Conceived and designed the experiments: IB JC GP OL CD HG DS JG. Performed the experiments: OL SF MS JG NB. Analyzed the data: GP CD AJ JG. Wrote the paper: IB JC LC OL CD BS BB IP GG JG. Other: Contributed to the pharmaceutical organization and planning of the study drug: CG. Project manager of this multi-centric trial: CD. Enrolled volunteers: BS GP BB IP LC GG OL.
The authors have declared that no competing interests exist.
The objective was to compare the safety and cellular immunogenicity of intradermal versus intramuscular immunization with an HIV-lipopeptide candidate vaccine (LIPO-4) in healthy volunteers.
A randomized, open-label trial with 24 weeks of follow-up was conducted in France at six HIV-vaccine trial sites. Sixty-eight healthy 21– to 55–year-old HIV-uninfected subjects were randomized to receive the LIPO-4 vaccine (four HIV lipopeptides linked to a T-helper–stimulating epitope of tetanus-toxin protein) at weeks 0, 4 and 12, either intradermally (0.1 ml, 100 µg of each peptide) or intramuscularly (0.5 ml, 500 µg of each peptide). Comparative safety of both routes was evaluated. CD8+ T-cell immune responses to HIV epitopes (ELISpot interferon-γ assay) and tetanus toxin-specific CD4+ T-cell responses (lymphoproliferation) were assessed at baseline, two weeks after each injection, and at week 24.
No severe, serious or life-threatening adverse events were observed. Local pain was significantly more frequent after intramuscular injection, but local inflammatory reactions were more frequent after intradermal immunization. At weeks 2, 6, 14 and 24, the respective cumulative percentages of induced CD8+ T-cell responses to at least one HIV peptide were 9, 33, 39 and 52 (intradermal group) or 14, 20, 26 and 37 (intramuscular group), and induced tetanus toxin-specific CD4+ T-cell responses were 6, 27, 33 and 39 (intradermal), or 9, 46, 54 and 63 (intramuscular). In conclusion, intradermal LIPO-4 immunization was well tolerated, required one-fifth of the intramuscular dose, and induced similar HIV-specific CD8+ T-cell responses. Moreover, the immunization route influenced which antigen-specific T-cells (CD4+ or CD8+) were induced.
ClinicalTrials.gov
One of the greatest challenges in human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) research is to develop a vaccine that can prevent virus transmission or halt progression to AIDS. An effective HIV vaccine should induce neutralizing antibodies to protect against infection
Intradermal administration is expected to enhance antigen exposure to antigen-presenting cells, because skin harbors more macrophages and dendritic cells than muscle. Those cells incorporate antigens and migrate to draining lymph nodes to present antigen fragments to resting T lymphocytes, thereby initiating antigen-specific immune responses. Thus, the skin is an attractive site for vaccine delivery, achieving the most effective immunization with the smallest antigen load. Indeed, studies on intradermal injection of HBV, rabies and, more recently, influenza vaccines highlighted the potential of this route in improving immunogenicity
The protocol for this trial and supporting CONSORT checklist are available as supporting information; see
This multicenter, open-label, randomized, phase I-B trial was conducted at six HIV-vaccine trial sites in France. The protocol was reviewed and approved by the Pitié–Salpêtrière Hospital Ethics Committee (Paris, France) and all volunteers gave informed consent.
HIV-1-uninfected healthy volunteers, 21 through 55 years of age, were pre-screened according to the procedure established by the French National Agency for AIDS Research (ANRS) to select HIV-unexposed healthy volunteers presenting psychological and sociological stability, i.e. able to be followed and with no plan to move during the entire trial. The selection procedure consisted in medical and psychological exams and standard laboratory tests. Subjects with a pre-existing medical condition or abnormal laboratory values that could affect the standard or protocol safety or immunogenicity evaluations were excluded. HIV-negative status was confirmed by both ELISA and Western blot tests. A committee composed of physicians specialized in clinical immunology, internal medicine, infectious diseases, psychiatrists and a sociologist accepted or not the candidate in the “ANRS HIV-uninfected volunteers network”. They were counseled regarding the uncertainty of efficacy of the trial vaccine and the need to avoid HIV infection and they did not receive financial incentive. Women were required to use effective contraception and have a negative pregnancy test prior to each vaccine injection.
Subjects were randomized to receive, at weeks 0, 4 and 12, the LIPO-4 vaccine injected intradermally (0.1 ml) or intramuscularly (0.5 ml) into the deltoid region of the nondominant arm. Solicited adverse events included the following local and systemic reactions: pain, erythema, induration, nodule, vesicle, papule, bulla and/or edema at the injection site; fever (at least 37.5°C), headache, malaise, nausea, diarrhea, rash, myalgia and/or arthralgia. Participants were seen 3 days and 2 weeks after each vaccination for safety assessment. Vaccination-site induration was measured 3 days postinjection. Information on adverse events was collected throughout the trial. Adverse events were codified for their relationship to the study product (none, possibly, probably or certainly related) and assigned a severity grade. Immune responses were assessed at weeks 0 (before injection), 2, 6, 14 and 24.
LIPO-4 vaccine contains four HIV sequences (Gag77–85, Pol342–354, Pol476–484 and Nef68–82), covalently linked to a T-helper–stimulating tetanus toxin (TT) peptide, TT830–843 (
The amino acid sequences correspond to the four antigenic segments of HIV proteins (red) covalently linked to the T-helper–stimulating epitope of tetanus toxin (TT, black) and linker residues (green). The lines under the HIV sequences represent 12 previously reported CD8+ T-cell–stimulating epitopes (peptide numbers in brown); the HLA class-I molecules (A or B in blue) restricting the T-cell responses are also indicated.
The primary objective was to determine whether intradermal versus intramuscular LIPO-4 vaccine injection resulted in significantly different percentages of participants experiencing any grade 2 or higher adverse event, possibly, probably or certainly related to the vaccination. Secondary endpoints were the evaluation of T-cell immune responses.
Safety Assessment. All subjects were observed for 30 minutes following vaccine administration to check for immediate local and/or systemic reactions. A self-monitoring diary card was given to all subjects to record any local and/or systemic reaction occurring during the post-injection period and any adverse event occurring during the entire study period. Clinical evaluations were performed at days 3 and 14 post-injection, where the study staff reviewed the diary card and followed up on any adverse event that had occurred since injection. All local and systemic adverse events were recorded, regardless of severity. The adverse event severity-grading scale was defined as follows: mild (grade 1) with no limitation of activities and no medical intervention; moderate (grade 2) with mild-to-moderate limitation of activities and no or minimal medical intervention; severe (grade 3) with marked limitation of activities and medical intervention required; and potentially life-threatening (grade 4). Local symptoms occurring at injection-site (e.g., erythema, induration or edema) were categorized as grade 1 if <15×15 cm; grade 2 if ≥15×15 cm; grade 3 for ulceration, superinfection or superficial phlebitis; and grade 4 for skin necrosis . Physicians graded severity, using self-monitoring diary cards filled by the volunteer. All events were coded in MedDRA terms and then linked to MedDRA system-organ class and preferred terms for reporting. The Events Validation Committee reviewed all events occurring during the study for severity and relationship to LIPO-4. Stopping rules were prespecified for safety concerns, such as grade 3 or 4 adverse events.
Short overlapping peptides spanning the four lipopeptide sequences and known to be optimal CD8+ T-lymphocyte–stimulating epitopes were synthesized by NeoMPS (Strasbourg, France) and tested in ELISpot interferon-γ (IFNγ) assays. The compositions of the 12 peptides used are shown in
A one-step stimulation strategy (cultured ELISpot) was used to amplify the CD8+ T-cell responses before ELISpot assay
The proliferation of each subject's fresh PBMC during 7 days of incubation in complete medium with phytohemagglutinin (PHA; 1 µg/ml), purified protein derivative (1 µg/ml), TT peptide (1 µg/ml) and staphylococcal enterotoxin B (0.1 µg/ml) was determined. [3H]Thymidine uptake was expressed as a stimulation index: mean test counts/minute (cpm) of quadruplicate wells/mean background cpm. An index ≥3 was considered positive.
Due to their structure, lipopeptides are known to induce a CD8+ T-cell response, although they also elicit a CD4+ T-cell response
The null hypothesis was that the intradermal and intramuscular routes would each have a 45% possibly, probably or certainly vaccination-related grade 2 or higher adverse event rate, excluding local pain, i.e., that observed in an ANRS pilot trial using LIPO-4 vaccine (unpublished data). To detect a between-arm difference of 45 versus 80% in a two group continuity corrected chi square test with 80% power and a two-sided significance level of 0.05, 35 patients per arm suffice.
Randomization was stratified by the participant's HLA-type, able to induce CD8 immune responses against 0–1 peptide, 2–5 peptides or 6–11 peptides, respectively. Randomization lists were generated with a block size of 4 using the statistical program SAS (SAS system for Windows version 8; SAS, Cary, North Carolina, United States). The three lists were generated by the trial statistician, who had no involvement in enrolment, follow-up, or assessment of participants.
Individuals were randomized by computer-generated lists, which were maintained centrally so no center knew the treatment allocation of any participant prior to randomization.
A central coordinating office was responsible for validation of participant eligibility, randomization, data collection, and monitoring. Once the screening process was completed, and in order of enrolment, each participant was assigned to the intradermal or intramuscular route by a centralized process, according to computer-generated random lists. The result of individual randomization was faxed and e-mailed by the central office to the trial center.
In this open-label study regarding safety assessment, all laboratory analyses were performed by individuals blinded to administration route assignments. Clinical staff and volunteers were aware of route assignments.
The primary outcome was the proportion of subjects experiencing any grade 2 adverse event possibly, probably or certainly vaccination-related, injection-site pain excluded (as specified in the protocol). The proportion including injection-site pain was also analyzed. CD4+ T-cell proliferation and CD8+ T-cell IFNγ secretion were analyzed based on a priori assessable assays at each time, independently of the vaccination route. Primary and secondary endpoints were analyzed based on intention-to-treat (using the same definitions of assessable as for immunological data) with Fisher's exact test with small numbers of expected frequencies (<5) or chi-square test for percentages and 95% exact confidence intervals, using the statistical program SAS.
The first enrollment was on 17 August 2004, and the last study visit was on 28 June 2005. Sixty-eight subjects were enrolled and randomly assigned to one of the two treatment arms: 33 received intradermal injections and 35 intramuscular injections (
Characteristic | Intradermal (N = 33) | Intramuscular (N = 35) | Total (N = 68) |
Sex, no. (%) | |||
Male | 28 (85) | 23 (66) | 51 (75) |
Female | 5 (15) | 12 (34) | 17 (25) |
Age, years, no. (%) | |||
21–30 | 2 (6) | 2 (6) | 4 (6) |
31–40 | 9 (27) | 7 (20) | 16 (24) |
41–50 | 16 (48) | 14 (40) | 30 (44) |
51–55 | 6 (18) | 12 (34) | 18 (26) |
Median [range] | 44 [28–56] | 48 [27–56] | 47 [27–56] |
Vaccinations received, no. (%) | |||
Day 0 | 33 (100) | 35 (100) | 68 (100) |
Week 4 | 33 (100) | 35 (100) | 68 (100) |
Week 12 | 22 (67) | 22 (63) | 44 (65) |
No grade-3 or -4 adverse events were reported during the study (
Intradermal (N = 33) | Intramuscular (N = 35) | |
All Reactions | ||
Any | 33 (100) | 34 (97) |
Grade 2 |
11 (33) | 11 (31) |
Grade 2 (pain excluded) |
11 (33) | 6 (17) |
Local Symptoms | ||
Any | 33 (100) | 32 (91) |
Grade 2 | 8 (24) | 7 (20) |
Injection-site pain | ||
Any | 9 (27) | 28 (80) |
Grade 2 | 0 | 6 (17) |
Injection-site erythema | ||
Any | 32 (97) | 26 (74) |
Grade 2 | 3 (9) | 0 |
Injection-site induration | ||
Any | 29 (88) | 10 (29) |
Grade 2 | 1 (3) | 0 |
Injection-site pruritus | ||
Any | 24 (73) | 4 (11) |
Grade 2 | 5 (15) | 0 |
Injection-site edema | ||
Any | 4 (12) | 4 (11) |
Grade 2 | 1 (3) | 1 (3) |
Other local symptoms | ||
Any | 14 (42) | 9 (26) |
Grade 2 |
2 (6) | 0 |
Systemic Reactions | ||
Any | 17 (52) | 18 (51) |
Grade 2 | 5 (15) | 5 (14) |
Myalgia | ||
Grade 1 | 3 (9) | 4 (11) |
Headache | ||
Any | 3 (9) | 4 (11) |
Grade 2 | 0 | 1 (3) |
Nausea | ||
Any | 2 (6) | 4 (11) |
Grade 2 | 1 (3) | 1 (3) |
Asthenia | ||
Any | 3 (9) | 3 (9) |
Grade 2 | 2 (6) | 1 (3) |
Diarrhea | ||
Any | 1 (3) | 2 (6) |
Grade 2 | 1 (3) | 0 |
Arthralgia | ||
Grade 1 | 2 (6) | 0 |
Other systemic symptoms | ||
Any | 6 (18) | 10 (29) |
Grade 2 |
1 (3) | 3 (9) |
Tabulated values are numbers (percentages) of volunteers experiencing at least one reaction after any vaccination. Percentages do not add up to 100 because some volunteers had >1 symptom and/or reaction.
All reactions resolved without sequelae within a median of 56 hours (interquartile range 9–304 hours).
95% confidence interval in intradermal (ID) arm:18 to 52% and in intramuscular (IM) arm: 17 to 49%; ID vs. IM P = 0.87.
95% confidence interval in ID arm:18 to 52% and in IM arm: 7 to 34%; ID vs. IM P = 0.12.
Other grade-2 local symptoms in the ID arm: local myalgia, injection-site vesicles.
Other grade-2 systemic reactions in the IM arm: conjunctivitis, fatigue and rhinitis; in the intradermal arm: flu-like syndrome.
Few responses were seen before vaccination (5 and 2 subjects in intradermal and intramuscular groups respectively, against 1 peptide in 6 cases, and 2 peptides in 1 case) and in most cases, these responses were no more detected after vaccination excepted in one case. Specific cellular responses mounted by PBMC from 17/33 intradermally vaccinated subjects were positive: 11, 3 and 3 generated CD8+ T cells against one, two or more peptides, respectively (
Panel A, all responders (N = 68), and Panel B, responders given 3 injections (N = 44°. For each responder, the magnitude of the response against individual HIV peptides, expressed as the total spot-forming units per million peripheral blood mononuclear cells (SFU/106 PBMC), evaluated at times 1–4 corresponding to weeks 2, 6, 14 and 24.
Panel A, all volunteers (N = 68), and Panel B, volunteers who received 3 injections (N = 44). The numbers of volunteers who had mounted responses to at least one HIV peptide at weeks 2, 6, 14, 24 are given above the columns: after intradermal (ID, crosshatched) or intramuscular (IM, empty) injections.
Ten CD8+ T-cell–stimulating HIV peptides, among the 12 sequences spanning the vaccine's four lipopeptides, were recognized by T cells from vaccinated volunteers: peptide 24 by one intramuscularly vaccinated subject; peptide 13 by six subjects (three per group); peptides 22, 78 and 87, by twelve subjects (seven and five subjects from the intradermal and intramuscular arms, respectively); peptides 29, 64, 65, 66 and 67, by twenty-three subjects (13 and 10 subjects immunized intradermally and intramuscularly, respectively).
Lipopeptides TT–Nef and TT–Pol1, which contain several epitopes unlike lipopeptides TT–Gag and TT–Pol2, were able to induce most IFNγ-secreting CD8+ T-cell responses. Unsurprisingly, HLA-B7 supertype (HLA-B7/-35/-51)-restricted epitopes were preferentially recognized, reflecting their high representation (5/12 epitopes) in LIPO-4.
Proliferative responses of PBMC from 13/33 (39%; 95% CI, 23–58) intradermally immunized volunteers were mostly obtained after at least two LIPO-4 injections (two, seven and four subjects responded after one, two or three injections, respectively) (
Responses were evaluated at weeks 2, 6, 14 and 24 as follows: CD8+ T-cell responses to HIV peptides, assessed with ELISpot IFNγ (total SFU/106 PBMC defined as total SFU of positive responses minus the background: nonresponder empty; <100 crosshatched; 100–<500 solid squares; 500+ dotted); CD4+ T-cell proliferation against TT peptide (stimulation index: <3 empty; 3–<5 crosshatched; 5–<10 dotted; >10 solid) and induration present 48–72 hours postinjection (diameter (mm): none open; <5 crosshatched; 5–<10 dotted; >10 solid). Subjects in each panel are ordered as follows: CD8+ T-cell nonresponders (ELISpot IFNγ), CD4+ T-cell responders (proliferation) and, finally, CD4+ T-cell responders. Injections were given at weeks 0 and 4 to all 68 subjects and also at week 12 to 44 subjects ND, not done; NA, not applicable.
Strategies to improve vaccine immunogenicity include increasing antigen content and/or the number of immunizations, adding adjuvant and/or alternative immunization routes. The efficacy of vaccine delivery to the dermal compartment, one of the body's most immunocompetent sites, is probably attributable to contact between deposited antigen and abundant professional antigen-presenting cells. Recent results describe lipopeptide penetration into dendritic cells and, once internalized, their processing via the cross-presentation pathway into peptides that are presented to CD8+ T lymphocytes
Herein, the outcome of intradermal antigen delivery was evaluated in terms of safety, CD4+ and CD8+ T-cell immune responses, and injection-site reactions after each immunization, in comparison to classical intramuscular injection. As in previous clinical trials evaluating HIV lipopeptides
The optimal way to measure antigen-specific T cells remains a matter of debate, especially since recent observations suggest that different assays might measure different T-cell responses. Although the detection of ex vivo IFNγ-producing cells using the ELISpot assay might be useful for identifying immunogenic vaccines, it may be less informative for the identification of a protective immune response
Our findings indicate that HIV-specific T-cell responses were induced in vaccinated subjects. CD8+ T-cell IFNγ secretion in response to at least one HIV peptide at weeks 2, 6, 14 and 24, expressed as cumulative percentages, was, respectively, 9, 33, 39 and 52, for intradermally injected volunteers and 14, 20, 26 and 37, for intramuscularly injected volunteers. In contrast, the respective percentages of TT peptide-specific CD4+ T-cell proliferation were 6, 27, 33 and 39 (intradermal arm) and 9, 46, 54 and 63 (intramuscular arm). To our knowledge, these are the first data suggesting that the immunization route might affect the CD4+ and CD8+ T-cell phenotypes.
Primary immunization encounters an immune system naive to the vaccine antigen. When the antigen is readministered in booster shots, antigen-specific, cellular delayed-type hypersensitivity reactions are triggered. Therefore, we scored skin responses during the first 72 hours after antigen administration. The nature of skin responses is not completely understood: no obvious relationship was observed with lymphocyte proliferation, CD8+ induction, delayed hypersensitivity responses and/or intensity or durability of the skin reactions, as shown
In conclusion, our results demonstrate that intradermally injected HIV lipopeptides induce complementary CD4+ and CD8+ T-cell responses. Moreover, intradermal injection of 20% of the intramuscular dose of LIPO-4 vaccine was at least as immunogenic as the full intramuscular dose, and would be a valid dose-sparing strategy, although the study was not powered to demonstrate non-inferiority. However, reduced intramuscular doses have not yet been evaluated and a study comparing 3 doses of HIV lipopeptides versus placebo in non-infected volunteers is currently ongoing (ClinicalTrial.gov identifier: NCT00121758). Our observations also suggest that combining intradermal and intramuscular vaccination routes might be a new approach to improve induction of cellular immunity and warrants further evaluation.
Trial protocol.
(0.55 MB DOC)
CONSORT checklist.
(0.05 MB DOC)
The authors thank the unpaid volunteers for the generous gift of their time, the participating clinicians at each study site, the members of the Events Validation Committee and the Scientific Committee, and Janet Jacobson for editorial assistance.
Presented in part at AIDS Vaccine 2006 International Conference, Amsterdam, The Netherlands, 29 August-1 September, Abstract P11-16.
Events Validation Committee: B. Milpied, J.-D. Lelièvre, L. Mouthon, S. Grabar, C. Picard. Scientific Committee: O. Launay, J.-G. Guillet, B. Bonnet, I. Bourgault-Villada, L. Cuzin, C. Desaint, C. Durier, H. Gahery, C. Guérin, N. Guérin, G. Gonzalez-Canali, J.-P. Lévy, G. Pialoux, I. Poizot-Martin, V. Rieux, M. Robain, B. Rouveix, D. Salmon, B. Silbermann. The following VAC16 Study Group members enrolled volunteers in this study: E. Chakvetadze, L. Slama, G. Pialoux (Hôpital Tenon, Paris); B. Silbermann, Z. Absi, B. Phung, O. Launay (Hôpital Cochin, Paris); P. Morineau-Le Houssine, B. Bonnet (Centre Hospitalo-Universitaire Hôtel-Dieu, Nantes); M. Obadia, L. Cuzin, A Sommet (Hôpital Purpan, Toulouse); M.-P. Drogoul, I. Poizot-Martin, (Hôpital Sainte-Marguerite, Marseille); P. Castiel, G. Gonzalez-Canali (Hôpital Européen Georges Pompidou, Paris)