Immunogenicity and safety of an HIV-1 lipopeptide vaccine in healthy adults: a phase 2 placebo-controlled ANRS trial
Salmon-Céron, Dominiquea,*; Durier, Christineb,*; Desaint, Corinnec; Cuzin, Lised; Surenaud, Mathieue; Hamouda, Nadine Bene; Lelièvre, Jean-Danielf; Bonnet, Bénédicteg; Pialoux, Gillesh; Poizot-Martin, Isabellei; Aboulker, Jean-Pierreb; Lévy, Yvesf; Launay, Odilea,c; for the ANRS VAC18 trial group
aUniversité Paris Descartes, Assistance Publique Hôpitaux de Paris (APHP), Hôpital Cochin, Paris, France
bInserm SC10, Villejuif, France
cCIC de Vaccinologie Cochin-Pasteur, Paris, France
dHôpital Purpan, Toulouse, France
eInstitut Cochin, Université Paris Descartes, CNRS (UMR 8104), Inserm U567, Paris, France
fHôpital Henri Mondor, Université Paris-Est, Créteil, France
gHôpital Hôtel Dieu, Nantes, France
hHôpital Tenon, Paris, France
iHôpital Sainte-Marguerite, Marseille, France
jANRS, Paris, France.
*D.S.-C. and C.D. contributed equally to the writing of this article.
Received 25 March, 2010
Revised 25 May, 2010
Accepted 6 June, 2010
Correspondence to Professor Dominique Salmon-Céron, Infectious Diseases, Internal Medicine Department, APHP, Cochin Hospital, 27 rue du Fbg Saint-Jacques, 75014 Paris, France. E-mail: firstname.lastname@example.org
Background: French National Agency for Research on AIDS and Viral Hepatitis's HIV-LIPO-5 vaccine includes five HIV-1 peptides, containing multiple CD8+ and CD4+ T-cell epitopes and coupled to a palmitoyl tail. Whether HIV-LIPO-5 immunogenicity varies with the dose is unknown.
Methods: HIV-negative volunteers were randomized to receive HIV-LIPO-5 vaccine at 50 μg/lipopeptide (N = 32), 150 μg/lipopeptide (N = 32), 500 μg/lipopeptide (N = 33) or placebo (N = 34) at weeks 0, 4, 12 and 24. HIV-1-specific CD8+ (interferon-γ ELISpot on peripheral blood mononuclear cells cultured for 12 days) and CD4+ responses (peripheral blood mononuclear cell lymphoproliferation) were assessed at baseline, after each injection and at week 48.
Results: Local reactions were dose-dependent but no differences in systemic reactions appeared between groups. Sustained (at least on two separate occasions) CD8+ response rates to at least one given HIV-1 pool were obtained in 22 of 32 (69%), 21 of 33 (64%) and 21 of 34 (62%) individuals for LIPO-5 50, 150 and 500 groups, respectively (P ≤ 0.0001 for all comparisons to the placebo). Cumulative CD4+ response rates were obtained in 15 of 32 (47%), 18 of 33 (55%) and 15 of 34 (44%) individuals (P < 0.0001 for all comparisons to placebo). At week 48, CD8+ responses persisted in 47 of 91 (52%) HIV-LIPO-5 recipients.
Conclusion: Doses of 50, 150 and 500 μg of French National Agency for Research on AIDS and Viral Hepatitis's HIV-LIPO-5 vaccine were able to elicit HIV-specific sustained CD8+ and CD4+ T-cell responses in healthy adults. Safety is good and all doses appear appropriate in further ‘prime-boost’ trials.
The development of a safe and effective preventive HIV-1 vaccine remains a global priority. Faced with the lack of efficacy of immunogens designed to elicit the production of neutralizing antibodies, research has focused on induction of T-cell responses.
Since 1994, the French National Agency for Research on AIDS and Viral Hepatitis (ANRS) has pursued a vaccine strategy based on the utilization of HIV lipopeptides. Lipopeptides are large synthetic peptides coupled with a palmitoyl lipid tail. The lipid moiety facilitates peptide entry into antigen-presenting dendritic cells, thereby allowing efficient processing, and expression of CD4+ as well as CD8+ T-cell epitopes at the cell surface [1,2]. The choice of a series of long HIV-1 peptides allows immune responses to be directed toward several regions of different HIV-1 proteins rich in conserved epitopes.
Previous phase 1 studies have shown that HIV lipopeptides at a dose of 500 μg/lipopeptide elicit HIV-specific T-cell responses in HIV-uninfected volunteers [3–6] and HIV-infected patients [7–9] and induce long-term specific immune responses in healthy volunteers . A meta-analysis  of lipopeptide trials also showed that lipopeptide vaccine safety is acceptable in humans.
However, it is unknown whether immunogenicity of HIV-1 lipopeptides varies with the dose. In the current work, we used HIV-LIPO-5 ANRS vaccine, which contains five HIV-1 sequences from Gag, Pol and Nef proteins. The objective of this trial was to evaluate the cellular immunogenicity and safety of three doses of HIV-LIPO-5, versus placebo in human healthy volunteers, to select the lowest dose able to induce cellular immunogenicity that could be used in combination with other HIV vaccine candidates in further trials.
ANRS HIV-LIPO-5 is a mixture of an equal weight of five HIV-1 peptides linked with palmitic acid through an amide bound to the C-terminus of each peptide, as previously detailed . HIV-LIPO-5 includes the five following HIV-1 sequences: Gag 17–35, 253–284; Pol 325–355; Nef 66–97, 116–145 (Table 1). The vaccine, produced by Sanofi Pasteur (Lyon, France), was formulated in three lots corresponding to three doses: lot S3903F4 (500 μg of each peptide), lot S3903F3 (150 μg) and lot S3903F2 (50 μg). Each vaccine dose was lyophilized in a single-dose 7 ml vial and stored at −20°C. Prior to use, each vial was thawed at room temperature and reconstituted with 1 ml of a 5% isotonic glucose Tris–HCl (20 mmol/l) solution, also used as placebo in a prefilled syringe of 2 ml.
HIV-uninfected volunteers, from 21 to 55 years of age, at low risk of HIV infection, were prescreened according to the procedure established by the ANRS, as previously described . All participants provided written informed consent.
ANRS VAC18 was a multicenter, randomized, double-blind, phase 2 trial conducted at six HIV vaccine trial sites in France. The protocol was reviewed and approved by the Pitié Salpêtrière Hospital Ethics Committee (Paris, France). Participants were randomized to receive, intramuscularly in the deltoid muscle, four injections of three doses of HIV-LIPO-5 vaccine (50, 150 or 500 μg of each lipopeptide) or placebo at weeks 0, 4, 12 and 24.
All participants were observed for 30 min following vaccine administration to check for immediate local, systemic or both reactions. Participants were seen 2 weeks after each vaccination for safety assessment (solicited local and systemic reactions). Information on adverse events was collected throughout the trial.
Solicited adverse events included the following local and systemic reactions: pain, erythema, induration, nodule and edema at the injection site; and fever (at least 37.5°C), headache, vomiting, nausea, diarrhea, rash, myalgia and arthritis/arthralgia. In addition to complete blood count, platelets together with CD4+ and CD8+ cell counts, antibodies against DNA, nuclear, smooth muscle, mitochondrial and cardiolipin were examined at screening and weeks 12, 26 and 48. An Events Validation Committee reviewed all grade ≥2 events and all ophthalmological, neurological or autoimmune events, whatever the grade. An independent Data and Safety Monitoring Board periodically reviewed the unblinded data from the trial.
Assessment of immunogenicity
Immune responses were assessed at weeks 0 (before injection), 2, 6, 14, 26 and 48 on fresh peripheral blood mononuclear cells (PBMCs) within 24 h.
HIV-1 peptides used for assessment of CD4+ and CD8+ cell responses
All peptides were synthesized by NeoMPS (Strasbourg, France). Nine pools, N1A, N1B, N2A, N2B, N2C, G3, G2A, G2B, and P, presented in the Table 1 including 77 peptides of 8–11 amino acids were used for CD8+ T-cell responses analysis. Control peptides from influenza virus (16 peptides), Epstein–Barr virus (EBV) (16 peptides) and cytomegalovirus (CMV) (10 peptides) were also used. Seven long peptides, N1, N2, G2, G3, P1, P2 and P3, were used for CD4+ T-cell response analysis. These peptides from HIV-1 Nef, Gag and Pol proteins correspond to sequences included in HIV-LIPO-5, except for the Pol lipopeptide sequence, which was subdivided into peptides of 17–30 amino acids (P1, P2 and P3).
Interferon-gamma secretion by CD8+ T cells detected by ELISpot assay
To amplify the CD8+ T-cell responses, a one-step stimulation strategy was used (for details, see Ref. ). Briefly, 4 × 106 fresh PBMCs were incubated with pools of HIV peptides containing a maximum of 11 epitopes (2 μg/ml of each). After washing, cells were cultured and 10 IU/ml of interleukin-2 (IL-2) was added at days 2 and 7, and cells were collected at day 12 for an interferon-gamma (IFNγ) ELISpot assay. Negative controls consisted of PBMCs incubated in medium alone. To be assessable, a given cell line had to have a positive response to one pool of CMV, EBV or influenza virus peptides (see Table 2), or to phytohemagglutinin (PHA). The criteria for definition of a positive ELISpot response was defined, as done in a recent HIV vaccine trial by the same group , as a mean of triplicate wells exceeding 100 spot-forming units (SFUs)/106 PBMCs and at least three-fold higher than the negative control (unstimulated wells). The common ELISpot positivity criterion (>55 SFUs/106 PBMCs and at least four-fold higher than the background SFUs, by Merck Research Laboratories, Boston, Massachusetts, USA) was used to cross-validate responses detected at baseline. Response to a vaccine pool of HIV peptides was considered as positive if detected at least twice throughout the follow-up and if not detected at baseline before vaccination.
CD4+ T-cell proliferation
Fresh PBMCs (105 cells/well) were cultured in complete medium alone (background) or with 1 μg/ml of one of the soluble long peptides, in quadruplicate (Table 1.). Proliferation was determined after a 7-day culture by adding 1 μCi/well of [3H]thymidine (Amersham, Buckinghamshire, UK). The capacity of the PBMCs to proliferate in vitro was controlled with PHA (1 μg/ml), purified protein derivative (1 μg/ml) and staphylococcal enterotoxin B (0.1 μg/ml). [3H]Thymidine uptake was expressed as a stimulation index defined as the ratio of the median stimulated count per minute (cpm) to median unstimulated cpm and as a net cpm after subtraction of unstimulated cpm. An index of at least 3 and net cpm above 3000 were considered positive.
The primary endpoint was the rate of volunteers with CD8+ T-cell-sustained response defined as positive response against a given HIV-1 peptide pool at least on two separate occasions from week 2 to week 26. Secondary endpoints were the rates of volunteers presenting after vaccination: CD4+ T-cell proliferative response against at least one HIV-1 long peptide; sustained CD8+ T-cell polyepitopic response, that is, response to more than one of the five HIV-1 regions present in HIV-LIPO-5 detected at least twice from week 2 to week 26; durable CD8+ T-cell response detected at week 48, 6 months after the last vaccination and the rate of grade ≥2 local and systemic reactions.
This study was designed to show superiority of HIV-LIPO-5 over placebo in eliciting CD8+ T-cell-sustained response. A sample size of 30 participants per arm was used to detect differences for the primary endpoint with 80% overall power and 0.05 overall type I error, allowing three pairwise comparisons of LIPO-5 arms versus placebo (Bonferroni–Holm correction). The hypothesis was that CD8+ T-cell-sustained response rates in placebo and HIV-LIPO-5 recipients were 10 and 50%, respectively . Thirty-three volunteers were enrolled per arm, assuming that 10% of the participants would be nonassessable. Randomization was generated with the help of a computer by the trial statistician and was concealed to participants, clinical investigators, laboratory personnel and central coordinating office.
All analyses were based on an intent-to-treat approach that included all volunteers in their initial randomization group. Statistical comparisons were carried out using Wilcoxon, χ2 or Fisher exact tests, wherever appropriate. Statistical analyses were performed using Statistical Analysis System (SAS) version 9.1 (SAS Institute Inc., Cary, North Carolina, USA).
Recruitment and follow-up
The first enrolment began on 20 December 2005, and the last study visit was on 9 November 2007. Of the 226 participants screened, 132 participants were enrolled and randomly assigned to placebo (n = 32, placebo group), 50 μg (n = 32, LIPO-5 50 group), 150 μg (n = 33, LIPO-5 150 group) and 500 μg (n = 34, LIPO-5 500 group) of each lipopeptide (Fig. 1). One did not receive any injection and was excluded from the analysis. Fifty-seven (43.5%) participants were women, of median age 43.5 years (range 22.8–54.9 years) and median BMI 23.7 kg/m2 (range 17.3–34.5 kg/m2). Overall, 115 of 131 (87.8%) participants completed all four injections, with 129 (98.5%) completing follow-up to week 48. Sixteen participants discontinued vaccinations for the following reasons: autoimmune antibody detection after the third vaccination in six participants, noncompliance in four participants and adverse event in six participants, that is, peripheral neuropathy following metronidazole ingestion, which occurred 6 months after placebo injection, transient proteinuria (placebo), axonal diabetic neuropathy detected by a mild pain in extremity 2 weeks after vaccination, confirmed by electromyography and resolving in 2 weeks (LIPO-5 150), vagal malaise, generalized erythema and uterus fibroma (LIPO-5 500).
Table 3 shows frequencies of local symptoms and systemic clinical reactions possibly, probably or certainly related to vaccinations.
The incidence of local reactions was significantly higher in the LIPO-5 groups as compared with placebo. Most (98%) local reactions were mild and resolved spontaneously within a few days. They appeared to be dose-dependent as numbers of reported events were 24, 94, 110 and 169 in placebo, LIPO-5 50, LIPO-5 150 and LIPO-5 500 groups, respectively (P = 0.0315, R2 = 0.94 in linear regression on logarithmic dose). Systemic reactions, including headache, migraine, influenza, arthralgia and fever, were generally mild, although one vaccinee of the LIPO-5 500 group experienced a grade 3 systemic reaction (headache) that appeared 2 days after vaccination and resolved within 3 days.
Overall, similar percentages of participants – 40.6% [95% confidence interval (CI) 23.7–59.4], 31.3% (95% CI 16.1–50.0), 30.3% (95% CI 15.6–48.7) and 32.4% (95% CI 17.4–50.5) in placebo, LIPO-5 50, LIPO-5 150 and LIPO-5 500 groups, respectively – experienced a grade ≥2 adverse event judged possibly, probably or certainly to be vaccination associated (P = 0.43, 0.38 and 0.49 for comparisons of placebo with LIPO-5 50, LIPO-5 150 and LIPO-5 500, respectively).
There were nonsignificant trends or abnormalities in hematologic, renal or hepatic profiles or in CD4+ T-cell counts in the four groups. Positive autoantibodies were noted in five of 32 (15.6%, 95% CI 5.3–32.8) volunteers in the placebo arm versus eight of 99 (8.1%, 3.5–15.3) volunteers in the lipopeptide arms (P = 0.30). There were no clinical signs and no pattern of association of any type of autoantibodies with the vaccination. Seven serious adverse events were observed during the study: four in individuals receiving placebo (hypereosinophilia, paresthesia, rhabdomyolysis and proteinuria), one in the LIPO-5 150 group (elevation of phosphocreatine) and two in the LIPO-5 500 group (malaise and uterus fibroma).
Anti-HIV-1-specific CD8+ T-cell responses
Few responses were seen before vaccination (66 of 1125 pools tested) and, as shown in the Fig. 2, most of these responses were also detected after vaccination. These peptide-specific responses were not considered positive after vaccination but the volunteer remained in the analysis for the other peptide pools.
The rates of CD8+ responders in the three LIPO-5 groups were reached at week 6 (following two boosts of vaccine) and maintained at weeks 14 (following the third boost) and 26 (following the fourth boost) (Table 4).
The primary endpoint rate of CD8+ sustained response, at least at two evaluations between week 2 and week 26, was 68.8% (95% CI 50.0–83.9), 63.6% (95% CI 45.1–79.6) and 61.8% (95% CI 43.6–77.8) for the LIPO-5 50, LIPO-5 150 and LIPO-5 500 groups, respectively [Fig. 3a, P ≤ 0.0001 for each vaccine group as compared with 15.6% (95% CI 5.3–32.8) of responses in the placebo group]. All three comparisons remain significant with Bonferroni–Holm adjustment. As the 12-day culture ELISpot assay, gave an inherent background of IFNγ responses after stimulation with HIV peptide pools, we performed a logistic regression model after adjustment for baseline response. We found that comparisons between placebo and LIPO-5 groups were highly significant (P = 0.0032, 0.036 and 0.016 for low, medium and high-dose LIPO-5, respectively). Response lasted 6 months after the last vaccination in 45.8% (13/29), 34.5% (10/29) and 30.3% (10/33) for the low, medium and high-dose LIPO-5 groups [P = 0.0006, 0.0067 and 0.0146 for respective comparisons with only 6.5% (2/31) responses still detectable in the placebo group] (Fig. 3a).
The three doses of vaccine induced comparable polyepitopic responses. Globally, 32.3% LIPO-5 recipients (95% CI, 23.3–42.5) developed responses to at least two of the five HIV epitopes contained in the vaccine as compared to 0% (0–10.9) of placebo recipients (P < 0.0001, Fisher test) (Fig. 3b). Sixty one percent of responders (n = 39/64 volunteers) developed responses to at least one peptide pool included in Gag 253-284; 36% (n = 23) in Nef 116-145, 36% (n = 23) in Nef 66-97 and 33% (n = 21) in Pol 325-355 (Fig. 3c).
The magnitude and the kinetics of the response were similar in the three vaccine groups and peaked at week 6 (P = 0.004, for comparison with placebo group) with a median of 967 spot-forming cells (SFCs)/106 PBMCs (interquartile range = 393–2698), whereas lower magnitude appeared at weeks 2, 26 and 48 (median 720, 792 and 820, respectively) (Fig. 3d). Finally, although the study was not powered to compare responses between the three vaccine groups, there were no apparent differences between vaccinated groups in terms of rate or magnitude of IFNγ response.
CD4+ T-cell proliferative responses
Percentages of volunteers who developed CD4+ cell responses were at most 28.6% (28/98) and 30.5% (29/95) at weeks 6 and 14 in the LIPO-5 groups compared with 0% (0/31 and 0/29) in the placebo group, respectively (P = 0.0008 and 0.0007) (Table 4).
The cumulative percentages between week 2 and week 26 were 46.9% (95% CI 29.1–65.3), 54.6% (95% CI 36.4–71.9) and 44.1% (95% CI 27.2–62.1) LIPO-5 50, LIPO-5 150 and LIPO-5 500 groups, respectively [P < 0.0001 between LIPO-5 and placebo groups, 6.3% (95% CI 0.8–20.8) of controls with detectable responses]. With criterion used for lymphoproliferation in early HIV lipopeptides trials, that is, stimulation index greater than 3, 80 of 99 (81%) LIPO-5 recipients developed HIV-specific response, which is similar to results obtained in ANRS VAC04 trial using Nef 66–97, Nef 117–147, Nef 182–205, Gag 183–214, Gag 253–284 and Env 303–335 lipopeptides . At week 48, response was maintained in 13.8% (13/94) of HIV-LIPO-5 recipients. The majority of responses were induced to at least one peptide in Gag 253–284 for 36 of 48 (75%) then in Pol 325–355 for 22 (46%) and in Nef 66–97 for 13 (27%) HIV-LIPO-5 responders (Fig. 4a). Responses were most frequent at week 6 for Pol 325–355, and at week 6 or week 14 for Gag 253–284. Data showed no correlation between CD4 stimulation indexes and CD8 IFNγ SFCs (Fig. 4b).
In this placebo-controlled trial, almost 100 HIV-negative volunteers received different dosages of HIV-LIPO-5 vaccine. Globally, results show that the three dosages of vaccine were well tolerated and induced the generation of sustained CD8+ and CD4+ T-cell responses in around 70 and 50% of volunteers, respectively.
The objective of this trial was to determine the minimal dose of lipopeptide that might induce anti-HIV cellular immunity. For doing this, we chose to use cultured IFNγ ELISpot assay, a sensitive method to detect vaccine-elicited memory T-cell response as previously reported in other vaccination trials [13,14]. By more conventional criteria, previous phase I/II studies [4–6,8,15] have shown that lipopeptides do not induce ex-vivo T-cell responses assessed by IFNγ ELISpot assay. However, this latter test does not allow the detection of vaccine-specific effector memory T cells capable to expand under antigenic stimulation. This was recently confirmed by Winstone et al.  who showed that cultured assays are a valuable tool for studying the vaccine-induced frequencies of HIV-1-specific T cells. Moreover, it is clear from experimentation in nonhuman primates and from human clinical trials that ex-vivo ELISpot assays are not a reliable guide to study protection against simian immunodeficiency virus (SIV) or HIV infection as exemplified recently by the lack of efficacy of the Merck HIV adenovirus 5 vaccine despite a good immunogenic profile attested by ex-vivo ELISpot assays . The work of others also suggests that cultured assays detecting memory T cells rather than ex-vivo ELISpot assays are a correlate of immunity in SIV infection  and in malaria [19,20]. In malaria studies, it was shown that T-cell responses detected by cultured IFNγ ELISpot assay, but not by ex-vivo IFNγ ELISpot assay, correlated with protection against natural infection, and controlled malaria challenge in healthy individuals receiving prime-boost vaccination regimens. Indeed, the ex-vivo and cultured IFNγ ELISpot assays do not detect the same T-cell subsets . IFNγ-secreting cells detected by ex-vivo assay can be natural killer cells, whereas it is largely plausible that cytokine-producing cells induced after few days of culture with antigen have more chance of being the product of T-cell activation .
The specificity of T-cell responses was tested using pools of optimal HIV peptides (8–11 amino acids). Results strongly suggest that vaccination elicited CD8+ T-cell responses in a high number of volunteers (62–68%). These responses were sustained at least at two separate time points against a given HIV-1 CD8+ epitope pool. We also showed that these responses persisted in the majority of volunteers (48–55%) 6 months after vaccination, suggesting that HIV-LIPO-5 may expand long-lived and memory T cells. One limitation inherent to the high sensitivity of the culture assay is the detection of responses directed to some peptide pools with PBMCs from the placebo recipients. However, characteristics of those responses, which probably correspond to cross-reactivities against irrelevant antigens, differ significantly from those detected in vaccinated individuals in magnitude and durability.
We found that lymphoproliferative CD4+ T-cell responses to soluble long peptides were elicited in about 50% of vaccinees of the three lipopeptide groups compared with 6% of placebo recipients. At all time points, CD4+ T-cell responses were directed predominantly to Gag 253–284, followed by a response directed to Pol 325–355, and to a lesser degree to Nef 116–145. At the late detection time (week 48), we still detected responses directed to Gag 253–284, Nef 116–145 and Gag 17–35. These results confirm and extend data from previous ANRS studies performed in a lower number of healthy volunteers immunized with a mixture of six lipopeptides (500 μg each), injected intramuscularly (weeks 0, 4 16 and 48), with and without adjuvant .
In agreement with previous data [5,10], the current results showed maximal IFNγ secretion at weeks 6, 14 and 26. The five lipopeptides can induce the expansion of T cells secreting IFNγ and again the CD8+ epitopes from region Gag 253–284 were the most frequently recognized, followed by epitopes from Nef and Pol regions. This point is important for vaccinal purposes, considering that the anti-Gag p24 T-cell responses are generally described as the most efficient in decreasing viremia during HIV-1 infection [23,24].
With regard to safety of ANRS HIV-LIPO-5 vaccine, systemic reactions and changes in laboratory data were rare and not significantly enhanced in volunteers vaccinated with lipopeptides when compared with individuals of the placebo group. No grade 4 systemic event was seen in any of the groups. As requested by the regulatory authorities after the occurrence of an unexplained demyelinizing event in the HVTN042 trial combining HIV-1 lipopeptides and an HIV-1 ALVAC candidate (vCP1452) , we performed very close monitoring and found no evidence of vaccine-related abnormalities. Moreover, similar low rates of autoantibodies were observed in the four groups without clinical consequences in any case. Altogether, in the current study, we only observed mild local reactions that were dose-dependent, as previously reported . We conclude that HIV-LIPO-5 vaccine was well tolerated in HIV-uninfected humans and our results agree with those of previous phase 1 and phase 2 vaccine trials of HIV-1 lipopeptides performed in seronegative volunteers [3,5,11,25].
In this placebo-controlled trial, we showed that HIV-1 lipopeptides are able to induce in uninfected individuals anti-HIV T-cell responses that are sustained and polyepitopic. Combined with previous phase I/II studies [3–12,25], these new results contribute to provide a large set of data about the safety and immunogenicity of these candidate vaccines. Three doses of 50, 150 and 500 μg of lipopeptides were of similar efficiency in inducing CD4+ and CD8+ T-cell responses and globally equivalent specificities were recognized by stimulated T cells. This indicates that the lower dose is sufficient for the induction of a helper response and that higher doses do not have the capacity to significantly enhance this response. As local reactions are dose-dependent, unlike induction of cellular immune responses, which were similar for all lipopeptide doses, the lower dose appears appropriate to be used in further trials combining HIV-1 lipopeptides with modified vaccinia virus Ankara vaccines. However, the correlation between in-vitro tests and the prediction of in-vivo immunogenicity or protection remains unsettled and precludes drawing strong conclusions about which vaccine dose is appropriate. Studies to better characterize the phenotype and function of vaccine-specific T cells using multiparametric flow cytometry assay (IL-2/IFNγ/tumor necrosis factor-alpha) are ongoing. We investigate cytokine production of specific CD4+ and CD8+ T cells with five pools of HIV (Gag, Pol and Nef) 15-mers overlapping 11 amino acids.
Recent results from the Thai RV-144 trial  demonstrated that a ‘prime-boost’ strategy combining a recombinant Canary Pox (ALVAC) vector and a peptide-based vaccine (AIDSVAX gp120) might confer a level of protection in acquisition of HIV-1 infection. This study raises also the issue of the durability of this protection and suggests that repeated boost of vaccine might be necessary to maintain long-term protection. The HIV-LIPO5 ANRS vaccine was designed as a component of a ‘prime-boost’ strategy and could be used in combination with envelope protein vaccines such as those used in the Thai trial. In this regard, the demonstration that several boosts of low doses of HIV-LIPO-5 vaccine could be safely administered has important safety and cost implications.
Financial support was provided by French National Agency for Research on AIDS and Hepatitis (ANRS); Sanofi Pasteur provided HIV-LIPO-5 vaccine.
The authors fully acknowledge Jean-Gérard Guillet who developed this strategy in which he spent more than 15 years of his life. They acknowledge the participation of all the study volunteers, the support of colleagues (Benjamin Silbermann, Linda Belarbi, Paula Duchet-Niedziolka, Bao Phung, Zeina Absi, Camille Fontaine, Laurence Slama, Tuna Lukiana, Eka Chakvetadze, Mikael Berdah, Martine Obadia, Marie-Pierre Drogoul-Vey, Pascale Morineau) at the participating sites, at the ANRS (Véronique Rieux) and Dr Bernard Weill for reviewing autoantibody events.
The authors are grateful to:
1. Immunology group: A. Jackson, C. Flys, Y. Hénin and J. Choppin [Institut Cochin, Université Paris Descartes, CNRS (UMR 8104) Inserm, U567, Paris].
2. Coordinating pharmacy: C. Guérin and V. Dufau [Assistance Publique Hôpitaux de Paris (APHP), Hôpital Cochin, Paris].
3. Data and Safety Monitoring Board: S. Herson, E. Oksenhendler, B. Varet, C. Bazin, F. Carrat, M. Labetoulle, P. Pinel and P. Wermersch.
4. Events Validation Committee: B. Milpied, D. Rey, L. Moachon and C. Picard-Dahan.
5. Sanofi Pasteur: M. Renaud and C. Méric.
Authors contributions: D.S-C. conceived and designed the study, cocoordinated the study, interpreted the data and wrote the manuscript; C. Durier conceived and designed the study, did the statistical analysis, analyzed and interpreted the data and wrote the manuscript; C. Desaint conceived and designed the study, managed the clinical trial, interpreted the data and wrote the manuscript; L.C. cocoordinated the study, collected and interpreted the data and provided critical revisions to the manuscript; M.S. and N.B. collected the immunological data, interpreted the data and wrote the manuscript; J-D.L., B.B., G.P., I.P-M. collected the data and provided critical revisions to the manuscript; J-P.A. conceived and designed the study, analyzed and interpreted the data and provided critical revisions to the manuscript; Y.L. conceived and designed the study, interpreted the data and wrote the manuscript; O.L. conceived and designed the study, co-coordinated the study, interpreted the data and wrote the manuscript. All of them approved the final version of the manuscript.
Trials registration: ClinicalTrials.gov Identifier – NCT00121758.
There are no conflicts of interest.
Presented in part: AIDS Vaccine 2009 Conference; 19–22 October 2009; Paris, France [abstract #OA04-01].
1. Andrieu M, Loing E, Desoutter JF, Connan F, Choppin J, Gras-Masse H, et al
. Endocytosis of an HIV-derived lipopeptide into human dendritic cells followed by class I-restricted CD8+
T lymphocyte activation. Eur J Immunol 2000; 30:3256–3265.
2. Andrieu M, Desoutter JF, Loing E, Gaston J, Hanau D, Guillet JG, et al
. Two human immunodeficiency virus vaccinal lipopeptides follow different cross-presentation pathways in human dendritic cells. J Virol 2003; 77:1564–1570.
3. Gahery-Segard H, Pialoux G, Charmeteau B, Sermet S, Poncelet H, Raux M, et al
. Multiepitopic B- and T-cell responses induced in humans by a human immunodeficiency virus type 1 lipopeptide vaccine. J Virol 2000; 74:1694–1703.
4. Pialoux G, Gahery-Segard H, Sermet S, Poncelet H, Fournier S, Gerard L, et al
. Lipopeptides induce cell-mediated anti-HIV immune responses in seronegative volunteers. AIDS 2001; 15:1239–1249.
5. Salmon D, Gahery H, Silbermann B, Jackson A, Mazarin V, Souag N, et al
. Safety and immunogenicity of HIV lipopeptides associated or not to a live HIV recombinant canarypox (ALVAC-HIV, vCP1452) in non-HIV-infected volunteers (ANRSVAC10)
[abstract #57]. In: AIDS Vaccine
; 30 August–1 September 2004; Lausanne, Switzerland; 2004.
6. Launay O, Durier C, Desaint C, Silbermann B, Jackson A, Pialoux G, et al
. Cellular immune responses induced with dose sparing intradermal administration of HIV vaccine to HIV uninfected volunteers in the ARNS VAC 16 Trial. PLoS One 2007; 2:e725.
7. Lévy Y, Gahéry-Ségard H, Durier C, Lascaux AS, Goujard C, Meiffrédy V, et al
. Immunological and virological efficacy of a therapeutic immunization combined with interleukin-2 in chronically HIV-1 infected patients. AIDS 2005; 19:279–286.
8. Gahery H, Daniel N, Charmeteau B, Ourth L, Jackson A, Andrieu M, et al
. New CD4+
T cell responses induced in chronically HIV type-1-infected patients after immunizations with an HIV type 1 lipopeptide vaccine. AIDS Res Hum Retroviruses 2006; 22:684–694.
9. Pialoux G, Quercia RP, Gahery H, Daniel N, Slama L, Girard PM, et al
. Immunological responses and long-term treatment interruption after human immunodeficiency virus type 1 (HIV-1) lipopeptide immunization of HIV-1-infected patients: the LIPTHERA study. Clin Vaccine Immunol 2008; 15:562–568.
10. Gahery-Segard H, Pialoux G, Figueiredo S, Igea C, Surenaud M, et al
. Long-term specific immune responses induced in humans by a human immunodeficiency virus type 1 lipopeptide vaccine: characterization of CD8+
-T-cell epitopes recognized. J Virol 2003; 77:11220–11231.
11. Durier C, Launay O, Meiffrédy V, Saïdi Y, Salmon D, Lévy Y, et al
. Clinical safety of HIV lipopeptides used as vaccines in healthy volunteers and HIV-infected adults. AIDS 2006; 20:1039–1049.
12. Klinguer C, David D, Kouach M, Wieruszeski JM, Tartar A, Marzin D, et al
. Characterization of a multilipopeptide mixture used as an HIV-1 vaccine candidate. Vaccine 1999; 18:259–267.
13. Tassignon J, Burny W, Dahmani S, Zhou L, Stordeur P, Byl B, et al
. Monitoring of cellular responses after vaccination against tetanus toxoid: comparison of the measurement of IFN-γ production by ELISA, ELISPOT, flow cytometry and real-time PCR. J Immunol Methods 2005; 305:188–198.
14. Goonetilleke N, Moore S, Dally L, Winstone N, Cebere I, Mahmoud A, et al
. Induction of multifunctional human immunodeficiency virus type 1 (HIV-1)-specific T cells capable of proliferation in healthy subjects by using a prime-boost regimen of DNA- and modified vaccinia virus Ankara-vectored vaccines expressing HIV-1 Gag coupled to CD8+
T-cell epitopes. J Virol 2006; 80:4717–4728.
15. Frey S, Peiperl L, Baden L, McElrath J, Wright P, Goepfert P, et al
. Immunogenicity of ANRS LIPO-5 alone, Sanofi-Pasteur ALVAC-HIV(vCP1452) alone, and ALVAC HIV Prime/LIPO-5 boost in healthy, HIV-1 uninfected adult participants
[abstract #P09B-21]. Antivir Ther
2006; 11 (Suppl 2)
16. Winstone N, Guimarães-Walker A, Roberts J, Brown D, Loach V, Goonetilleke N, et al
. Increased detection of proliferating, polyfunctional, HIV-1-specific T cells in DNA-modified vaccinia virus Ankara-vaccinated human volunteers by cultured IFN-gamma ELISPOT assay. Eur J Immunol 2009; 39:975–985.
17. Buchbinder SP, Mehrotra DV, Duerr A, Fitzgerald DW, Mogg R, Li D, et al
. Efficacy assessment of a cell-mediated immunity HIV-1 vaccine (the Step Study): a double-blind, randomised, placebo-controlled, test-of-concept trial. Lancet 2008; 372:1881–1893.
18. Sun Y, Schmitz JE, Buzby AP, Barker BR, Rao SS, Xu L, et al
. Virus-specific cellular immune correlates of survival in vaccinated monkeys after simian immunodeficiency virus challenge. J Virol 2006; 80:10950–10956.
19. Reece WH, Pinder M, Gothard PK, Milligan P, Bojang K, Doherty T, et al
. A CD4(+) T-cell immune response to a conserved epitope in the circumsporozoite protein correlates with protection from natural Plasmodium falciparum
infection and disease. Nat Med 2004; 10:406–410.
20. Keating SM, Bejon P, Berthoud T, Vuola JM, Todryk S, Webster DP, et al
. Durable human memory T cells quantifiable by cultured enzyme-linked immunospot assays are induced by heterologous prime boost immunization and correlate with protection against malaria. J Immunol 2005; 175:5675–5680.
21. Flanagan K, Lee E, Gravenor M, Reece W, Urban B, Doherty T, et al
. Unique T cell effector functions elicited by Plasmodium falciparum
in Malaria-exposed Africans tested by three T cell assays. J Immunol 2001; 167:4729–4737.
22. Desombere I, Clement F, Rigole H, Leroux-Roels G. The duration of in vitro stimulation with recall antigens determines the subset distribution of interferon-g producing lymphoid cells: a kinetic analysis using the interferon-g secretion assay. J Immunol Meth 2005; 301:124–139.
23. Kiepiela P, Ngumbela K, Thobakgale C, Ramduth D, Honeyborne I, Moodley E, et al
T-cell responses to different HIV proteins have discordant associations with viral load. Nat Med 2007; 13:46–53.
24. Rolland M, Heckerman D, Deng W, Rousseau CM, Coovadia H, Bishop K, et al
. Broad and Gag-biased HIV-1 epitope repertoires are associated with lower viral loads. PLoS One 2008; 3:e1424.
25. Frey S, Peiperl L, Baden L, McElrath J, Wright P, Goepfert P, et al
. Safety of ANRS LIPO-5 alone, sanofi-pasteur ALVAC-HIV(VCP1452) alone, and ALVAC HIV prime/LIPO-5 boost in healthy, HIV-1 uninfected adult participants
[abstract #300P]. In: AIDS Vaccine
; 6–9 September 2005; Montréal, Québec, Canada; 2005. p. 122.
26. Rerks-Ngarm S, Pitisuttithum P, Nitayaphan S, Kaewkungwal J, Chiu J, Paris R, et al
. Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. N Engl J Med 2009; 361:2209–2220.
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