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Evaluation of SIV-lipopeptide immunizations administered by the intradermal route in their ability to induce antigen specific T-cell responses in rhesus macaques

Zoe Coutsinos, Pascale Villefroy, Helene Gras-Masse, Jean-Gerard Guillet, Isabelle Bourgault-Villada
DOI: http://dx.doi.org/10.1016/j.femsim.2004.09.003 357-366 First published online: 1 March 2005

Abstract

Numerous clinical and experimental observations have shown that cellular immunity, in particular CD8+ T-lymphocytes, plays an important role in the control of HIV infection. We have focused on a lipopeptide vaccination strategy that has been shown to induce polyepitopic T-cell responses in both animals and humans, in order to deliver simian immunodeficiency virus (SIV) antigens to rhesus macaques. Given the relevance of antigen administration route in the development of an effective cellular immune response, this study was designed to assess SIV lipopeptide immunizations administered either by the intradermal (ID) or the intramuscular (IM) routes in their ability to elicit GAG and NEF multispecific T-lymphocytes in the rhesus macaque. Antigen specific T-cell responses were observed between 7 and 11 weeks following vaccination in both groups. Macaques immunized by the IM route yielded antigen-specific IFN-γ secreting lymphocytes in response to no more than two pools of peptides derived from SIV-NEF. In contrast, among the four ID-immunized macaques, two presented multi-specific T-cell responses to as many as four pools of SIV-NEF and/or GAG peptides. Responses persisted 16 weeks following the vaccination protocol in one of the ID-vaccinated macaques. The induction of such responses is of great clinical relevance in the development of an effective HIV vaccine. Given the crucial role of CD8+ T-lymphocytes in HIV/SIV containment, vaccination through the intradermal route should merit high consideration in the development of an AIDS vaccine.

Keywords
  • Intradermal route of immunization
  • SIV-lipopeptide vaccination
  • Induction of T-lymphocytes

1 Introduction

Faced with the human immunodeficiency virus (HIV) pandemic, one of the international priorities has been to define and put forth effective methods destined to fight and prevent this pathogen. The introduction of highly active antiretroviral therapy (HAART) has improved the prognosis of HIV-infected patients [1]. This treatment decreases viral load and controls viral replication with a consequence in prolonging patients' lives. Nevertheless, antiretroviral therapy is not accessible to patients worldwide and, till now, there exists no cure for HIV persistence. Therefore, an effective vaccine will undoubtedly play a very substantial role as a weapon against HIV.

There are many challenges which have been encountered in the search of a vaccine against HIV. The very properties of this virus such as its antigenic diversity, hypervariability and mutation frequency have hampered vaccine development. HIV also uses a number of natural immune evasion strategies to achieve persistent replication and elude the immune system [2]. An appropriate vaccine must therefore target, in particular, the most highly conserved viral epitopes for mounting an effective cellular immune response. Numerous clinical and experimental observations have in fact shown that cellular immunity plays an important role in the control of HIV/SIV infections particularly in containing viral replication. Virus specific cytotoxic T-lymphocytes (CTL) have been shown to control primary viremia in humans [3,4] where decline of viral load coincided with the increase of virus specific CTL in the peripheral blood. In chronically infected individuals, CTL have also been shown to control viral replication [57].

The functional importance of virus specific CTL responses was confirmed in the macaque model by in vivo depletion of CD8+ T-lymphocytes in both primary and chronically SIV-infected animals [810]; a rapid and dramatic increase in viremia was detected in the CD8+ depleted macaques. Moreover, vaccines that elicit potent CTL responses reduce viral replication and slow clinical disease progression following pathogenic viral challenge [1117].

The specificity as well as the breadth of the CTL responses are factors that are primordial in controlling viral spread. Indeed, patients with a reduced breadth of potential CTL epitopes seem to have a reduced capacity to control viral replication [1820]. Macaque vaccination studies suggest that single viral epitope-specific CTL response induced by immunization may not be sufficient in blocking SIV infection [21]. In fact, selection of virus variants and emergence of virus-escape mutants can be observed when the induced CTL response is limited to a single epitope [22,23]. Therefore, the induction of CTL responses by vaccination should be as broad and as multispecific as possible in order to avoid the selection of viral variants and viral mutants.

With the aim of inducing multispecific CTL in rhesus macaques, we have focused on a lipopeptide vaccine approach [24]. Lipopeptide vaccination has been shown to induce CTL responses in both macaques [17,22,25,26] and humans [27,28]. We were particularly interested in evaluating the potential of the intradermal route to stimulate broad T-cell responses prior to the initiation of human lipopeptide vaccine trial using this route. Indeed, the skin is an attractive target since it plays an important role in immunological host defenses particularly the Langerhans cells residing in the epidermis and the dermal dendritic cells residing in the dermis. The relevance of the ID route of inoculation in the induction of cellular immune responses has already been demonstrated in numerous animal models using various immunogens [2933]. Therefore, the present study was designed to assess lipopeptide immunizations administered by the ID route in their ability to elicit GAG and NEF multispecific CD8 T-lymphocytes in the rhesus macaque.

2 Materials and methods

2.1 Lipopeptides and related peptides

The synthesis of the seven lipopeptides used to vaccinate the macaques has previously been described [34]. Briefly, five NEF and two GAG lipopeptides whose amino acid sequences where derived, respectively, from the NEF and GAG proteins of molecular clone BK28, were synthesized by solid-phase synthesis and contained at the C-terminal position an enantiomerically pure Nε-palmitoyl-lysylamide (Table 1). The lipopeptides, purified by reverse-phase HPLC, were formulated as mixed micelles. Short overlapping peptides (Table 2) as well as seven long peptides whose sequence corresponded to the peptide moiety of the lipopeptides used in the immunizations were purchased from Neosystem (Strasbourg, France).

View this table:
Table 1

Lipopeptides and corresponding long peptide sequences

LipopeptideLong peptide Lipopeptide sequence
N1Nef 101–126SVRPKVPLRAMTYKLAIDMSHFIKEK K(PAM)NH2
N2Nef 125–147EKGGLEGIYYSARRHRILDMYLE K(PAM)NH2
N3Nef 155–178DWQDYTSGPGIRYPKTFGWLWKLV K(PAM)NH2
N4Nef 201–225SKWDDPWGEVLAWKFDPTLAYTYEA K(PAM)NH2
N5Nef 221–247YTYEAYARYPEELEASQACQRKRLEEG K(PAM)NH2
G1Gag 165–195KFGAEVVPGFQALSEGCTPYDINQMLNCVGD K(PAM)NH2
G2Gag 246–281QIQWMYRQQNPIPVGNIYRRWIQLGLQKCVRMYNPTN K(PAM)NH2
  • Amino acid positions are identified with reference to the sequence of molecular clone BK28.

  • Sequences were modified in the C-terminal position by a palmitoyl-lysylamide group, K(PAM)NH2.

View this table:
Table 2

Pool constitution and sequenses of peptides used for in vitro stimulation

Tested poolsPool 1Pool 2Pool 3Pool 4Pool 5Pool 6Pool 7Pool 8Pool 9Pool 10
Pool constitution101–109126–134156–165201–211214–223221–235166–185250–257257–266268–276
106–113129–136168–177205–213210–219225–240176–195270–277259–267269–277
110–119136–144165–174211–219203–211232–247249–257261–269270–278
116–124128–136157–165215–225206–215251–259262–270250–259
103–111128–137169–178201–210215–223255–263263–271253–262
108–115133–141166–174205–212204–212256–264267–275262–271
112–119158–167266–275
116–126162–171
167–175
Corresponding long peptideNEF 101–126NEF 125–147NEF 155–178NEF 201–225NEF 221–247GAG 165–195GAG 246–281

2.2 Animals and experimental protocol

A group of male rhesus macaques (Macaca mulatta) (95643, 95647, 9493 and 9497) were immunized intradermally (ID) with a mixture of seven lipopeptides (500 µg of each lipopeptide) on weeks 0, 3 and 6. The lipopeptides were resuspended in a total volume of 1 ml of isotonic saline solution. The three ID immunizations were done as follows: the animals' backs were shaven and the immunogen was administered at 10 injection sites (100 µl per injection). The needle was inserted into the epidermis at an angle parallel to the long axis of the back and care was taken to ensure that the entire bevel penetrated the skin. The injected solution raised a small bleb. Another group of macaques (94327, 95571 and 9491) were immunized intramuscularly with the same lipopeptide preparation. These animals received three IM injections at the upper anterolateral portion of the thigh muscle. The macaques were selected irrespective of their MHC type. Care of non-human primates was in accordance with European guidelines.

2.3 Isolation of peripheral blood mononuclear cells

Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation through lymphocyte separation medium (Pharmacia, Uppsala, Sweden). The cells were then washed twice in culture medium (RPMI 1640, 100 U ml−1 penicillin, 100 µg ml−1 streptomycin, 2 mM l-glutamine, 1% non-essential amino acids, 1 mM sodium pyruvate, 10 mM HEPES buffer, 20 µM β-mercaptoethanol) supplemented with 10% heat-inactivated fetal calf serum (FCS). The isolated PBMC were immediately used for testing.

2.4 Generation of in vitro cultured effector cells (short term cell lines)

Following an over-night incubation with 10 µg ml−1 of each of the seven long peptides (the peptide equivalent of the lipopeptides used in the immunizations) in the above mentioned culture medium, PBMC were washed and resuspended at 2 ×106 ml−1 in 24 well microtiter plates (Costar, Cambridge, MA). Human rIL2 (10 IU ml−1, Boehringer Mannheim, Germany) was added at days 3 and 7 and cells were tested 12 days after restimulation in an ELISpot-IFNγ assay. PBMC cultivated in presence of IL2 and without any peptide were used as control of anti-peptide specificity of T-cell activity.

With respect to IFNγ ELISpot assay sensitivity, Russel et al. [35] recently demonstrated that the total HIV-specific IFNγ-secreting cells in HIV infected patients are present in frequencies at least 1 log greater than those induced by vaccination of HIV-uninfected subjects. In this regard, IFNγ ELISpot assay done after one in vitro stimulation was appropriate in the present study to identify frequencies of circulating effector T-cells.

2.5 ELISPOT-IFNγ assay

Nitrocellulose-backed microplates (96 wells; Millipore) were coated overnight at 4 °C with murine anti-human IFNγ monoclonal antibody (0.05 µg; Mabtech, Nacka, Sweden). The wells were washed six times with PBST (PBS containing 0.05% Tween 20) and saturated with culture medium supplemented with 10% FCS for 2 h at 37 °C in 5% CO2. Duplicate aliquots of short term cell lines were plated (2 ×105/well) and incubated with pools of overlapping short peptides (5 µg ml−1) for 24 h at 37 °C in 5% CO2. Ten distinct peptide pools containing a combined total of 59 short overlapping peptides representing the entire 5 SIV-NEF and 2 SIV-GAG sequences used to vaccinate the animals were tested (Table 2). Peptide pools, which stimulated IFNγ production above our criteria of positivity in pre-immune macaques, were excluded from the assay (data not shown).

Negative control wells, which were used to determine positivity threshold contained fresh PBMC without any peptide in the presence of 10% FCS. Positive controls were obtained by activating PBMC with 50 ng ml−1 phorbol myristate acetate and 500 ng ml−1 ionomycine (Sigma–Aldrich Chimie SARL) (2000 cells/well). Following incubation of PBMC, the cells were removed and the wells were washed six times with PBST. Murine anti-human biotinylated anti-IFNγ (0.1 µg; Mabtech, Nacka, Sweden) was added into each well. After a 2 h incubation at 37 °C, the plates were washed six times with PBST. Spot forming cells (SFC) were revealed by adding successively alkaline-phosphatase labeled extravidin (2.5 µg ml−1; Sigma) and tetrazolium nitro-blue substrate (Bio-Rad) with incubation times of 1 h and 30 min, respectively, at 37 °C. Finally, the wells were washed with distilled water and air-dried. Spots, each of which corresponds to an IFNγ-secreting cell, were counted using a transmitted-light stereomicroscope connected to a camera, using an image analyzing software (KS ELISPOT System, Carl Zeiss Vision, Germany). The results were expressed as the number of antigen specific IFNγ SFC/106 PBMC. For each experiment, a positivity threshold was defined as being the mean value SFC of the control wells to which was added 2.5 times the mean standard deviation.

2.6 Antigen-specific proliferation assay

Triplicate cultures of fresh PBMC (2 ×105/well in 96-well microplates) were performed in complete culture medium. Various concentrations of the seven long peptides (1, 5 and 10 µg ml−1), medium alone (negative control), or 10 µg of concanavalin A (ConA) ml−1 (positive control) was added. Cultures were incubated at 37 °C in a 5% humidified atmosphere for 96 h, pulsed with 1 µCi of [3H] thymidine (Amersham)/well and harvested after 18 h. The mean radioactivity (counts per minute) of the antigen containing cultures was divided by the mean radioactivity of the medium only control cultures to derive a proliferation index.

3 Results

3.1 Immune responses following intradermal lipopeptide immunizations

Assessment of antigen specific immune responses was performed prior to immunization, after each consecutive lipopeptide administration, between weeks 7 and 11 and on week 16 following the last lipopeptide injection. Detectable responses using a sensitive IFNγ ELISpot assay began to appear after the third immunization (data not shown). Fig. 1 shows the antigen-specific responses that were observed between weeks 7 and 11 in the macaques vaccinated by the intradermal route. Up to four NEF and/or GAG peptide pools were recognized by IFNγ-secreting T-cells in these animals. Macaque 95643 had IFNγ-secreting lymphocytes in response to four pools of short peptides derived from both SIV-NEF (pools 3 and 4 contained in NEF 155–178 and NEF 201–225, respectively) and -GAG (pools 9 and 10 from GAG 246–281) proteins. Macaque 9493 had a better overall response yielding high frequencies of IFNγ-secreting cells to peptide pools derived from four out of the seven long peptides (pool 5–8 included respectively in long peptides NEF 201–225, NEF 221–247, GAG 165–195 and GAG 246–281) whose lipopeptide equivalents were used for the immunizations.

Figure 1

ELISpot-IFNγ responses of intradermally immunized macaques evaluated 7–11 weeks following the third lipopeptide immunization. Four macaques (95647, 9497, 9493 and 95643) were immunized with a mixture of seven SIV-lipopeptides. T-cell effector responses, tested against ten pools of overlapping peptides derived from SIV-NEF and GAG proteins, were evaluated by performing a sensitive IFNγ-ELISpot assay. Results are expressed as number of IFNγ spot-forming cells (SFC) per 106 PBMC. Positivity threshold was defined as being the mean value SFC of the control wells to which was added 2.5 times the standard deviation (horizontal dotted line). A result was considered non-interpretable when there was an elevated variability of the ELISPOT IFNγ SFC counts within the different wells of a particular sample. *non interpretable.

In the third responding macaque (9497), a positive response was identified against NEF-specific peptide pool 4, corresponding to short peptides derived from SIV-NEF 201–225.

Finally, macaque 95647 showed no significant response to any of the tested peptide pools as compared with the response to medium alone which was markedly elevated.

All controls of anti-peptide specificity of T-lymphocytes obtained by cultivating effector cells only in the presence of IL2 were negative (data not shown).

3.2 Immune responses following intramuscular lipopeptide immunizations

As in the ID-vaccinated group, IM-immunized macaques showed detectable antigen-specific responses following the third lipopeptide injection (data not shown). Between 7 and 11 weeks post-vaccination, PBMC from two out of three monkeys immunized by the IM route contained T-cells that secreted IFNγ in response to either one or two peptide pools (Fig. 2). Macaque 94571 responded only to pool 6 which contained a mixture of short peptides derived from SIV-NEF 221–247. Macaque 94327 yielded antigen-specific IFNγ-secreting lymphocytes in response to peptide pools 3 and 4, corresponding respectively to SIV-NEF 155–178 and SIV-NEF 201–225. None of the SIV-GAG derived peptide pools (pools 7–10) was able to stimulate a significant number of IFNγ producing T-lymphocytes in any of the macaques vaccinates by the IM route. As for macaque 9491, no significant response was detected to any of the tested pools.

Figure 2

ELISpot-IFNγ responses of intramuscularly immunized macaques evaluated 7–11 weeks following the third lipopeptide immunization. Three macaques (94327, 9491 and 94571) were immunized with a mixture of seven SIV-lipopeptides. T-cell effector responses, tested against ten pools of overlapping peptides derived from SIV-NEF and GAG proteins, were evaluated by performing a sensitive IFNγ-ELISpot assay. Results are expressed as number of IFNγ spot-forming cells (SFC) per 106 PBMC. Positivity threshold was defined as being the mean value SFC of the control wells to which was added 2.5 times the standard deviation (horizontal dotted line). A result was considered non-interpretable when there was an elevated variability of the ELISPOT IFNγ SFC counts within the different wells of a particular sample. *non interpretable.

3.3 Lymphoproliferative responses in macaques following lipopeptide vaccination

Lymphoproliferative responses were systematically evaluated in both intradermally and intramuscularly SIV-lipopeptide-vaccinated macaques following each immunization. Prior to vaccination no proliferation was found against any of the seven long peptides tested. Out of the four ID-injected macaques, only 95643 had significant proliferative responses against long peptides N1, N4 and G2, as of the second lipopeptide administration. Fig. 3 depicts the proliferative responses of this animal two weeks following the third immunization. Peptides N4 and G2 stimulated the highest responses followed by peptide N1. Intramuscular injections of lipopeptides did not induce any detectable proliferative responses against the immunizing peptides.

Figure 3

Lymphoproliferative responses of macaque 95643. PBMC of macaque 95643 were collected 2 weeks after the third SIV-lipopeptide injection. Cells (2 ×105) were cultured with various concentrations (1, 5 and 10 µg/ml) of SIV long peptides and proliferation was evaluated at day-4 by [3H]thymidine incorporation. The long peptides included NEF 101–126 (N1), NEF 125–147 (N2), NEF 155–178 (N3), NEF 201–225 (N4), NEF 221–247 (N5), GAG 165–195 (G1) and GAG 246–281 (G2). The capacity of PBMC to proliferate in vitro was tested in independent culture with 10 µg/ml of ConA. PBMC strongly proliferated in response to this antigen (data not shown). A positive index was defined as an index of ≥3.

3.4 Fine characterization of one NEF and one GAG epitope in an ID-immunized macaque

A further analysis of the IFNγ-secreting capacity of PBMC isolated from the ID immunized macaques was conducted 16 weeks post-vaccination. The number of IFNγ-producing cells detected at this time point, in each of the animals, was reduced to non-significant levels (data not shown) except for macaque 95643 vaccinated by the ID route. Indeed, the response of this animal persisted at least four months after immunization and was still multispecific. A fine characterization of two peptide pools (derived either from SIV-NEF 201–225 or SIV-GAG 246–281) which were previously recognized by IFNγ-secreting T-lymphocytes on week 7 and still maintained this capacity at week 16 is shown in Fig. 4. The minimal epitope, within the 25-mer sequence of N4 (NEF 201–225), able to stimulate IFNγ secretion was identified as NEF 211–219. Likewise, a mixture of short peptides GAG 263–271 and GAG 267–275 within G2 (GAG 246–281) significantly stimulated IFNγ secretion in effector cells.

Figure 4

Fine characterization of the polyepitopic responses in intradermally immunized macaque 95643. IFNγ-secreting capacity of PBMC isolated from ID-immunized macaque 95643 was conducted 16 weeks post-vaccination by performing an IFNγ-ELISpot assay. Results are expressed as number of IFNγ spot-forming cells (SFC) per 106 PBMC. Positivity threshold was defined as being the mean value SFC of the control wells to which was added 2.5 times the standard deviation (horizontal dotted line). A: Pools H, I, J and K are included in pool 4 (derived from SIV-NEF 201–225) recognized by effector cells at week 7. B: Pools L, M and N are included in pool 9 (derived from SIV-GAG 246–281) recognized by effector cells at week 7.

4 Discussion

Given that the route of immunization can play an important role in the development of an effective immune response, because of antigen targeting to different antigen presenting cells, the present study was designed to assess lipopeptide immunizations administered by the intradermal route in their ability to elicit specific T-lymphocytes in the rhesus macaque. For this purpose, macaques were vaccinated intradermally with a mixture of seven lipopeptides, five of which were derived from SIV-NEF and two from SIV-GAG. A second group of macaques was immunized intramuscularly with the same preparation. The intramuscular route of administration was explored in this context given that this route is, thus far, exclusively used in all human HIV-lipopeptide vaccine trials. The NEF and GAG proteins were selected for their high immunogenicity and because they include a large number of CTL epitopes previously determined in SIV infected macaque or analog to epitopic peptides defined in HIV-infected patients [3638].

Our results indicate that lipopeptides injected intradermally in rhesus macaques elicited T-cell responses in three out of the four immunized macaques. These responses were multispecific in two animals that had T-cells directed against four pools of peptides derived from NEF and/or GAG proteins. Negative controls obtained by stimulating PBMC only in presence of IL2 did not exhibit any activity, thereby eliminating a non-antigen specific lymphokine activated killer activity.

The ELISpot assay presented in this study makes use of a large panel of short peptides compatible with MHC class I presentation. Indeed, the TCR on CD8+ T-cells recognizes epitopes between 8 and 11 amino acids in length and restricted by class I MHC and the TCR on CD4+ T-cells can recognize epitopes of around 13–25 amino acids that are restricted by class II MHC. Thus, it is appropriate to conclude that the responses observed in intradermally vaccinated macaques 9497 and 95643 are most probably mediated by CD8+ cells since these two animals had T-cell responses directed against pools containing exclusively short peptides. As for the responses of macaque 9493, they were likely mediated by CD8+ T-cells for pools 5 and 8, however, for pools 6 and 7 the presence of CD4+ T-cell responses cannot be excluded given that the length of the tested peptides in these pools were between 15 and 20 amino acids, compatible with both classes I and II presentation.

In order to evaluate the accurate kinetics of the T-cell response, the immune response was quantified at several time points following each lipopeptide immunization. Detectable responses began to appear after the third lipopeptide injection. Strong cell-mediated responses were measurable between 7 and 11 weeks following the immunization protocol and began waning off thereafter to undetectable levels at week 16 with the exception of one intradermally inoculated macaque (95643). Interestingly, the T-cell responses of this animal on week 16 were directed against pools of short peptides included in long peptides NEF 201–225 and GAG 246–281, confirming the CD8+ nature of these T-cell responses. It is well known that a successful CD8+ mediated memory immune response requires the help of antigen specific T CD4+ helper cells [39]. As the lipopeptides used for immunization in this study can be presented by MHC class II molecules, the persistence in time of the immune response of macaque 95643 is most probably associated to the activation of CD4+ T helper cells. Moreover, out of the vaccinated macaques, only 95643 had a positive proliferative response against three long peptides (NEF 101–126, NEF 201–225 and GAG 246–281). Nevertheless, given that depletion experiments were not performed in this study, we are unable to formally conclude whether the observed proliferative responses were exclusively due to CD4+ T-lymphocytes and whether the latter were, in this case, critical for the induction of memory CD8+ specific T-cells.

The potential of the ID route to induce broad immune responses is likely attributable to the immunologically potent dermis, which contains dermal dendritic cells involved in the initiation of immune responses [40]. Indeed, these cells have strong antigen-uptake properties in their immature state. As they mature, their antigen-processing and antigen-presentation functions increase [41]. In our system, the delivery of lipopeptides in close proximity to the dermal dendritic cells through ID injection could facilitate the antigen uptake process. Indeed, in vitro fluorescent confocal microscopy studies revealed that lipopeptides can be internalized into dendritic cells [42]. Once internalized, lipopeptides are processed by the exogenous pathway allowing the presentation of degraded peptides by MHC class II molecules. Although the exact processing and presentation of lipopeptides leading to the association with MHC class I molecules have not been fully elucidated, a fraction of the lipopeptide was observed translocating directly into the cytoplasm. This was followed by cross-presentation and release of the peptide into the cytosol leading to an association of a short peptide to MHC class I molecules [42]. It is therefore conceivable that the ID route favors the targeting of lipopeptides to dermal dendritic cells and presentation of the processed antigens both to CD4+ and CD8+ T-lymphocytes.

This study moreover shows that SIV lipopeptides administered intramuscularly are capable of inducing T-cells recognizing pools of peptides derived from NEF protein in 2/3 immunized macaques. In term of response breadth, the macaques vaccinated intramuscularly developed a T-cell response against only one or two peptide pools, whereas up to four peptide pools were recognized by T-lymphocytes of intradermally vaccinated animals. This observation is compatible with the presence of small number of antigen-presenting cells in the muscle [43]. It should be noted that the frequencies of macaques that respond to immunization do not appear to significantly differ between the two arms of the study: 3/4 in ID group and 2/3 in IM group, respectively. These groups are statistically equivalent and we are therefore unable to draw comparative conclusions between intradermal and intramuscular routes in terms of efficacy of immune response induction.

The efficacy of intradermal immunization has previously been reported in human clinical studies of hepatitis B and rabies vaccines [4447]. Likewise, in numerous animal models, it has been shown that ID administrations can stimulate both strong antibody and cell mediated immune responses [29,30,32,33,48]. Moreover, intradermal injections have been shown to require one tenth of an intramuscular dose to elicit an equivalent antibody response and seroconversion rate [29].

In conclusion, this study validates the ID route as immunogenic for the induction of T-cell responses using lipopeptides in rhesus macaques. It furthermore demonstrates the ability of SIV-derived lipopeptides to induce broad immune responses when injected by the intradermal route. Breadth of T-cell responses likely influences the effectiveness of T-cells which play a primordial role in the control of viral load during primary HIV/SIV infection, as well as during chronic disease. Therefore, the induction of multiepitopic T-cell responses in vivo through intradermal lipopeptide injection is of great clinical relevance in the development of an efficient HIV vaccine. The high immunogenicity of intradermally injected lipopeptides as well as the ease of administration render this route of administration very promising in this context. This pre-clinical study was primordial prior to the setup of a Phase I multi-centric human volunteer trial using HIV derived lipopeptides currently underway which evaluates the potency of ID route to induce specific T-cell responses.

Acknowledgements

This work was supported by funding from the Agence Nationale de Recherche sur le SIDA (ANRS) and the Pasteur Institute of Lille. ZC was initially supported by a fellowship from the ANRS and subsequently from the ECS (Ensemble Contre le SIDA). We thank Bruno Hurtrel for handling and care of the macaques.

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View Abstract