Immunization with an HIV-1 immunogen induces CD4+ and CD8+ HIV-1-specific polyfunctional responses in patients with chronic HIV-1 infection receiving antiretroviral therapy

Vaccine (2008) 26, 2738—2745

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Immunization with an HIV-1 immunogen induces CD4+ and CD8+ HIV-1-specific polyfunctional responses in patients with chronic HIV-1 infection receiving antiretroviral therapy L. Valor a,1, J. Navarro a,1, J. Carbone a,1, C. Rodr´ıguez-S´ ainz a,1, opez b, E. Fern´ andez-Cruz a,∗,1 , J. Gil a,1, F. L´ for STIR-2102 Team a b

´n’’, Madrid, Spain Division of Immunology, University General Hospital ‘‘Gregorio Mara˜ no Nufarm 21, Madrid, Spain

Received 7 December 2007; received in revised form 29 February 2008; accepted 12 March 2008 Available online 1 April 2008

KEYWORDS HIV-1 immunogen; CD4+ and CD8+ HIV-1-specific lymphoproliferative responses; Cytotoxic T lymphocyte (CTL) precursors; Therapeutic vaccination

Summary Development of polyfunctional T lymphocyte responses is critical in the immunological response against HIV-1. Fifty-four HIV-1 infected patients receiving antiretroviral treatment (ART) and immunization with an HIV-1 immunogen or placebo, periodically every 3 months throughout a period of 36 months, were evaluated for the purposes of analysing the development of HIV-1-specific CD4+ and CD8+ responses. A significant increase of proliferating and IFN-␥ producing CD8+ HIV-1-specific T cells, of HIV-1-specific precursor frequencies for CD8+ and for CD4+ T cells and of Gag/pol-specific memory CTL precursors (CTLp) was observed in the immunogen group in comparison to placebo. IL-2 intracellular expression and IFN-␥ and TNF-␣ co-expression in HIV-1-specific CD8+ T cells were also substantially increased in the immunized group. A negative correlation between viral load and CD3+ CD4+ CFSElow HIV-1-specific lymphoproliferative response and frequency of Gag/pol-specific CTLp was solely observed in the HIV-1 immunogen group. Long-term immunization in patients receiving ART helps to develop HIV-1-specific polyfunctional T cell responses. © 2008 Elsevier Ltd. All rights reserved.

∗ Corresponding author at: Division of Immunology, University General Hospital ‘‘Gregorio Mara˜ ´n’’, Doctor Esquerdo, 46, 28007 Madrid, no Spain. Tel.: +34 91 5868423; fax: +34 91 5866698. E-mail address: [email protected] (E. Fern´ andez-Cruz). 1 See Appendix A.

0264-410X/$ — see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2008.03.019

CD4+ and CD8+ HIV-1 responses induced by an HIV-1 immunogen

Introduction In the natural course of HIV-1 infection there is a gradual increase in HIV-1 RNA viremia jointly with an impairment on functional HIV-1-specific CD4+ and CD8+ T cell responses that ultimately leads to a state of immunodeficiency and disease progression in HIV-1 infected individuals. Recent studies suggest that the induction of HIV-1-specific CD4+ T helper cells and polyfunctional CD8+ T cells could play a role in controlling HIV-1 replication and spread in patients with HIV-1 infection [1—4]. Several mechanisms have been associated with the CD8+ T cells driven antiviral effect, such as the ability to eliminate HIV-1 infected cells by cytotoxic T cells (CTL) and the production of cytokines (IFN-␥, TNF-␣, IL-2) and ␤-chemokines (MIP-1␣, MIP-1␤ and RANTES) with antiviral activity [5—8]. Currently, we are lacking consistent evidence that antiretroviral therapy is able to induce and maintain in the long-term HIV-1-specific immunological responses in patients with HIV-1 chronic infection. During the last decade, new approaches such as immunization with different modalities of therapeutic vaccines have been evaluated in patients with HIV-1 chronic infection to assess their ability to induce sustained anti-HIV-1-specific immunological responses [9—11]. We have previously reported sustained augmentation of HIV-1-specific T cell proliferation in patients with HIV-1 chronic infection receiving long-term antiretroviral treatment (ART) in combination with an HIV-1 virion-based therapeutic vaccine (STIR-2102 study) [12,13]. Few studies have evaluated both CD4+ and polyfunctional CD8+ T cell responses after long-term therapeutic vaccination [14—16]. The aim of the present study was to evaluate in 54 patients who had participated in study STIR-2102, the effect of long-term immunization with an HIV-1 immunogen to induce HIV-1-specific CD4+ and polyfunctional CD8+ T cell responses.

Materials and methods

2739 value 2.30 log10 copies/ml equals undetectable viral load (199 copies/ml) as defined in protocol of STIR-2102 [12]. The pre-ART VL baseline of the substudy’s population (n = 54) and the pre-ART VL baseline of the total population (n = 243) of patients from STIR-2102 were equivalent [baseline pre-ART VL of total population, median (range): 4.15 (2.30—5.72) log10 copies/ml vs. substudy population: 3.95 (2.30—5.78) log10 copies/ml. Median difference (95% CI): 0.06 (−0.17—0.32); Wilcoxon rank (Mann—Whitney) p = 0.575]. The pre-ART VL baseline of IFA group, median (range): 4.35 (2.30—4.88) log10 copies/ml was not significant different from INM group: 4.06 (3.27—5.00) log10 copies/ml. Median difference (95% CI): 0.05 (−0.47 to 0.59); Wilcoxon rank-test, p = 0.290. In addition, the pre-immunization VL baseline for both treatment groups (IFA vs. IMN) were equivalent: median (range) 2.30 (2.30—3.28) log10 copies/ml vs. 2.30 (2.30—3.76) log10 copies/ml. Median difference (95% CI): 0 (0—0); Wilcoxon rank-test, p = 0.99. Post-immunization VL for IFA group was: median (range) 2.30 (2.30—3.99) log10 copies/ml, and for IMN group: median (range) 2.30 (2.30—3.85) log10 copies/ml. Median difference (95% CI): 0 (0—0.14); Wilcoxon rank-test, p = 0.242. Peripheral blood mononuclear cells (PBMC) from HIV-1 infected adults were evaluated retrospectively at baseline (pre-immunization) and at month (m) 36 post-immunization using distinct functional immunological assays. Prior to the commencement of the study, Institutional Review Board approval by each participant hospital was obtained. Furthermore, informed consents from all participants jointly with the review and approval of the protocol by the Spanish Agency of Medicament and Sanitary Products were also obtained prior to the initiation of the study STIR-2102.

HIV-1-specific CD8+ and CD4+ T cells proliferation by CFSE (5,6 carboxyfluorescein-diacetate-succinimidyl-ester) loading assay

Study design and selected population Fifty-four HIV-1 chronically infected adults who had participated in the STIR-2102 trial have been subject to retrospective analysis for the evaluation of CD4+ and CD8+ HIV-1-specific polyfunctional responses [12]. All the patients included in this study were recruited from four Hospitals in Madrid participating in STIR-2102 trial. STIR-2102 is a multicenter, randomized, double-blind, placebo-controlled phase II clinical trial of HIV-1 immunogen (Remune) plus ART vs. ART alone in asymptomatic, ART na¨ıve, HIV-1 infected subjects with CD4+ T cell counts between 300 and 700 × 106 l−1 . At week-6, prior to randomization, na¨ıve patients started ART (baseline pre-ART) and at day 0 (baseline pre-immunization) patients were randomized in equal proportions to either the HIV-1 immunogen or IFA arm [12]. HIV-1 immunogen (IMN), an inactivated gp120 depleted HIV-1 virion in Incomplete Freund’s Adjuvant (IFA), or placebo (IFA) was administered intramuscularly every 3 months throughout a period of 36 months of ART [12,13]. Plasma levels of HIV-1 RNA (VL) were determined using the Amplicor assay (Hoffmann La Roche, Nutley, NJ). The

We have previously evaluated the lymphoproliferative responses (LPR) to HIV-1 antigens (Gp120 depleted HIV-1HZ321 antigen clade A envelope and clade G gag HIV-1 Immunogen; IRC) by the classical 3 H-thymidine incorporation assay, and we have found that LPR was significantly enhanced in the HIV-1 immunogen group compared to the IFA group. Strong LPR was clearly established after approximately 15—18 months post-immunization and was maintained up to month 36 [12]. In this study, we have further evaluated the in vitro lymphoproliferative response of PBMC in 54 patients, by examining the percentages of proliferating CD4+ and CD8+ HIV-1-specific T cells by CFSE assay which is equivalent to the classical 3 H-thymidine incorporation assay and have significant advantages in terms of the ability to gate on a specific population of lymphocytes [17,18]. PBMC were CFSE loaded (FITC labeled; Molecular Probes, Eugene, OR) as described elsewhere [8,16]. Thereafter, were cultured with medium alone (negative control), phytohemagglutinin (PHA, positive control), HIV-1HZ321 antigens, rVV Gag/pol (WR-Gag) and vvluc. After 6 days of incubation at 37 ◦ C and 5% CO2 , cells were collected and stained with anti-CD3 mAb (APC), antiCD8 (PerCP) and anti-CD4 (PE) (BD Biosciences). A total of

2740

L. Valor et al. expressed as spot-forming cells (SFC)/106 PBMC, was calculated after subtracting negative control values (RPMI medium for HIV-1HZ321 antigens and rVV-luc for recombinant vaccinias).

CD4+ and CD8+ T cell intracellular staining for IL-2, IFN-␥ and TNF-␣ expression

Figure 1 Representative sample shown here, demonstrates from one immunized patient the PBMC labelled with CFSE and then stimulated with PHA (a) and with HIV-1HZ321 antigen (b). Events were evaluated in a CD3+ gate.

100,000 events in a lymphocyte gate were analysed by flow cytometry. CD3+ , CD8+ and CD4+ CFSElow were evaluated simultaneously, gating on CD3+ T cells and measuring sequentially proliferating CD8+ and CD4+ subsets within the CD3+ subset. The percentages of CD4+ , CD8+ T cells shown in Table 1 correspond to proliferating cells which were analysed separately [8]. PHA was used for estimation of precursor frequency [19,20]. Briefly, PHA polyclonal stimulation of T cells results in cell division with distinct CFSE fluorescence peaks, allowing determination of the mean CFSE fluorescence for each generation. These values are used to calculate the average number of cell divisions in cells stimulated with antigen. The absolute number of precursors for the antigen positive cells was estimated by dividing the number of PHA positive cells by 2x (x is the average number of cell divisions) and then dividing this value by the total number of cells analysed (Fig. 1). Net percentages of CFSElow and net precursor frequencies were calculated subtracting the negative control values (RPMI medium for HIV-1HZ321 antigens and rVV-luc for recombinant vaccinias). Control experiments in healthy individuals (n = 10) showed that stimulation with HIV-1 antigens was not able to induce greater than 0.2% of CFSElow T cells. Consequently, LPR scoring greater than 0.2% of CD3+ CFSElow cells were deemed positive.

ELISpot assay for IFN-␥ producing T cells to HIV-1 antigens ELISpot assays were performed as described elsewhere [21] using the appropriate stimuli: RPMI medium alone (negative control), PHA (Sigma Chemical Co., St. Louis, MO) at 1 ␮g/ml (positive control); HIV-1HZ321 antigens at 10 ␮g/ml and recombinant vaccinia (rVVs) expressing Gag/pol HIV-1 antigens (specific stimuli) and rVVs expressing env (controlspecific stimuli) or luc as vaccinia negative control [22]. Stimuli were added to PBMC at a multiplicity of infection (MOI) of 5 PFU/cell. Spots were counted using a dissection microscope. Number of antigen-specific T cells,

PBMC were resuspended in RPMI-1640 plus 10% FCS containing 50 IU IL-2 [23—25], anti-CD28 and anti-CD49 mAb (1 ␮g/ml, BD Biosciences) and then stimulated in RPMI 1640 culture medium for 6 h with HIV-1HZ321 antigens at 10 ␮g/ml or rVVs encoding either Gag/pol or luc at a MOI of 5 PFU/cell. Brefeldin A at 10 ␮g/ml (BFA, Sigma Chemical Co., St. Louis, MO) was added for the last 12 h. PBMC incubated with PHA at 1 ␮g/ml or medium alone served as positive and negative control, respectively. After incubation, cells were washed with PBS/BSA, fixed with PFA 4%, permeabilized with FACSperm (BD Biosciences) and stained with anti-CD3 APC, anti-CD8 PerCP, anti-IFN-␥ FITC and antiTNF-␣ or anti-IL-2 PE (BD Biosciences). Data acquisition was performed on a FACScalibur flow cytometer (Becton Dickinson). IFN-␥ and TNF-␣ or IL-2 producing cells were analysed within the CD3+ /CD8+ or CD3+ /CD8− gated populations. Background values have been subtracted to all data.

Quantification of HIV-1-specific cytotoxic T cell activity (CTL) and frequencies of HIV-1-specific cytotoxic T cell precursors (CTLp) Frequencies of HIV-1-specific memory cytotoxic precursors were determined using standard methods of limiting dilution analysis (LDA) [26,27]. The CTLp frequencies were calculated using the single-hit Poisson model. Results of CTLp are presented as number of CTLp/106 input PBMC. Standard CTL culture assay was performed as previously reported [28]. To assay HIV-1-specific CTL activity, autologous B-LCL were infected with rVV expressing HIV-1 gene products (Gag/pol and env) and with rVV-luc for control at MOI of 5 PFU/cell and labelled with Na2 51 CrO4 (Amersham Pharmacia Biotec, UK) to be used as target cell [22]. Target cells (5 × 103 ) were added to each effector cell containing well. Unlabelled B-LCL infected with vv-luc was added at 20 times the number of cold targets cells. 50 ␮l of supernatant from each well were harvested and mixed with 150 ␮l of scintillation liquid (Hisafe III, EGG Wallac, Turku, Finland). Finally, radioactivity was measured in a ␤-counter (Microbeta 1450, EGG Wallac, Turku, Finland). Results are expressed in Lytic Units (LU) per 106 input blasts, as the number of effector cells able to lyse 20% of targets (calculated by a regression line) with background vaccinia control CTL activity subtracted.

Statistical analysis Treatment arms were compared by an exact Mann—Whitney test as immunological variables did not exhibit a normal distribution. Also, correlations between all variables were calculated using the non-parametric Spearman Rho test.

Percentages and precursor frequencies of proliferating PBMC to HIV-1HZ321 antigen in the immunogen group vs. IFA group pre- and post-immunization %CFSElow CD3+ Median

(a) Baseline IMN IFA Median difference p Post-immunization IMN IFA Median difference p

0.03 0.7 −0.03

2.8 0.6 2.0

%CFSElow CD3+ CD4+ Range

0—1.3 0—1.2 — 0.875 0.1—23.3 0—4.3 — 0.083

95% CIa

(b) Baseline IMN (×1000) IFA (×1000) Median difference p Post-immunization IMN (×1000) IFA (×1000) Median difference p

Range

0—1.3 0—1.2 −1.2 to 1.2

0.02 0.4 0

0—1.4 0—1.0 — 1.000

0.5—8.4 0.2—2.5 −0.1 to 6.8

3.9 1.1 2.7

0—19.0 0—5.3 — 0.053

CD3+ Precursors Median

Median

%CFSElow CD3+ CD8+ 95% CIa

0—1.4 0—1.0 −1.0 to 1.4

0.8—9.7 0.2—4.2 −0.05 to 7.3

CD4+ Precursors

Range

0 1.4 −1.4

0—0.7 0—3.2 — 0.232

3.9 1.3 2.5

0—35.4 0—7.4 — 0.056

95% CIa

0—0.7 0—3.2 −3.2 to 0.6

1.2—17.8 0.5—4 −0.3 to 12.5

Median

Median

Range

0 1.0 −0.9

0—0.8 0—2.7 — 0.143

2.9 0.6 2.3

0—29.5 0—3.7 — 0.035

95% CIa

0—0.8 0—2.7 −2.7 to 0

0.6—8.2 0.2—1.8 0.07—6.5

CD8+ Precursors

Range

0 1.1 −1.1

0—0 0—3.9 — 0.107

2.8 1.1 2.2

0—29.8 0—7.4 — 0.054

95% CIa

Median

0—0 0-3.9 −3.9 to 0

0 2.8 −2.6

0—2.0 0—2.9 — 0.143

5.6 1.6 3.8

0—44.4 0—9.8 — 0.031

1.5—16.8 0.4—2.6 −0.2 to 14.0

Range

CD4+ and CD8+ HIV-1 responses induced by an HIV-1 immunogen

Table 1

95% CIa

0—2.0 0—2.9 −2.9 to 0

2.2—16.6 0—3.8 0.3—11.5

IMN = HIV-1 immunogen. a 95% CI lower and upper limit of IMN and IFA held at minimum and maximum of sample.

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L. Valor et al.

Table 2 Comparison between T cell effector responses measured by IFN-gamma ELISpot assay to HIV-1HZ321 antigen in the immunogen group vs. IFA group pre- and post-immunization Baseline

IMN IFA Median difference p

Post-immunization a

Median

Range

95% CI

35 115 −65

15—110 0—255 — 0.393

15—110 0—255 −220 to 35

Median

Range

95% CIa

410 75 355

95—875 0—100 — 0.003

206.4—824.4 4—100 205—705

IMN = HIV-1 immunogen. a 95% CI lower and upper limit of IMN and IFA held at minimum and maximum of sample.

Results were considered statistically significant when the p-value was inferior to 0.05. Statistical analysis was performed using SPSS (Statistical Product and Service Solutions) software.

Results HIV-1 immunogen induced CD4+ and CD8+ -specific lymphoproliferative responses to HIV-1 antigens compared to placebo We have evaluated HIV-1-specific lymphoproliferative responses in patients immunized with the HIV-1 immunogen or IFA, by examining the percentages of proliferating CD4+ and CD8+ HIV-1-specific T cells and the frequency of HIV-1-specific T-cell precursors by CFSE assay. Table 1 is demonstrative of the fact that the immunogen group developed higher proliferative response of CD8+ T cells against HIV-1 antigens and higher numbers of HIV-1-specific CD8+ CTLp. Likewise, there was a trend towards higher proliferative response of HIV-1-specific CD4+ T cells and higher numbers of HIV-1-specific CD4+ T cell precursors. In the immunogen group there was a negative correlation between CD3+ CFSElow and CD3+ CD4+ CFSElow HIV-1-specific lymphoproliferative responses and the log viral load (r = −0.696 and r = −0.684, respectively; p < 0.05). However, in the placebo group CD3+ CFSElow and CD3+ CD4+ CFSElow HIV-1-specific lymphoproliferative responses correlated positively with log viral load (r = 0.672 and r = 0.407, respectively; NS).

IFN-␥ production (ELISpot) was augmented in the immunogen group Post-immunization, the number of IFN-␥ producing T cells against HIV-1HZ321 antigens in the immunogen group was significantly increased compared to IFA group (Table 2). Post-immunization, the number of IFN-␥ producing T cells against WR-Gag antigens was elevated in the IMN group compared to IFA [median (range) 572.5 (60—3205) SFC/106 PBMC vs. 190 (65—2225) SFC/106 PBMC, respectively; p = 0.024]. No significant differences in the number of IFN-␥ producing T cells against vv-env were observed between both groups [IMN: 52.5 (0—1675) SFC/106 PBMC vs. IFA: 25 (0—920) SFC/106 PBMC; p = 0.841].

Cytokine intracellular expression in CD4+ and CD8+ T cells specific to HIV-1 antigens was upregulated in the HIV-1 immunogen group When PBMC from patients immunized with the HIV-1 immunogen were stimulated with HIVHZ321 antigens there was a significant increase of IFN-␥ and IL-2 producing CD4+ T cells (0.39 ± 0.05% of total CD4+ T cells: 0.10% IL-2 producing cells plus 0.23% IFN-␥ producing cells plus 0.06% cells co-expressing both IL-2 and IFN-␥) compared to IFA group (p = 0.02). Additionally, in this group we observed a significant increase of IFN-␥ producing CD8+ T cells (0.19% of total CD8+ T cells: 0.03% IL-2 producing cells plus 0.11% IFN-␥ producing cells plus 0.05% cells co-expressing both cytokines) compared to placebo (p < 0.05). Furthermore, when PBMC were stimulated with WR-Gag only the immunogen group showed a significant increase in CD8+ IFN-␥ producing cells (0.34% of total CD8+ T cells: 0.32% IFN-␥ producing cells and 0.02% cells co-expressing both IFN-␥ and IL-2; p < 0.05). Moreover, in this group more than 80% of IFN-␥+ Gag-specific CD8+ T cells co-expressed TNF-␣ (Fig. 2).

Enhanced CTL activity and increased CTL precursors in the HIV-1 immunogen group compared to IFA group Fig. 3a shows that the pre-immunization mean levels of Gag/pol-specific LU and of env-specific LU from patients [immunogen (n = 27) and IFA (n = 27) groups] were marginal (2.3 ± 1 LU/106 PBMC and 1.3 ± 0.9 LU/106 PBMC, respectively). However, the mean levels of Gag/polspecific cytotoxicity post-immunization were significantly increased in the immunogen group as compared to placebo group (Gag/pol-specific LU = 20.2 ± 10.1 LU/106 PBMC vs. 4.3 ± 2.6 LU/106 PBMC, respectively; p < 0.05). Fig. 3b shows the quantification of the frequencies of HIV-1-specific memory CTL precursors. The baseline mean levels of Gag/pol-specific memory CTLp and of env-specific memory CTLp were 171 cell/106 PBMC Gag/pol-specific CTLp and 144 cell/106 PBMC env-specific CTLp, respectively. Mean levels of Gag/pol-specific memory CTLp post-immunization were strongly increased in the immunogen group in comparison to the IFA group (1963 ± 672 cell/106 PBMC vs. 185 ± 48 cell/106 PBMC; p < 0.05). Only the immunogen group showed a significantly negative correlation between CTLp frequency for Gag/pol and log viral load (HIV-

CD4+ and CD8+ HIV-1 responses induced by an HIV-1 immunogen

2743

Figure 2 Analysis of HIV-1-specific IFN-␥ and IL-2 secreting CD4+ and CD8+ T cells after stimulation with antigens. One representative profile of two patients [patient immunized with HIV-1 immunogen (IMN) and patient from the placebo group (IFA)] is shown for PHA or HIV-1HZ321 antigens CD4+ and CD8+ T cells responses.

1 immunogen: r = −0.783; p = 0.003 and IFA: r = −0.134; p = 0.694) (Fig. 2).

Discussion

Figure 3 (a) Cytotoxicity of specific Gag/pol and envspecific T cells at baseline and post-immunization expressed as LU/106 PBMC. (b) Frequency of Gag/pol HIV-1-specific cytotoxic T cell precursors at baseline and post-immunization, expressed as CTLp/106 PBMC.

These results show that enhanced HIV-1-specific CD4+ T cell responses were elicited after long-term therapeutic immunization with antiretroviral therapy in patients with HIV-1 chronic infection. Our data confirm other reports that have evidenced that therapeutic immunization induces CD4+ Tspecific helper responses [29—32]. Since baseline CD4+ T cell count has been observed to impact on the response to immune-based therapies all the HIV-1 seropositive subjects recruited in this study were asymptomatic, ART na¨ıve, who had CD4+ T cell counts between 300 and 700 × 106 l−1 [12,33—35]. A recent study suggests that it is extremely problematic to augment fully functional HIV-1-specific CD4+ and CD8+ effector T cell responses by therapeutic immunization in patients with advanced chronic HIV-1 infection who initiated ART with low CD4+ T cell counts (median absolute CD4+ T-cell count was less than 300 cells/␮l) [36]. This study underscores the importance of early initiation of ART for the outcome of immune-based therapies and therapeutic vaccines [36]. Furthermore, we have previously evaluated periodically every 3 months throughout a period of 36 months the lymphoproliferative responses to HIV-1 antigens and we have found that in the HIV-1 immunogen group strong LPR was clearly established after approximately 15 months post-immunization and was maintained up to month 36 [12]. This suggests the importance of longterm immunization in HIV-1 infected patients with gradual destruction of HIV-1-specific CD4+ T helper cells that leads to continuous impairment on functional HIV-1-specific CD4+ and CD8+ T cell responses. IFN-␥ production by CD8+ T lymphocytes is a good marker of cytotoxic activity in healthy controls. Antigen-specific CD8+ T lymphocytes producing both IFN-␥ and TNF-␣ have

2744 been also associated with cytotoxic activity in HIV-1 infected patients [7]. However, HIV-1 infected patients who have a defect on IFN-␥ production by CD8+ T cells shows an impairment on HIV-1-specific cytotoxicity [30]. In this study the group of patients immunized with the HIV-1 immunogen had an expansion of CD8+ HIV-1 Gag/pol-specific precursors that can proliferate and secrete IFN-␥ after WR-Gag-specific stimulation. Also in this group there was an augmentation of the subset of Gag/pol-specific CD8+ T lymphocytes coexpressing both IFN-␥ and TNF-␣. Furthermore, a robust Gag/pol-specific cytotoxicity was observed only in the group of patients immunized with the HIV-1 immunogen. Patients who did not show an expansion of CD3+ CD4+ CFSElow neither showed an increase in the frequency of CD8+ HIV-1 Gag/polspecific precursors nor in the cytotoxic activity (data not shown). A dependence on helper activity for the expansion of HIV-1-specific CD8+ T cells has been described [16]. During the course of progression of HIV-1 infection there is a continuous impairment on functional HIV-1-specific CD4+ and CD8+ T cell responses jointly with a poor control of viral load. In this cohort of chronically HIV-1 positive individuals long-term treated with antiretrovirals in combination with an HIV-1 virion-based therapeutic vaccine, we observed a sustained augmentation of HIV-1-specific immunological responses (increase of CD3+ CD4+ CFSElow HIV-1-specific LPR, HIV-1-specific CD3+ T cell precursors and Gag/pol-specific memory CTLp) and an inverse relationship between viral load and HIV-1-specific proliferative responses. This would be consistent with other reports which have shown that the preservation of the proliferation capacity of both HIV1-specific CD4+ and CD8+ T cells and the ability of CD8+ T cells to co-express both IFN-␥ and IL-2 were generally associated with an apparently effective immune response in patients with HIV-1 infection [8,23,37]. However, based on the sample size of this substudy we cannot conclude whether vaccination has a potential beneficial effect in the group of patients with HIV-1-specific proliferative responses. Further studies are warranted to elucidate whether longterm enhancement of HIV-1-specific CD4+ and polyfunctional CD8+ responses could be associated with immune control in patients for whom functional immunological impairment has been prevented with earlier initiation of ART.

Acknowledgements This work was supported by Fondo de Investigaci´ on Sanitaria (FIS) Grant: PI06/1255 and Fundaci´ on para la Investigaci´ on y Prevenci´ on del SIDA en Espa˜ na (FIPSE) Grants: 36412/03 and 36562/06.

Appendix A. STIR 2102 Team M. Desco (Unidad de Cirugia y Medicina Experimental. Hospi´n, Madrid. Spain), tal General Universitario Gregorio Mara˜ no J. Rodr´ıguez-Molina (Department of Immunology, Hospital ´n, Madrid, Spain); E. General Universitario Gregorio Mara˜ no Bouza, J. P´ erez Molina (Department of Microbiology, Hospi´n, Madrid, Spain); tal General Universitario Gregorio Mara˜ no J.M. Pe˜ na (Hospital La Paz, Madrid, Spain); D. Podzamczer, E. Ferrer (Ciudad Sanitaria Bellvitge, Barcelona, Spain); I. Oca˜ na, M. D´ıaz (Hospital Vall D’Hebron, Barcelona, Spain);

L. Valor et al. B. Clotet, A. Jou, G. Sirera (Hospital Germans Trias i Pujol, Badalona, Spain); R. Rubio, F. Pulido (Hospital 12 de Octubre, Madrid, Spain); P. Viciana (Hospital Virgen del Rocio, Sevilla, Spain); J.A. Maradona (Hospital Central de Asturias, Oviedo, Spain); R. Blazquez (Hospital Morales Meseguer, Murcia, Spain); C. Barros (Hospital de Mostoles, Madrid, Spain); S. Moreno, C. Quereda (Hospital Ramon y Cajal, Madrid, Spain); J.M. Gatell (Hospital Clinic i Provincial, Barcelona, Spain); J. Gonz´ alez-Lahoz (Instituto de Salud Carlos III, Madrid, Spain).

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