[From fundamental immunology to development of vaccinology]. / De l'immunologie fondamentale au développement de la vaccinologie.

In the last ten years research in vaccinology has been developed in the world to conceive new vaccine approaches against infections like HIV/AIDS. Jean-Gérard Guillet is a pioneer in the development of new vaccine strategies. From the first results he obtained in the late 80's on the presentation of antigenic peptides to T cells, he axed his work on the study of induction mechanisms of T cell mediated immune responses. The selection of antigenic peptides and the search to enhance antigen immunogenicity led him to elaborate lipopeptides as new vaccine formulae. The efficacy of these preparations was tested in animal models (mouse, macaque) and, thereafter, in humans with clinical trials promoted by the French National Agency for AIDS and viral hepatitis (ANRS). The study of T-cell induced responses in vaccinated volunteers was implemented following the creation of two facilities, an immuno-monitoring platform and the Clinical Investigation Centre Cochin-Pasteur, a structure specialized in vaccinology.

New CD4+ and CD8+ T cell responses induced in chronically HIV type-1-infected patients after immunizations with an HIV type 1 lipopeptide vaccine.

We showed that an anti-HIV lipopeptide vaccine injected to HIV-uninfected volunteers was well tolerated and able to induce a specific CD4(+) and CD8(+) T cell responses. The same vaccine was injected in HIV-1 chronically infected patients controlled by HAART to evaluate its immunogenicity. In this trial, 24 patients were immunized three times with a mixture of six lipopeptides (Nef 66-97, Nef 117-147, Nef 182-205, Gag 183-214, Gag 253-284, and Env 303-335) at 0, 3, and 6 weeks. We studied the HIV-1-specific CD4(+) T cell proliferative responses. The IFN-gamma secretion by activated CD8(+) T cells was evaluated, using an ex vivo ELISpot assay and 60 CD8(+) T cell epitopes derived from the vaccine. Before immunization (W0), anti-HIV CD4(+) T cell responses to Gag, Nef, and Env large peptides were detected in 7/23 (30%) analyzable patients. After three injections, 17/23 (74%) patients had a proliferative response and 16 of them induced new specific CD4(+) T cell responses. At W0, CD8(+) T cell responses to HIV-1 epitopes were detected in 6/23 (26%) patients. After vaccination, 16/23 (70%) patients showed CD8(+) T cell responses and 13 of these patients induced new T cell responses to 25 different HIV-1 epitopes. These HIV-1 epitopes were detected in patients with various HLA class I molecules (HLA-A2, -A3/A11, -A24, -B7 superfamily, -B8), as found in the majority of the white population. Lipopeptides induce new anti-HIV T cell responses in vaccinated infected patients and could be used as a new immunotherapy strategy. The majority of these responders induced specific new CD4(+) and CD8(+) T cell responses.

Cross-clade conservation of HIV type 1 Nef immunodominant regions recognized by CD8+ T cells of HIV type 1 CRF02_AG-infected Ivorian (West Africa).

Most HIV vaccine trials in the world are conducted with clade B while most circulating viral strains in Africa are non-B subtypes. We determined whether CD8+ T cells from HIV-1 intersubtype CRF02_AG-infected Ivorian individuals were able to recognize clade B epitopes. CD8+ T cell responses of nine HIV-1 intersubtype CRF02_AG-infected Ivorian patients and nine HIV-1 subtype B-infected French patients were studied using pools of HIV-1 clade B peptides (110 well-defined HIV CD8+ T cell epitopes) in an ELISPOT IFN-gamma assay. There was no difference in the number of recognized peptide pools between Ivorian and French cohorts (mean of four pools in both cases). Ivorian individuals had generated CD8+ T cell responses cross-reactive against HIV-1 subtype B and some individual peptides had been identified. Furthermore, sequence analysis of nef HIV genes of the Ivorian patients and nef cloning in two patients revealed very few variations between HIV- 1 intersubtype CRF02_AG and subtype B in nef immunodominant regions included in HIV clade B lipopeptide vaccines, currently tested in France.

HIV preventive vaccine research at the ANRS: the lipopeptide vaccine approach.

The HIV (human immunodeficiency virus)/AIDS epidemic is of unprecedented gravity and is spreading rapidly, notably in the most disadvantaged regions of the world. The search for a preventive vaccine is thus an absolute priority. For over 10 years the ANRS (Agence Nationale de Recherches sur le SIDA) has been committed to an original programme combining basic science and clinical research. The HIV preventive vaccine research programme includes upstream research for the definition of immunogens, animal models, and clinical research to evaluate candidate vaccines. In 2004, most researchers believed that it should be possible to obtain partial vaccine protection through the induction of a strong and multiepitopic cellular response. Since 1992, 15 phase I and II clinical trials have been established with the aim of evaluating the safety of candidate vaccines and their capacity to induce cellular immune responses. The candidate vaccines tested were recombinant canarypox viruses (ALVAC) containing sequences coding for certain viral proteins, utilised alone or combined with other immunogens (whole or truncated envelope proteins). An original strategy, based on the use of lipopeptides, is also under development. These vaccines comprise synthetic fragments of HIV proteins associated with lipids that facilitate the induction of a cellular immune response. These approaches have within a short time allowed the assessment of a prime-boost strategy combining a viral vector and lipopeptides.

In situ detection of antigen-specific tumor-infiltrating lymphocytes using newly designed tetramers.

We described a new process for the design of HLA tetramers using soluble MHC class I molecules purified from Epstein Barr Virus-transformed B cells. This method does not rely on genetic engineering and presents a significant advantage in view of the polymorphism of MHC class I molecules because tetramers can be produced with any HLA molecule. Here, we showed that our HLA-A*0201 tetramers provided experimental results similar to those obtained with tetramers made with recombinant MHC molecules. Moreover, they can be used to efficiently identify peptide-specific T cells from ex vivo PBMCs as well as from lymphocytes infiltrating human tumor. This innovative and simple method could be widely adopted, specially in diagnostic procedures for monitoring peptide-based immunotherapy.

Long-term CD4(+) and CD8(+) T-cell responses induced in HIV-uninfected volunteers following intradermal or intramuscular administration of an HIV-lipopeptide vaccine (ANRS VAC16).

BACKGROUND: We have shown that the intradermal (ID) administration of an HIV-1 lipopeptide candidate vaccine (LIPO-4) is well tolerated in healthy volunteers, with one fifth the IM dose delivered by this route inducing HIV-1-specific CD8(+) T-cell responses of a magnitude and quality similar to those achieved by IM administration. In this long-term follow-up, we aimed to investigate the sustainability and epitopic breadth of the immune responses induced. METHODS: In a prospective multicentre trial, 68 healthy volunteers were randomised to receive, at weeks 0, 4 and 12, either a 0.5 ml IM (500 µg of each lipopeptide; 35 volunteers) dose or a 0.1 ml ID (100 µg of each lipopeptide; 33 volunteers) dose of the LIPO-4 vaccine, in the deltoid region of the non-dominant arm. All 68 volunteers received the first two vaccinations, and 44 volunteers in the ID group and 22 in the IM group received the third. We describe here the long-term CD8(+) and CD4(+) T-cell immune responses, up to 48 weeks after the first immunisation. RESULTS: Response frequency was highest at week 14 for CD4(+) T cells, at 85% (28/33) for the IM group and 61% (20/33) for the ID group (p=0.027), and at week 48 for CD8(+) T cells, at 36% (12/33) for the ID group and 31% (11/35) for the IM group (p=0.67). Response rates tended to be lower for volunteers receiving the third vaccination boost, whether IM or ID. Finally, we also observed a striking change in the specificity of the CD8(+) T-cell responses induced shortly (2 weeks) or several months (48 weeks) after LIPO-4 vaccination. CONCLUSION: Lipopeptide vaccines elicited sustainable CD4(+) and CD8(+) T-cell responses, following IM or ID administration. CD8(+) T-cell responses had shifted and expanded to different epitopes after one year of follow-up. These results should facilitate the design of the next generation of prime-boost trials with repeated doses of lipopeptide vaccines.

Recognition of human proinsulin leader sequence by class I-restricted T-cells in HLA-A*0201 transgenic mice and in human type 1 diabetes.

OBJECTIVE: A restricted region of proinsulin located in the B chain and adjacent region of C-peptide has been shown to contain numerous candidate epitopes recognized by CD8(+) T-cells. Our objective is to characterize HLA class I-restricted epitopes located within the preproinsulin leader sequence. RESEARCH DESIGN AND METHODS: Seven 8- to 11-mer preproinsulin peptides carrying anchoring residues for HLA-A1, -A2, -A24, and -B8 were selected from databases. HLA-A2-restricted peptides were tested for immunogenicity in transgenic mice expressing a chimeric HLA-A*0201/beta2-microglobulin molecule. The peptides were studied for binding to purified HLA class I molecules, selected for carrying COOH-terminal residues generated by proteasome digestion in vitro and tested for recognition by human lymphocytes using an ex vivo interferon-gamma (IFN-gamma) ELISpot assay. RESULTS: Five HLA-A2-restricted peptides were immunogenic in transgenic mice. Murine T-cell clones specific for these peptides were cytotoxic against cells transfected with the preproinsulin gene. They were recognized by peripheral blood mononuclear cells (PBMCs) from 17 of 21 HLA-A2 type 1 diabetic patients. PBMCs from 25 of 38 HLA-A1, -A2, -A24, or -B8 patients produced IFN-gamma in response to six preproinsulin peptides covering residues 2-25 within the preproinsulin region. In most patients, the response was against several class I-restricted peptides. T-cells recognizing preproinsulin peptide were characterized as CD8(+) T-cells by staining with peptide/HLA-A2 tetramers. CONCLUSIONS: We defined class I-restricted epitopes located within the leader sequence of human preproinsulin through in vivo (transgenic mice) and ex vivo (diabetic patients) assays, illustrating the possible role of preproinsulin-specific CD8(+) T-cells in human type 1 diabetes.

Blood CD8+ T cell responses against myelin determinants in multiple sclerosis and healthy individuals.

Patients with multiple sclerosis (MS) display significant peripheral blood CD8(+) T cell receptor biases, suggesting clonal selection. Our objective was to identify relevant myelin-derived peptides capable of eliciting responses of fresh blood CD8+ T cells in MS patients. We focused our analysis on the HLA supertypes (HLA-A3, -A2, -B7, -B27, -B44) predominant in a patient cohort. Three myelin protein (MBP, PLP and MOG) sequences were screened for HLA binding motifs and peptides were tested for their binding to HLA molecules. The cellular responses of 27 MS patients and 19 age- and sex-matched healthy controls (HC) were tested in IFN-gamma ELISPOT assays only detecting pre-committed CD8+ T cells. Sixty-nine new epitopes elicited positive responses, with MOG-derived peptides being the most immunogenic and peptides binding to HLA-A3 being the most frequent. However, MS patients and HC displayed the same frequency of autoreactive cells. The epitopes inducing the strongest responses were not those with the highest HLA binding, suggesting an effective thymic selection in MS patients. Our data extend the concept that the frequency of myelin-reactive T cells in MS patient blood is not increased compared to HC. The description of this set of myelin-derived peptides (MHC class I restricted, recognized by CD8+ T cells) offers new tools to explore the CD8+ cell role in MS.

Cellular immune responses induced with dose-sparing intradermal administration of HIV vaccine to HIV-uninfected volunteers in the ANRS VAC16 trial.

OBJECTIVE: 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. METHODOLOGY: 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 microg of each peptide) or intramuscularly (0.5 ml, 500 microg of each peptide). Comparative safety of both routes was evaluated. CD8+ T-cell immune responses to HIV epitopes (ELISpot interferon-gamma assay) and tetanus toxin-specific CD4+ T-cell responses (lymphoproliferation) were assessed at baseline, two weeks after each injection, and at week 24. RESULTS AND CONCLUSION: 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. TRIAL REGISTRATION: ClinicalTrials.gov NCT00121121.

Influence of dominant HIV-1 epitopes on HLA-A3/peptide complex formation.

The binding of peptides to MHC class I molecules induces MHC/peptide complexes that have specific conformational features. Little is known about the molecular and structural bases required for an optimal MHC/peptide association able to induce a dominant T cell response. We sought to characterize the interaction between purified HLA-A3 molecules and four well known CD8 epitopes from HIV-1 proteins. To define the characteristics of HLA-peptide complex formation and to identify potential structural changes, we used biochemical assays that detect well formed complexes. We tested the amplitude, stability, and kinetic parameters of the interaction between HLA-A3, peptides, and anti-HLA mAbs. Our results show that the four epitopes Nef73-82, Pol325-333, Env37-46, and Gag20-28 bind strongly to HLA-A3 molecules and form very stable complexes that are detected with differential patterns of mAb reactivity. The most striking result is the nonrecognition of the HLA-A3/Gag20-28 complex by the A11.1M mAb specific to HLA-A3/-A11 alleles. To explain this observation, from the data published on HLA-A11 crystallographic structure, we propose molecular models of the HLA-A3 molecule complexed with Nef73-82, Pol325-333, and Gag20-28 epitopes. In the HLA-A3/Gag20-28 complex, we suggest that Arg at position P1 of the peptide may push the alpha2 helix residue Trp-167 of HLA-A3 and affect mAb recognition. Such observations may have great implications for T cell antigen receptor recognition and the immunogenicity of HLA/peptide complexes.

Recognition of a subregion of human proinsulin by class I-restricted T cells in type 1 diabetic patients.

Proinsulin is a key autoantigen in type 1 diabetes. Evidence in the mouse has underscored the importance of the insulin B chain region in autoimmunity to pancreatic beta cells. In man, a majority of proteasome cleavage sites are predicted by proteasome cleavage algorithms within this region. To study CD8+ T cell responses to the insulin B chain and adjacent C peptide, we selected 8- to 11-mer peptides according to proteasome cleavage patterns obtained by digestion of two peptides covering proinsulin residues 28 to 64. We studied their binding to purified HLA class I molecules and their recognition by T cells from diabetic patients. Peripheral blood mononuclear cells from 17 of 19 recent-onset and 12 of 13 long-standing type 1 diabetic patients produced IFN-gamma in response to proinsulin peptides as shown by using an ELISPOT assay. In most patients, the response was against several class I-restricted peptides. Nine peptides were recognized within the proinsulin region covering residues 34 to 61. Four yielded a high frequency of recognition in HLA-A1 and -B8 patients. Three peptides located in the proinsulin region 41-51 were shown to bind several HLA molecules and to be recognized in a high percentage of diabetic patients.

Automatic sequence design of major histocompatibility complex class I binding peptides impairing CD8+ T cell recognition.

An automatic protein design procedure was used to compute amino acid sequences of peptides likely to bind the HLA-A2 major histocompatibility complex (MHC) class I allele. The only information used by the procedure are a structural template, a rotamer library, and a well established classical empirical force field. The calculations are performed on six different templates from x-ray structures of HLA-A0201-peptide complexes. Each template consists of the bound peptide backbone and the full atomic coordinates of the MHC protein. Sequences within 2 kcal/mol of the minimum energy sequence are computed for each template, and the sequences from all the templates are combined and ranked by their energies. The five lowest energy peptide sequences and five other low energy sequences re-ranked on the basis of their similarity to peptides known to bind the same MHC allele are chemically synthesized and tested for their ability to bind and form stable complexes with the HLA-A2 molecule. The most efficient binders are also tested for inhibition of the T cell receptor recognition of two known CD8(+) T effectors. Results show that all 10 peptides bind the expected MHC protein. The six strongest binders also form stable HLA-A2-peptide complexes, albeit to varying degrees, and three peptides display significant inhibition of CD8(+) T cell recognition. These results are rationalized in light of our knowledge of the three-dimensional structures of the HLA-A2-peptide and HLA-A2-peptide-T cell receptor complexes.

Adenovirus hexon protein is a potent adjuvant for activation of a cellular immune response.

The capacity of recombinant adenoviruses (rAd) to induce immunization against their transgene products has been well documented. In the present study, we evaluated the vaccinal adjuvant role of rAd independently of its vector function. BALB/c mice received one subcutaneous injection of a mixture of six lipopeptides (LP6) used as a model immunogen, along with AdE1 degrees (10(9) particles), a first-generation rAd empty vector. Although coinjected with a suboptimal dose of lipopeptides, AdE1 degrees significantly improved the effectiveness of the vaccination, even in the absence of booster immunization. In contrast to mice that received LP6 alone or LP6 plus a mock adjuvant, mice injected with AdE1 degrees plus LP6 developed both a polyspecific T-helper type 1 response and an effector CD8 T-cell response specific to at least two class I-restricted epitopes. The helper response was still observed when immunization was performed using LP6 plus a mixture of soluble capsid components released from detergent-disrupted virions. When mice were immunized with LP6 and each individual capsid component, i.e., hexon, penton base, or fiber, the results obtained suggested that hexon protein was responsible for the adjuvant effect exerted by disrupted Ad particles on the helper response to the immunogen. Our results thus have some important implications not only in vaccinology but also for gene therapy using rAd vectors.

Tumor-associated antigen human chorionic gonadotropin beta contains numerous antigenic determinants recognized by in vitro-induced CD8+ and CD4+ T lymphocytes.

The beta subunit of human chorionic gonadotropin (hCG beta) is markedly overexpressed by neoplastic cells of differing histological origin including those present in colon, breast, prostate and bladder tumors. We have previously shown that some patients with hCG beta-producing urothelial tumors have circulating T cells that proliferate in response to hCG beta. To make a comprehensive study of hCG beta as a potential target for cancer immunotherapy, we investigated whether hCG beta peptides could induce CD4+ or CD8+ T-cell responses in vitro. By stimulating peripheral blood mononuclear cells (PBMCs) from three donors with mixtures of overlapping 16-mer synthetic peptides analogous to portions of either the hCG beta 20-71 or the hCG beta 102-129 region, we established six CD4+ T-cell lines that proliferated specifically in response to five distinct determinants located within these two hCG beta regions. Three antigenic determinants (hCG beta 52-67, 106-121 and 114-125) were presented by HLA-DR molecules, while the two other antigenic determinants (hCG beta 48-63 and 56-67) were presented by HLA-DQ molecules. Interestingly, one T-cell line specific for peptide hCG beta 106-121 recognized hCG beta peptides comprising, at position 117, either an alanine or an aspartic acid residue, with the latter residue being present within the protein expressed by some tumor cells. In addition, three other hCG beta-derived peptides that exhibited HLA-A*0201 binding ability were able to stimulate CD8+ cytotoxic T cells from two HLA-A*0201 donors. These three immunogenic peptides corresponded to regions hCG beta 40-48, hCG beta 44-52 and hCG beta 75-84. Our results indicate that the tumor-associated antigen hCG beta possesses numerous antigenic determinants liable to stimulate CD4+ and CD8+ T lymphocytes, and might thus be an effective target antigen for the immunotherapy of hCG beta-producing tumors.

Trypanosoma cruzi down-regulates lipopolysaccharide-induced MHC class I on human dendritic cells and impairs antigen presentation to specific CD8(+) T lymphocytes.

Trypanosoma cruzi, the etiological agent of Chagas' disease, may persist for many years in its mammalian host. This suggests escape from the immune response and particularly a suboptimal CD8(+) T cell response, since these cells are involved in infection control. In this report, we show that T. cruzi inhibits the lipopolysaccharide (LPS)-induced up-regulation of MHC class I molecules at the surface of human dendritic cells (DC). To further investigate the functional consequences of this inhibition, a trypomastigote surface antigen-derived peptide (TSA-1(514-522) peptide) was selected for its stable binding to HLA-A*0201 molecules and used to generate a primary T. cruzi-specific human CD8(+) T cell line in vitro. We observed that DC infected with T. cruzi or treated with T. cruzi-conditioned medium (TCM) had a weaker capacity to present this peptide to the specific CD8(+) T cell line as shown in an IFN-gamma ELISPOT assay. Interestingly, T. cruzi or TCM also reduced the antigen presentation capacity of DC to CD8(+) T cell lines specific for the influenza virus M(58-66) or HIV RT(476-484) epitopes. This dysfunction appears to be linked essentially to reduced MHC class I molecule expression since the stimulation of the RT(476-484) peptide-specific CD8(+) T cell line was shown to depend mainly on the MHC class I-TCR interaction and not on the co-stimulatory signals which, however, were also inhibited by T. cruzi. This impairment of DC function may represent a novel mechanism reducing in vivo the host's ability to combat efficiently T. cruzi infection.