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Cytokine and gene transcription profiles of immune responses elicited by HIV lipopeptide vaccine in HIV-negative volunteers

Richert, Lauraa,b,*; Hue, Sophiec,d,e,f,*; Hocini, Hakimc,f; Raimbault, Mathieua,b; Lacabaratz, Christinec,d,f; Surenaud, Mathieuc,f; Wiedemann, Auréliec,d,f; Tisserand, Pascalinec,f; Durier, Christineg; Salmon, Dominiqueh; Lelièvre, Jean-Danielc,d,f,i; Chêne, Genevièvea,b,j; Thiébaut, Rodolphea,b,*; Lévy, Yvesc,d,f,i,*for the ANRS Vaccine NetworkVaccine Research Institute

doi: 10.1097/QAD.0b013e32835f5b60
Clinical Science

Objective: To dissect the biological mechanisms involved in the cellular responses to a candidate vaccine containing 5 HIV peptides coupled to a palmytoil tail (HIV-LIPO-5) in healthy volunteers, by using extensive immunogenicity assessments with different stimulation durations.

Design: Immunogenicity substudy of a randomized phase II prophylactic HIV vaccine trial (ANRS VAC 18).

Methods: HIV-LIPO-5 or placebo was administered at W0, W4, W12 and W24. Peripheral blood mononuclear cells from a subset of participants at W0 and W14 were stimulated with HIV-LIPO-5, Gag peptides contained in the vaccine and control peptides. ELISpot, lymphoproliferation, intracellular cytokine staining (ICS), cytokine multiplex and transcriptomic analyses were performed. Different time points and stimulation conditions were compared, controlling for test multiplicity.

Results: Cultured ELISpot and lymphoproliferation responses were detected at W14. Ex-vivo ICS showed mainly interleukin (IL)-2-producing cells. Secretion of interferon (IFN)-γ, tumour necrosis factor (TNF)-α, IL-5 and IL-13 increased significantly after culture and Gag stimulation at W14 compared to W0. Metallothionein genes were consistently overexpressed after HIV-LIPO-5 stimulation at W0 and W14. At W14, significant probes increased substantially, including IFN-γ, CXCL9, IL2RA, TNFAIP6, CCL3L1 and IL-6. Canonical pathway analyses indicated a role of interferon signalling genes in response to HIV-LIPO-5.

Conclusion: HIV-LIPO-5 vaccination elicited Th1 and Th2 memory precursor responses and a consistent modulation in gene expression. The response profile before vaccination suggests an adjuvant effect of the lipid tail of HIV-LIPO-5. Our combined immunogenicity analyses allowed to identify a specific signature profile of HIV-LIPO-5 and indicate that HIV-LIPO-5 could be further developed as a prime in heterologous prime-boost strategies.

aUniversity Bordeaux

bINSERM, ISPED, Centre INSERM U897-Epidemiologie-Biostatistique, Bordeaux


dUniversity Paris Est Créteil, Faculté de Médecine

eGroupe Henri-Mondor Albert-Chenevier, Immunologie biologique

fMondor Immunology Center, Créteil

gInserm SC10, Villejuif

hUniversity Paris Descartes-Hôpital Cochin, Paris

iGroupe Henri-Mondor Albert-Chenevier, Immunologie clinique, Créteil

jCHU de Bordeaux, Pole de sante publique, Bordeaux, France.

*Laura Richert, Sophie Hue, Rodolphe Thiébaut, and Yves Lévy contributed equally to the writing of this article.

Correspondence to Professor Yves Lévy, MD, PhD, Clinical immunopathology, Hôpital Henri Mondor, INSERM U955, Faculté de Médecine de Créteil, 51, Avenue du Maréchal de Lattre de Tassigny, F-94010 Créteil, France. Tel: +33 1 4981 2455; fax: +33 1 4981 2469; e-mail:

Received 5 September, 2012

Revised 21 November, 2012

Accepted 23 January, 2013

Preliminary results of this study were presented at the AIDS Vaccine Conference, 28 September–1 October 2010, Atlanta, Georgia, USA (abstract P14.02), and at the AIDS Vaccine Conference, 12–15 September 2011, Bangkok, Thailand (abstract P14.07).

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Website (

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The search for a well tolerated and effective vaccine strategy against HIV remains a high priority to control the global pandemic. Recently, the phase IIB RV 144 trial in healthy volunteers in Thailand showed that peptide candidate vaccines may be promising components of heterologous prime-boost regimens against HIV [1].

The current objective of HIV vaccine development is to induce both humoral and cellular immune responses [2,3]. In particular, T-cell responses seem to be important in controlling virus replication after HIV infection in humans [4,5] and after simian immunodeficiency virus (SIV) infection in macaques [6]. However, to date correlates of protection against HIV infection are not well known, and phase II vaccine trials can currently not rely on a surrogate immunogenicity marker to predict clinical vaccine efficacy. The ability to integrate immunogenicity data generated in vaccine trials is thus crucial for optimizing HIV vaccine development [7].

The ANRS HIV-LIPO-5 vaccine is a lipopeptide candidate vaccine including an equal-weight mix of five synthetic HIV-1 peptides (covering Gag, Pol and Nef sequences), each coupled to a palmytoil tail. The lipid component of the vaccine promotes uptake and processing of the antigen by dendritic cells (DCs). The current HIV-LIPO-5 candidate vaccine contains T-cell epitopes from Gag, Pol and Nef sequences and was designed to induce T-cell responses [8–10]. However, previous studies have shown that an HIV lipopeptide vaccine (HIV-LIPO-6, containing Env, Nef and Gag peptides) also elicited antibody responses [11]. HIV lipopeptide vaccines have a good safety profile in humans [12], and their development is pursued for therapeutic and prophylactic HIV vaccine strategies.

The immunogenicity of HIV-LIPO-5 in HIV-negative volunteers was recently evaluated in the ANRS VAC 18 trial, a randomized phase II trial comparing three different vaccine doses versus placebo [13]. The results of the core trial showed that all vaccine doses induced HIV-specific sustained CD4+ and CD8+ T-cell responses, assessed by lymphoproliferation and 12-day cultured IFN-γ ELISpot assays [13]. Both IFN-γ ELISpot and lymphoproliferative CD4+ T-cell responses were predominantly elicited by Gag peptide pools. In concordance with previous results of HIV lipopeptide vaccines [14–16], responses were detected using cultured in-vitro assays allowing for the expansion of vaccine-elicited memory T-cell responses [17,18]. Responses to ex-vivo ELISpot assays were weak, suggesting that HIV-LIPO-5 might primarily elicit memory precursor cells detectable in cultured assays.

To further characterize the immunogenicity profile of the HIV-LIPO-5 vaccine as a single component before proceeding to evaluation of heterologous prime-boost regimens, a larger spectrum of different immunological markers is needed. In the present study, we performed more extensive analyses of cellular responses induced by HIV-LIPO-5 in a subgroup of ANRS VAC18 trial participants. The objective of these analyses was to dissect the biological mechanisms involved in the cellular responses to HIV-LIPO-5, by using assays with different stimulation durations, including intracellular cytokine staining (ICS), multiplex cytokine and gene transcription microarray assays.

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Study design

The ANRS VAC18 trial was a phase II randomized, double-blinded, multicenter trial in France evaluating the safety and immunogenicity of HIV-LIPO-5 in HIV-negative volunteers. HIV-LIPO-5 includes the following HIV-1 sequences: Gag 17–35, 253–284; Pol 325–355; Nef 66–97, 116–145. One hundred and thirty-two volunteers were randomized to receive four intramuscular injections of different doses (50, 150 or 500 μg) of HIV-LIPO-5 (n = 99) or placebo (n = 33) at weeks 0, 4, 12 and 24. Randomization was stratified on the region (Paris region versus other regions). The trial was approved by the Ethics Committee ‘CCPPRB Pitié Salpêtrière’ (Paris, France), and written informed consent was obtained from all volunteers. Detailed methods of the core trial have been described elsewhere [13].

During the trial, fresh full blood samples from all participants were shipped from the clinical sites to the central immunology laboratory on the day of sampling. Peripheral blood mononuclear cells (PBMCs) were isolated from full blood by density gradient at the central laboratory and frozen.

The present substudy was based on samples from the PBMC repository of the trial. Given that previous studies have shown that HIV lipopeptide vaccines elicit antibody responses [11] but that the HIV-LIPO-5 vaccine does not contain Env, humoral responses were not assessed in the substudy. PBMC samples from volunteers in the Paris region were eligible for this substudy in order to include only samples with short shipment times of fresh full blood before PBMC isolation. Among the 84 trial participants in the Paris region having received at least one injection of the vaccine strategy, 10 per arm were randomly selected for inclusion of their samples in the substudy (Fig. 1). As there were no apparent differences in IFN-γ ELISpot response between the three HIV-LIPO-5 doses in the core trial, the active vaccine arms were pooled for the substudy. Substudy assessments were performed on W14 samples to target the time point with peak vaccine responses in the core trial [13].

Fig. 1

Fig. 1

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Immunogenicity assessments

Interferon-γ ELISpot

Twelve-day cultured IFN-γ ELISpot assays were performed as previously described [13]. Briefly, PBMCs were incubated with nine different HIV peptide pools (including 77 peptides of 8–11 amino acids, thereby primarily targeting CD8+ T-cell responses), cultured for 12 days and then harvested for the IFN-γ ELISpot assay. Magnitude of ELISpot response was summed over HIV peptide pools after background-subtraction.

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CD4+ T-cell responses were assessed in a 7-day culture lymphoproliferation assay using seven HIV long peptides, as previously described [13]. Median count per minute (cpm) was measured across quadruplicate wells. Net cpm was calculated by subtracting median unstimulated cpm from median stimulated cpm, and summed over HIV peptide stimulations.

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Intracellular cytokine staining

Among the 40 randomly selected substudy participants, 37 had sufficient PBMC volumes in the central repository for ICS measurements at W14, that is 2 weeks after the third vaccine administration. PBMCs were stimulated overnight by five HIV-1 peptide pools covering Gag, Pol and Nef (15-mers overlapping by 11 amino acids). Unstimulated cells served as negative controls. Following cell surface staining (anti-CD3 APC, anti-CD4 Pacific Blue and anti-CD8 APC-H7; BD Biosciences, San Jose, California, USA), PBMCs were permeabilized with Perm II Buffer (BD Biosciences), stained with anti-IFN-γ Fluorescein isothiocyanate, anti-tumour necrosis factor (TNF)-α PE-Cy7 and anti-interleukin (IL)2 PE (BD Biosciences), washed and stored until analysis. Data were acquired with an LSRII flow cytometer (Becton Dickinson, Franklin Lakes, New Jersey, USA) and background-subtracted before analysis.

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Multiplex cytokine measurements

Among the volunteers selected for ICS measurements, functional profiles were further characterized by multiplex cytokine measurements in 12 vaccine recipients with sufficient PBMC volumes in the repository before (W0) and after vaccination (W14).

PBMCs were cultured for 11 days with either HIV-LIPO-5; a pool of 15-mer Gag peptides included in HIV-LIPO-5 (Gag+); a pool of 15-mer Gag peptides not included in HIV-LIPO-5 (Gag−); or no stimulating peptide (unstimulated control). Peptide restimulation was performed at day 9, and supernatants were collected at day 11. A Luminex assay was used to quantify IFN-γ, IL-1β, IL-2, IL-5, IL-6, IL-10, IL-13, IL-17, IL-21 and TNF-α (MILLIPLEX MAP kit, Millipore, Molsheim, France). Data were acquired with the Bio-Plex 200 system (Bio-Rad, Marnes-la-Coquette, France).

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Microarray analysis

To capture early signatures of vaccine effects, gene transcription was assessed after 6-h and 24-h stimulations in the same PBMC samples using the same stimulating molecules as in the multiplex cytokine assay.

RNAs were purified, quantified and checked for integrity. In-vitro transcription was generated from 150 ng RNA, and cRNA was hybridized onto Illumina Human HT-12v4 Expression BeadChips (Illumina, San Diego, California, USA). Data acquisition was done by Illumina. Raw data were exported from Genomestudio on Bead Summary Data format [19]. Standard quality control thresholds were applied after preprocessing of signal intensity data.

The microarray data are accessible in the MIAME-compliant database Gene Expression Omnibus (,accessionnumberGSE39506).

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Statistical analyses

ELISpot, lymphoproliferation and ICS responses were compared between the vaccine and placebo groups at W14 by Wilcoxon rank-sum tests. ICS responses were analysed for each individual stimulating peptide pool as well as for the sum of stimulations by peptide pools belonging to HIV-LIPO-5, controlling test multiplicity by an adaptive false discovery rate (FDR) [20].

Statistical analyses of Luminex data were performed on fluorescence intensity and concentration (pg/ml), comparing results before versus after vaccination in HIV-LIPO-5 recipients by Wilcoxon signed-rank tests. Analyses of fluorescence intensity rather than concentration were considered the primary analyses, since conversion to concentration resulted in a loss of power (increase of data variability, reduction of number of observations). We evaluated the possibility that distributions of fluorescence intensity measurements may potentially be right-censured and left-censured due to lower detection and upper saturation limits. Given the small proportion (2–6%) of potentially censured data in our data set and hence the minimal impact expected on the results, we did not use statistical methods for censured data [21,22]. To account for dependency among statistical tests of different cytokines, we used dependent FDR-adjusted P values [23] with a cut-off of less than 0.10 for significance.

Gene transcription data were preprocessed [24–27] and corrected for a chip effect [28]. A selection procedure was applied to remove probes with Illumina detection P ≥ 0.01 in all samples used in a comparison of interest [29]. After selection, statistical comparisons were based on paired empirical Bayes moderated t-statistics (Limma package [26,30]), testing each stimulation condition separately against unstimulated controls at each time point and incubation duration. An adaptive FDR procedure was used to control for test multiplicity [31]. We then carried out canonical pathway and biological function analyses for significant genes with adaptive FDR-adjusted P < 0.05 and fold-change |FC| >1.5 in comparisons of HIV-LIPO-5 stimulations versus unstimulated controls.

Statistical analyses were done with SAS, version 9.2 (SAS Institute, Cary, North Carolina, USA) and R (version 2.14.1; The R Foundation for Statistical Computing, Vienna, Austria). Ingenuity software (version 12710793; Ingenuity Systems, Redwood City, California, USA) was used for gene pathway and function analyses.

Additional details on immunogenicity assays and statistical methods are reported in the supplemental digital content (see supplemental methods, supplemental digital content 1,

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Characteristics of the study population

The characteristics of the 37 substudy participants are shown in Table 1. The subset of 12 HIV-LIPO-5 recipients, whose samples were used for Luminex and gene expression measurements, had a median age of 45 years [interquartile range (IQR) 37–50] and two-thirds were male.

Table 1

Table 1

Overall, the baseline characteristics of the 37 volunteers, and of the subset of 12 volunteers, were similar to those of the remaining volunteers in the core trial [13]. All but one participant from an active vaccine arm had received the three planned vaccine or placebo doses by W12.

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T-cell responses after vaccination

The distributions of responses in the cultured IFN-γ ELISpot and the lymphoproliferation assays at W14 in this substudy were similar to those in the core trial (Table 1). In the substudy, median magnitude of ELISpot response at W14 was 855 spot forming units (SFU) (IQR 320–2652) in vaccinees and 323 SFU (IQR 152–735) in placebo recipients (P = 0.07). Median CD4+ T-cell lymphoproliferation responses were 1320 cpm (IQR 894–8061) and 348 cpm (IQR 69–690), respectively (P = 0.002).

ICS after overnight stimulation was performed at W14. In the 27 vaccinees, the percentages of CD4+ cytokine+ and CD8+ cytokine+ responding cells (i.e. cells producing at least one cytokine among IFN-γ, TNF-α and IL-2), summed over the HIV peptide pools included in the vaccine, were 0.20% (IQR 0.13–0.30%) and 0.18% (IQR 0.11–0.24%) (see Figure, Supplemental Digital Content 2, Analysis of the functional profile showed that responding CD4+ and CD8+ T cells were mainly single IL-2-producing cells.

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Th1 and Th2 cytokine responses in cultured multiplex assays

Next, we investigated more broadly and after 11-day culture the cytokine profile of vaccine-elicited responses in PBMCs using Luminex assays. Concentrations (pg/ml) of IL-5 and IL-13 increased significantly between W0 and W14 in the supernatants of PBMCs stimulated by Gag+ and by HIV-LIPO-5 (see Figure, Supplemental Digital Content 3, No significant differences were detected after stimulations with Gag peptides not included in the vaccine (Gag−).

In a second step, statistical analyses were based on fluorescence intensity. In addition to IL-5 and IL-13, IFN-γ and TNF-α fluorescence intensity increased significantly between W0 and W14 after stimulation by Gag+ (Fig. 2).

Fig. 2

Fig. 2

As expected, no difference was observed between unstimulated PBMCs and Gag+ stimulated PBMCs at W0. In contrast, when comparing HIV-LIPO-5 stimulation to no stimulation at W0, IFN-γ and IL-10 were significantly increased. At W14, significant differences were observed for IFN-γ, TNF-α, IL-5, IL-13, IL-6 and IL-10 following Gag+ or HIV-LIPO-5 stimulations, compared to no stimulation. There were no significant differences for IL-1β, IL-2, IL-17 or IL-21 in any comparisons.

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Modulation of gene expression by HIV-LIPO-5

At W0, we observed significant variations in gene transcription after 6-h (77 probes corresponding to 73 genes) and 24-h HIV-LIPO-5 stimulations (22 probes, 21 genes), compared to unstimulated samples. After vaccination (W14), the number of significant probes increased substantially to 1223 probes (1126 genes) after 6-h, and to 1415 probes (1278 genes) after 24-h stimulations (see Table, Supplemental Digital Content 4, The majority of significant probes after HIV-LIPO-5 stimulations at W0 also showed significant variation at W14 (Fig. 3). In contrast, stimulation with Gag peptides (6-h Gag+ stimulation) at W14 induced gene expression changes in two genes only. No significant variations in gene expression were detected after stimulations by Gag sequences not contained in the vaccine (Gag−).

Fig. 3

Fig. 3

Among differentially expressed genes at W0 and W14, those from the metallothionein family (MT1A, MT1E, MT1F, MT1G, MT1H, MT1M, MT1X, MT2A and MTE) were consistently overexpressed after HIV-LIPO-5 stimulation compared to unstimulated samples. Moreover, among genes with a significant variation at W14 after 24-h HIV-LIPO-5 stimulation, IFN-γ, CXCL9, IL2RA, TNFAIP6, CCL3L1 and IL-6 were overexpressed with a fold change |FC| >1.8.

Biological function analyses at W14 indicated changes in the expression of genes involved in activation of immune cells, including differentiation and activation of T cells, differentiation of antigen presenting cells, and motility of immune cells (see Table, supplemental digital content 5,; and Figure, supplemental digital content 6, These functions were not modulated at W0, suggesting a specific induction by vaccination.

Canonical pathways at W14 showed a variation in interferon signalling genes after 24-h HIV-LIPO-5 stimulation (Fig. 4a and b). Upregulated genes of the interferon pathway included IFN-γ signal transducer activator of transcription STAT1, suppressor of cytokine signalling 1, interferon-induced protein 35 (IFI35) and transporter 1, ATP-binding cassette, subfamily B (MDR/TAP) (Fig. 4b). IFN-γ upregulation was observed both after 6-h and more pronounced after 24-h stimulation, whereas a downregulation of IFN-γ receptor 1 (IFN-γ Rα) was present after 24 h. Analysis of transcription factors that may be responsible for gene expression changes indicated an activation of nuclear factor (NF)-kB complex and an inhibition of STAT3, ESR1, Nr1h and TP53 (see Figure; supplemental digital content 7,

Fig. 4

Fig. 4

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In the present study, we report the results of extensive functional analyses of cellular responses induced by the HIV-LIPO-5 vaccine, including cultured ELISpot, lymphoproliferation, ICS, multiplex cytokine and gene transcription microarray assays. We show that the combination of HIV antigens linked to a lipid tail induced broad functional cellular responses in HIV-negative volunteers.

HIV-LIPO-5 elicits only weak ex-vivo ELISpot responses in healthy volunteers, and cultured assays have been adopted in this study in order to detect vaccine-elicited memory T-cell responses [13]. Our results show that HIV-LIPO-5 induced memory T-cell precursors capable to respond in vitro to HIV-LIPO-5 itself or to Gag peptides belonging to the vaccine, as attested by proliferation, production of Th1 (IFN-γ, TNF-α)-related and Th2 (IL-5, IL-13)-related cytokines, and upregulation of gene transcription. Together with the absence of significant responses in ex-vivo assays, these findings suggest that vaccination by HIV-LIPO-5 induces memory precursors capable to expand under antigenic stimulation, but not effector cells.

Previous studies have shown that the cultured ELISpot technique allows to detect proliferating T-cell responses to other HIV vaccine strategies [32] and may predict in-vivo protection against Malaria [33,34]. In a recent study evaluating a CMV-vectored vaccine, CD8+ effector memory responses correlated with early control of SIV-infection in macaques [35].

Transcriptomic analysis of PBMCs stimulated with HIV-LIPO-5 showed variations in gene expression, to a small extent before vaccination and at a much higher level after vaccination. Transcription of metallothionein genes was observed in the presence of HIV-LIPO-5, but not with Gag peptides, before and after vaccination. In humans, four metallothionein isoforms (MT-1, MT-2, MT-3 and MT-4) and 17 metallothionein genes have been identified. Genes induced by HIV-LIPO-5 belonged to the 10 metallothionein genes known to be functional [36]. Metallothionein plays possible biological roles in cell proliferation and apoptosis, homeostasis of essential metals, cellular free radical scavenging, and metal detoxification. Increased metallothionein levels have been reported in-vitro during cell growth [37,38] and human CD4+ T-cell proliferation [39]. Elevated metallothionein levels during proliferation could be related to the increased demand for certain metals or protection from oxidative stress [40]. Metallothionein inducers, such as lipopolysaccharides, cytokines and TNFs, have been described [41]. However, the regulation of metallothioneins and their function are still not completely elucidated. Although we did not directly evaluate the effect of stimulation of PBMCs by the lipid component only, our results suggest that the palmytoil tail of HIV-LIPO-5 may enhance metallothionein gene transcription in vitro.

Furthermore, after vaccination, genes related to chemokine and cytokine functions were upregulated following HIV-LIPO-5 stimulation. This gene expression signature likely corresponds to a response primed in vivo by the vaccination. Our results support a model, in which genes involved in interferon signalling and antigen presentation pathways are strongly upregulated in the initial 24 h after HIV-LIPO-5 stimulation. Changes in gene expression observed only after vaccination are reminiscent of in-vitro studies of the presentation of lipopeptides by DCs, inducing CD4+ and CD8+ T-cell responses [8,42]. It was previously shown that cross-presentation of lipopeptides by human plasmacytoid DCs results in the activation of effector T-cell responses with IFN-γ secretion and cell proliferation [42].

Modulations in gene expression were associated with functional responses, attested by the cytokine profile of vaccine-elicited immune responses. Cultured multiplex cytokine assays showed a significant increase in cytokine secretion after vaccination, not only in response to HIV-LIPO-5 stimulation but also to stimulation by Gag+ peptides. The observed cytokine response to Gag+, that is to peptides belonging to the vaccine sequence without the lipid component, is likely HIV-specific. The contrastingly small variation in gene expression after shorter time Gag+ stimulation may be related to a low frequency of vaccine-specific T-cell precursors and to the lack of early variation in gene expression in the absence of an adjuvant such as the lipid tail.

From a methodological perspective, the number of significantly increased cytokines in the Luminex assay was greater when analyses were based on fluorescence intensity than on cytokine concentration. This suggests that comparisons based on concentration may have reduced statistical power, which is supported by the following observations: first, concentration values are missing if the raw fluorescence intensity is out of the range of the standard curve. Second, conversion to concentration resulted in larger variability of the data, with considerably higher variation coefficients (data not shown). We therefore suggest to base significance testing of Luminex results on fluorescence intensity rather than concentration.

Our combined results extend our knowledge on the immunogenicity of an epitope-based HIV-vaccine approach and on the interest of integrating a large array of immunological tests to untangle potential mechanisms of vaccine response. Gene expression analyses have been successfully used to identify an early signature predicting the immunogenicity of the yellow fever vaccine [43]. In the field of HIV vaccine research, only few reports on gene expression signatures in response to prophylactic HIV vaccines are available so far, and published cellular immunogenicity analyses are often restricted to ELISpot, proliferation assays and ICS. Gene expression analyses have been used to characterize the response to HIV/SIV viral vector vaccines, peptide vaccines and DNA vaccines in animal models [44,45]. Recently, gene expression and functional immunogenicity measurements were reported in healthy volunteers vaccinated by a canarypox virus HIV vaccine (ALVAC-HIV) alone or by a DC vaccine loaded with ALVAC-HIV [46]. To our knowledge, our study is the first to evaluate combined immunogenicity assessments from transcriptomic and functional assays in HIV peptide-vaccine recipients. Especially the cultured functional assays allowed us to deduce that the HIV-LIPO-5 vaccine primarily elicits memory precursor T-cells.

Despite our small sample size, Luminex and transcriptome assays allowed to capture significant changes after vaccination. This demonstrates that gene expression and cytokine quantification data have their value in the characterization of the immunogenicity profile of an HIV vaccine in humans. Our findings show that the different cellular assays are not redundant and measure distinct aspects of vaccine response. The use of comprehensive approaches to analyse immunogenicity data generated during HIV vaccine trials may lead to new biological insights and contribute to the search for correlates of protection, which should facilitate the successful development of HIV vaccination strategies.

In conclusion, we suggest that the lipid tail of HIV-LIPO-5 has an adjuvant effect eliciting a cellular response. Vaccination with HIV-LIPO-5 induces memory precursor responses in healthy volunteers, indicating that HIV lipopeptide vaccines could be further developed as prime components of heterologous prime-boost strategies.

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The authors wish to thank all the volunteers included in the trial and all investigators and members of the ANRS VAC 18 Trial Management Team, Scientific Committee and Independent Data Monitoring Committee. They also thank Benoît Liquet for discussions on the statistical aspects of the gene expression analyses.

Design and set up of the trial was done by C.D., D.S., Y.L.; participant recruitment and follow-up in the trial by J.D.L., D.S.; laboratory experiments by S.H., H.H., M.S., A.W., P.T., C.L.; statistical analyses by L.R., M.R.; overview of the statistical analyses by G.C., R.T.; interpretation of the results by L.R., S.H., H.H., M.R., C.L., G.C., R.T., Y.L.; and writing of the paper by L.R., S.H., H.H., R.T., Y.L. All authors reviewed and approved the final version of the manuscript.

Participating study sites were Paris Cochin (D. Salmon, O. Launay), Toulouse (L. Cuzin), Nantes (B. Bonnet), Tenon (G. Pialoux), Créteil Mondor (J.-D. Lelièvre), and Marseille (I. Poizot-Martin). Trial methodology and clinical trials unit: Inserm SC10, Paris (J.-P. Aboulker, C. Durier), Paris Cochin (C. Desaint).

This study was supported by a grant from ANRS ( The HIV-LIPO-5 vaccine was provided by Sanofi Pasteur (

L.R. receives a PhD grant financed by Sidaction (

The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Clinical trial registration:,identifierNCT00121758.

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Conflicts of interest

The authors declare that no conflicts of interest exist.

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cellular factors/cytokines; cellular immunity; clinical trial; HIV; lipopeptides; transcriptome; vaccine

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