assessments were scheduled about every 3 months, according to the timing of routine clinical monitoring, or, in the absence of signs of disease progression, every 6 months. The study was conducted in 8 Italian clinical sites (Policlinic of Modena, Modena; Arcispedale S. Anna, Ferrara; San Gallicano Dermatological Institute IRCCS, Rome; Policlinic of Bari, Bari; S. Raffaele Hospital, Milan; S. Maria Annunziata Hospital, Florence; Luigi Sacco Hospital, Milan; San Gerardo Hospital, Monza). All subjects signed an informed consent prior to enrollment. The study protocol was submitted and approved by the competent authorities (Director General of the Coordinating Clinical Center, S. Maria Annunziata Hospital, Florence) and by the Ethics Committees of each participating clinical center.

An HIV-1 B-clade Tat protein (GenBank accession no.: AAA44199.1) purchased from Diatheva (Fano, Italy) was used for anti-Tat Ab determinations. The protein was biologically active as determined by the rescue assay with HLM-1 cell line carrying a Tat-defective HIV provirus (1, 4) and/or by Tat uptake by monocyte-derived DC (MDDC) evaluated by intracellular staining for Tat in flow cytometry (36), a potency test that is used to release the Tat vaccine clinical lots.

Serum IgM, IgA, and IgG against B-clade Tat were assessed by enzyme-linked immunosorbent assay (ELISA), as previously described (35, 37). Briefly, 96-well microplates (Nunc-Immuno Plate MaxiSorp Surface; Nunc) were coated with Tat (100 ng/well) in 200 μL of 0.05 mol/L carbonate-buffer (pH 9.6), and incubated overnight at 4°C. Wells were washed 5 times with phosphate-buffered saline (PBS), pH 7.4, containing 0.05% Tween-20, by an automatic plate washer (Asys Hitech flexi wash). Wells were then saturated with PBS containing 1% bovine serum albumin (BSA) and 0.05% Tween-20 (Sigma) (blocking buffer) for 90 min at 37°C and then washed again as above. One hundred μL of patient serum samples diluted in blocking buffer at 1:100 for anti-Tat IgG detection or at 1:25 for anti-Tat IgM or IgA detection, were added to the wells and incubated at 37°C for 90 min. To correct for binding background, each sample was assessed in duplicate on Tat-coated wells and singly on the wells coated with Tat resuspension buffer. After washing, wells were saturated again with blocking buffer for 15 min at 37°C, washed again and then a goat anti-human IgG, IgM, or IgA horseradish peroxidase-conjugated secondary Ab (100 μL/well) (PIERCE-Thermo Scientific) was added to each well, and incubated for an additional 90 min at 37°C. Antigen-bound Abs were revealed by the addition of ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) solution (Roche Diagnostics) for 60 min at 37°C. Absorbance was measured at 405 nm using a microplate reader (BIO-TEK Instruments EL800). The assay was considered valid only when both the positive and negative controls were within ±10% of variation of the absorbance values recorded in previous 50 assays. For the cut-off calculation, both the optical density (OD) readings at 405 nm of the wells coated with Tat and the delta (Δ) value were utilized. The Δ value was obtained by subtracting the OD reading of the well coated with the buffer alone from the arithmetic mean of the OD values of the two wells coated with the Tat protein. Serum samples were considered positive when the sample OD at 405 nm and Δ values were ≥0.350 and ≥0.150, respectively, as previously described (37). Ab titers ≥25 for IgM and IgA, or ≥100 for IgG were considered positive.

Peripheral blood CD4⁺ and CD8⁺ T-cell counts were determined at the clinical sites by flow cytometry, using common Standard Operating Procedures (SOPs) according to standard national laboratory measurements.

HIV-1 DNA quantification was performed by SYBR green real-time polymerase chain reaction (qPCR), as described (33, 38). DNA was obtained from 1.6 mL of peripheral blood. After incubation for 45 min at 37°C in a lysis buffer (sodium dodecyl sulfate 5%, 8 M Urea, 0.3 M NaCl, 10 mM Tris–HCl pH 7.5, 10 mM EDTA pH 8.0), DNA was purified by phenol extraction followed by ethanol precipitation and RNase treatment, and amount and quality evaluated with a NanoDrop ND-1000 Spectrophotometer by assessing the ratio of absorbance at 260 nm and 280 nm (NanoDrop Technologies, Wilmington, DE, US). Real-time PCR was performed using the HIV-1 DNA quantitative PCR kit (Diatheva s.r.l., Fano, Italy), on a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, US). Each sample was analyzed in triplicate. Forward and reverse primers (5′-TAGCAGTGGCGCCCGA-3′ and 5′-TCTCTCTCCTTCTAGCCTCCGC-3′, respectively) were designed to amplify a 161 bp fragment derived from the 5′ long terminal repeat (LTR) U5 end to Gag-Pol start sequence of HIV-1 (GenBank accession no. AF003888). Primer specificity for HIV-1 group M was confirmed both in silico using BLAST (https://www.hiv.lanl.gov/content/sequence/HIV/COMPENDIUM/2012compendium.html), and by real-time PCR with different HIV-1 subtypes (B, C, F, CRF 01_AE, and CRF 02_AG), the reference strains A, D, H, and the complete DNA sequence of HIV-2 ROD (EU Programme EVA Centralized Facility for AIDS Reagents, NIBSC, UK) (38). Cross-reactivity with endogenous retroviral sequences was excluded by testing 150 HIV-1 negative blood donors (38). HIV-1 DNA copy number was estimated as described (38) using a standard curve comprising a 10-fold serial dilutions (10⁵ to 10¹) and 2 copies dilutions of a plasmid containing the 161 bp HIV target region, including the Primer Binding Sites (PBS plasmid). The standard curve was considered valid when the slope was between −3.50 and −3.32 (93–100% efficiency) and the minimum value of the coefficient of correlation (R²) was 0.98. The limit of quantification was 2 copies per μg of DNA, with a detection limit of 1 copy and a dynamic range of quantification of 5 orders of magnitude (10⁵ to 10¹). The reproducibility, assessed by calculating the mean coefficient of variation (CV%) for the threshold cycle (Ct) values, was determined as 1.4%, confirming quantification in the dynamic range. Results were expressed as log₁₀ copies/10⁶ CD4⁺ T cells, calculated as the ratio between copies/μg DNA and the CD4⁺ T-cell number present in 1.5 × 10⁵ white blood cells (WBC) using the following formula: [(copies/μg DNA)/(CD4⁺/WBC) × 150,000 WBC] × 10⁶ (33).

The HIV-1 viral load (VL) in the plasma of HIV-1-infected patients was quantitatively determined using a standardized RT-PCR (AmpliPrep/COBAS® TaqMan® HIV-1 Test, version 2.0; Roche Diagnostics) that gives a linear response from 20 to 10,000,000 HIV-1 RNA copies/mL. According to manufacturer's instructions Ct values above the quantitation limit or absence of Ct were both categorized as “undetectable VL.”

The lot-specific calibration constants provided with the COBAS® AmpliPrep/COBAS® TaqMan® HIV-1 Test were used, with the Amplilink software, to calculate the titer value for the specimens and controls below the limit of detection (≥95%) of the assay (i.e., between 1 and 20 copies/mL), based upon the HIV-1 RNA and HIV-1 Quantitation Standard (QS) RNA Ct values.

Descriptive statistics summarizing quantitative variables included mean, standard deviation, minimum and maximum; qualitative variables were presented as number and percentage. Kaplan-Meier method was used to assess the cumulative probability of anti-Tat Ab persistence in responding participants, by vaccine regimen, and compared by the Log-Rank test. Subjects who developed anti-Tat Abs within 24 weeks of the ISS T-002 trial were defined “responders.”

Longitudinal analysis for repeated measurements (including multiple measurements per year) was used to evaluate changes from baseline of CD4⁺ and CD8⁺ T-cell number, CD4⁺/CD8⁺ T-cell ratio and HIV-1 DNA load at each year of follow up, both overall and upon stratification.

Multivariate analysis of variance for repeated measures was used in order to evaluate the effect of anti-Tat humoral responses, vaccine treatment groups, residual viremia/viral blips, CD8⁺ T-cell counts, and cART on changes from baseline of CD4⁺ T-cell counts and HIV-1 DNA load.

A longitudinal regression analysis was performed using a random-effects regression model in order to evaluate CD4⁺ T-cell number and HIV-1 DNA load over time stratified by vaccine regimen, CD4⁺ or HIV-1 DNA quartiles at baseline.

To estimate the decay rate of HIV-1 DNA, a longitudinal analysis using a random-effects regression model was performed to determine the slope of the decay from a plot of log₁₀ DNA (copies/10⁶ CD4⁺ T cells) vs. time in days; the use of log plot assumes a first order kinetic decay (26).

Analysis of covariance (ANCOVA) was used to take into account the reversion to the mean (RTM) when patients were categorized for baseline CD4⁺ T-cell counts or proviral DNA quartiles (39). For this analysis, the CD4⁺ T-cell or proviral DNA measurements done at ISS T-002 baseline and at the last follow-up visit attended were used. Baseline CD4⁺ T-cell numbers or log₁₀ proviral DNA copies per million CD4⁺ T cells and quartiles were the model covariates.

A generalized estimating equation with adjustment for repeated measures in the same volunteers was performed in order to evaluate relationship between CD4⁺/CD8⁺ T-cell ratio, CD4⁺ T-cell number, CD8⁺ T-cell number and HIV-1 DNA load.

The Spearman Correlation Coefficient was used in order to test the correlation between CD4⁺/CD8⁺ T-cell ratio, CD4⁺ T cells, CD8⁺ T cells, and HIV-1 DNA load.

Statistical analyses were carried out at two-sided with a 0.05 significance level, using SAS® (Version 9.4, SAS Institute Inc., Cary, NC, USA) and STATA^tm version 8.2 (Stata Corporation, College Station, Texas, United States).

Ninety-three volunteers out of the 155 participants evaluated for the ISS T-002 trial agreed to enter the extended follow-up study (Supplementary Figure 1). One patient was excluded from all the analyses because of persistent non-compliance with cART. Five patients dropped out of the study: 1 after 24 weeks (discontinued cART), 1 after 36 weeks (relocated), 1 after 48 weeks (not compliant to study visits), 1 after 48 weeks (death due to lung tumor) and 1 after 84 weeks (lost to follow-up). Table 1 reports the baseline characteristics of the ISS T-002 EF-UP study participants and the comparison with ISS T-002 baseline. Seventy-seven out of the 92 evaluable volunteers were male (83.7%). With regard to vaccine regimen, 21 volunteers had received Tat at 7.5 μg, 3 × ; 20 Tat at 7.5 μg, 5 × ; 27 Tat at 30 μg, 3 × ; and 24 Tat at 30 μg, 5 × (Table 1). Seventy-eight percent of the patients had a CD4⁺ T-cell nadir >250 cells/μL. Volunteers entered the study at different time periods after the first immunization (median time 200 weeks) with a range comprised between week 144 and 304 (the week at study entry was taken as week 0 of the extended follow-up protocol). They were followed-up for a median of 120 weeks (range 24–132). The median time of follow-up since the first immunization was 316 weeks (range 185–412). At ISS T-002 EF-UP study entry, volunteers were in cART for a mean of 10 years.

Analysis of humoral responses indicated that anti-Tat Abs persisted for the entire period of follow-up in a high proportion of participants, particularly in the Tat 30 μg regimens (49% of the vaccinees) (Figure 1). In fact, as compared to the 7.5 μg regimens, volunteers vaccinated with the 30 μg Tat regimens showed persistent anti-Tat IgG and IgA Abs (log-rank Test p = 0.0179 and 0.0128, respectively, data not shown) and those who developed anti-Tat Abs of all 3 classes were more likely to maintain the Ab response to Tat (at least 1 class) [66 vs. 23% (2 classes) and 27% (1 class), Log-Rank Test p = 0.0007] (Supplementary Figure 2).

Analysis of immunological parameters of the 92 vaccinees was performed by years of follow-up, starting from the ISS T-002 study entry, that is, from year 1 to 8.

The statistically significant increase of CD4⁺ T-cell counts recorded since year 2 continued over time, reaching levels higher than 100 cells/μL over baseline values since year 5, gains which were maintained through year 8 (Figure 2A).

Increases of CD4⁺ T-cells were similar with non-nucleoside reverse transcriptase (NNRTI)/nucleoside reverse transcriptase (NRTI)- or PI-based treatments (data not shown).

Interestingly, subjects with CD4⁺ T-cell nadir ≤250 cells/μL had also statistically significant CD4⁺ T-cell increases from baseline, which were equal or even greater than those observed in vaccinees with a higher nadir (Supplementary Figure 3). This is of clinical relevance, since subjects with a nadir ≤250 cells/μL are known to respond insufficiently to cART (40, 41) or vaccination (42).

CD8⁺ T-cell levels remained stable throughout the 8 years of follow-up (Figure 2B). The increase of CD4⁺ T-cell counts and the maintenance of CD8⁺ T-cell number after vaccination resulted in a statistically significant increase of the CD4⁺/CD8⁺ T-cell ratio by year 4, which persisted through year 8 of follow-up (Figure 2C).

When the immunization regimen was considered, the greatest effects of Tat vaccination were observed with the Tat 30 μg, 3 ×, with increases of CD4⁺ T cells up to 165 cells/μL (Figure 3A), substantially stable levels of CD8⁺ T cells (Figure 3B), and significant increases of the CD4⁺/CD8⁺ T-cell ratio over time (Figure 3C).

In order to better evaluate Tat vaccine dose-effects on the increase of CD4⁺ T cells, a longitudinal regression analysis using a random-effect regression model was used for the four vaccine regimens. This analysis showed statistically significant increases of CD4⁺ T cells for all regimens with the highest increase in the two Tat 30 μg regimens, while CD4⁺ T-cell gains in the two Tat 7.5 μg regimens were more modest (Supplementary Figure 4 and Supplementary Data Sheet 1).

HIV proviral DNA in peripheral CD4⁺ T cells was evaluated longitudinally over the whole follow-up period. As shown in Figure 4A, HIV-1 DNA levels continued to decrease over time with changes from baseline becoming statistical significant from year 3, reaching a −82% mean reduction (−0.72 log₁₀ copies/10⁶ CD4⁺ T cells) at year 8 as compared to study entry. Although all vaccine regimens had very similar baseline HIV-1 DNA mean values, the Tat 30 μg, 3 × regimen was the most effective at reducing proviral DNA load, reaching at year 8 a decrease of −92% (−1.1 log₁₀ copies/10⁶ CD4⁺ T cells), followed by the Tat 30 μg, 5 × that showed a decrease of −79% (−0.7 log₁₀ copies/10⁶ CD4⁺ T cells), Tat 7.5 μg, 3 ×, which had very similar kinetics of proviral decay reaching at year 8 a decrease of −76% (−0.6 log₁₀ copies/10⁶ CD4⁺ T cells), while Tat 7.5 μg, 5 × was the least effective with a proviral DNA reduction of −70% (−0.5 log₁₀ copies/10⁶ CD4⁺ T cells, at year 8) (Figure 4B).

Moreover, although in the first 3 years of follow-up a significant reduction of HIV-1 DNA was seen only in vaccinees under PI-based regimens (33), since year 4 of follow-up both NNRTI/NRTI- and PI-based cART regimens were associated with similar, persistent and statistically significant reductions of proviral DNA (Supplementary Figure 5).

These results were confirmed in a longitudinal regression analysis using the random-effects regression model after stratification of the results according to the vaccine regimen. A marked reduction of HIV proviral DNA was again observed in all vaccine regimens, with the greatest decrease in the Tat 30 μg, 3 ×, followed by Tat 7.5 μg, 3 × and Tat 30 μg, 5 ×, while Tat 7.5 μg, 5 × showed the smallest HIV proviral DNA decay (Supplementary Figure 6 and Supplementary Data Sheet 1).

The random-effects regression model was then used to estimate the rate of HIV-1 DNA decay. As shown in Figure 5, a HIV-1 DNA half-life (t_1/2) of 3 years and a 90% reduction of the DNA reservoir in 10 years were estimated for the whole study population. However, these estimates varied markedly according to the vaccine regimen. In fact, t_1/2 and time to 90% reduction were shortest for the Tat 30 μg, 3 × group (t_1/2 = 2 years; 90% reduction = 8 years), followed by Tat 7.5 μg, 3 × (t_1/2 = 3 years; 90% reduction = 10 years), Tat 30 μg, 5 × (t_1/2 = 3 years; 90% reduction = 10 years), and Tat 7.5 μg, 5 × (t_1/2 = 4 years; 90% reduction = 15 years) (Figure 5).

The time required for proviral DNA eradication was calculated assuming the average frequency of lymphocytes harboring proviral DNA to be about 300/10⁶ (43) in a background of 10¹² total body lymphocytes (44). Accordingly, the eradication times were estimated to be 83 years for the whole population, and 66, 77, 83, 122 years for the 30 μg, 3 ×; 7.5 μg, 3 ×; 30 μg, 5 × and 7.5 μg, 5 × group, respectively (Figure 5).

The transient presence of detectable plasma VL in otherwise virologically suppressed individuals on ART appears to be associated with a slower decay of HIV proviral DNA (45–47). We therefore examined the relationship between the frequency and the level of intermittent residual viremia/viral blips and proviral DNA decay. To this purpose, regression analyses were made in vaccinees grouped by either the frequency of visits with complete virological suppression (<90 or ≥90% of visits with VL = 0) or by VL cut-off intervals (0; 1–40; 41–99; ≥100 RNA copies/mL) (see also Supplementary Table 1). A faster HIV DNA decay with a shorter proviral DNA half-life, time to 90% reduction and time to eradication, was observed in vaccinees with VL = 0 in ≥90% of visits as compared to <90% of visits (Figure 6A and Supplementary Data Sheet 1). When vaccinees were stratified according to VL cut-off classes, the fastest HIV DNA decay was observed in patients in the lowest VL class (VL = 0), whereas the slowest kinetics were observed in vaccinees experiencing blips above 100 RNA copies/mL; intermediate kinetics of DNA decay were observed in vaccinees with intermediate viremic measurements (Figure 6B and Supplementary Data Sheet 1). These data suggested replenishment of the HIV-1 proviral DNA reservoir by residual viremia and/or viral blips.

To evaluate whether the individual immunological status or size of latent HIV reservoir of the volunteers might have affected the response to Tat vaccination, the kinetics of CD4⁺ T-cell increases or proviral DNA decays were evaluated upon stratification by quartiles according to CD4⁺ T-cell number or proviral DNA at ISS T-002 study entry. The data were then analyzed by three models including (i) changes from baseline upon time by a longitudinal analysis for repeated measurements, (ii) variations of absolute CD4⁺ T-cell numbers or proviral DNA copies upon time by a random-effects linear regression model, and (iii) single paired pre-post vaccination measures by ANCOVA, to take into account the RTM effect.

The analysis of CD4⁺ T-cell changes from baseline over time showed that subjects with the lowest CD4⁺ T-cell counts at baseline (Q1: CD4⁺ T cells <493/μL) experienced very early (year 1) and statistically significant increases of CD4⁺ T cells, followed by those in Q2 (CD4⁺ T cells 493–600/μL) and in Q3 (CD4⁺ T cells: 601–734/μL) in whom statistically significant increases of CD4⁺ T cells were recorded since year 2 of follow-up, while the increase observed in Q4 (CD4⁺ T cells >734/μL) were not statistically significant (Figure 7).

The analysis of absolute CD4⁺ T-cell kinetics by longitudinal regression analysis showed slightly different results, with significant increases of CD4⁺ T cells for all quartiles, including Q4, although the greatest increases were observed in Q1, followed by Q2 and Q3 as compared to Q4 (Supplementary Figure 7 and Supplementary Data Sheet 1). In particular, Q1 showed the highest CD4⁺ T-cell increase, with the 95% CI of the Q1 regression curve transitioning into the 95% CI of the Q2 curve, suggesting positive effects of Tat vaccination in poor immunological responders.

When the RTM effect was taken into account by ANCOVA, the post-vaccination means of CD4⁺ T cells were not found to be significantly different among quartiles (data not shown), indicating that Tat vaccination has similar effects irrespective of the CD4⁺ T-cell number at study entry.

Similar but inverse relationships among quartiles were observed for proviral DNA decay. When proviral DNA kinetics were analyzed as changes from baseline over time, vaccinees with the highest proviral DNA load at study entry (Q4) experienced the earliest (year 1), progressive and most pronounced DNA load reduction (from −0.4 to −1.3 HIV-1 DNA log₁₀ copies/10⁶ CD4⁺ T-cells), followed by vaccinees in Q3 and Q2, in whom DNA reduction was less pronounced and became significant only since year 4; significant proviral DNA load increases were observed in Q1 from year 1 to 3, while reductions thereafter were not significant (Figure 8).

Conversely, when absolute proviral DNA copies stratified by baseline quartiles were analyzed by the random-effects regression model, a significant decay was observed in all quartiles including Q1, with the greatest decay for Q4 followed by Q3, while Q2 and Q1 showed the lowest proviral decay (Supplementary Figure 8 and Supplementary Data Sheet 1).

Finally, when the RTM effect was taken into account by ANCOVA, the post-vaccination means of proviral DNA (log₁₀) copies were not found to be significantly different among quartiles (data not shown), indicating similar effects of vaccination irrespective of the size of the reservoir at study entry.

The CD4⁺/CD8⁺ T-cell ratio has been indicated to correlate inversely with the HIV reservoir size (48, 49). Therefore, analyses were made to verify the presence of a correlation between proviral DNA decay and the CD4⁺/CD8⁺ T-cell ratio, CD4⁺ or CD8⁺ T-cell counts. A significant inverse relationship was observed between the variations over time of HIV proviral DNA and the CD4⁺/CD8⁺ T-cell ratio (both measured as changes from baseline; β = −0.3385; p = 0.0246) using a generalized estimating equation (GEE) with adjustment for repeated measures (Figure 9).

An inverse correlation was also observed with CD4⁺ T-cell changes from baseline (Spearmen correlation coefficient = −0.11034; p = 0.0005), although the GEE analysis did not reach statistical significance (Supplementary Figure 9A).

In contrast, a significant direct relationship between HIV proviral DNA and CD8⁺ T-cell changes from baseline (β = 0.0002; p = 0.0166) was revealed by the GEE analysis (Supplementary Figure 9B).

We next investigated the effect of anti-Tat humoral responses, vaccine treatment regimens, residual viremia/viral blips, cART, and CD8⁺ T-cells on the kinetics of CD4⁺ T-cell counts or HIV-1 DNA decay by a multivariate regression model, in which the effect of these variables were evaluated on the changes from baseline of CD4⁺ T-cell counts and HIV-1 DNA copy number (Tables 2, 3). For this analysis, VL was categorized in two groups according to the frequency of visits with undetectable viremia (i.e., ≥90 or <90% with VL = 0, see Figure 6A).

A statistically significant increase of CD4⁺ T-cell counts was observed at year 2 and then from year 4 to 6 post-vaccination in the subgroup of patients who developed anti-Tat Abs for all 3 classes (IgM, IgG, and IgA). A significant increase of the CD4⁺ T-cell counts was also found by year 4 to 6 post-vaccination in patients who developed only 2 anti-Tat Ab classes. In the patients who mounted only 1 Ab class (IgG or IGM or IgA), the increase of the CD4⁺ T-cell counts was significant only by year 5 and 6 post-vaccination. In the subgroup that did not develop anti-Tat Ab after vaccination, the CD4⁺ T-cell increase was, nonetheless, statistically significant from year 5 until year 8 (Table 2). As discussed later, these patients developed anti-Tat cellular responses after vaccination.

When the different vaccine regimens were taken into consideration, Tat 30 μg, 3 × was the most effective, inducing early (year 1) statistically significant and durable CD4⁺ T-cell increases, followed by 30 μg 5 ×, and 7.5 μg, 5 × that were similarly effective, but with significant increases only since year 4 or 5 (Table 2), whereas the 7.5 μg, 3 × vaccine regimen was the least effective, showing a statistically significant CD4⁺ T-cell increase only at year 5.

The evaluation of residual viremia/viral blips indicated that vaccinees with VL = 0 in more than 90% visits experienced CD4⁺ T-cell increases that became significant at years 4, 5, and 6, and were higher as compared to vaccinees with VL = 0 in <90% visits, who had, however, a significant CD4⁺ T-cell increase at year 2 and then from year 4 to 8 post-vaccination (Table 2).

Finally, no statistically significant differences were observed between PI-based and NNRTI-based regimens on CD4⁺ T-cell counts (Table 2).

Concerning HIV DNA decay, as shown in Table 3, the earliest, most pronounced, durable and statistically significant HIV-1 DNA reduction occurred in vaccinees with all or at least 2 classes of anti-Tat Abs, starting at year 1 or 3 after vaccination; in contrast, in vaccinees positive for a single anti-Tat Ab class or negative for all Ab classes the decrease was less prominent but remained statistically significant from year 6 to 8 post-vaccination (Table 3).

When the vaccine regimens were taken into consideration, Tat 30 μg, 3 × was again the most effective, showing the most pronounced and durable HIV-1 DNA decrease since year 3 post-vaccination, although vaccinees in the other regimens also showed durable, but less pronounced, and significant decreases with early decay kinetics (since year 1 or 2)(Table 3).

When residual viremia/viral blips were evaluated, vaccinees with VL = 0 in more than 90% visits experienced a pronounced HIV DNA decay starting from year 4, whereas in vaccinees with VL = 0 in <90% visits, DNA decay occurred earlier (year 2) but was much less prominent (Table 3). Moreover, CD8⁺ T-cell changes from baseline were significantly and directly related to proviral DNA variations (Table 3).

Finally, non-statistically significant differences were observed between PI-based and NNRTI-based regimens, both showing a significant and similar HIV proviral DNA reduction in association with Tat vaccination (Table 3).

MuM hold shares in Diatheva SrL. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.