Impaired cytotoxic T lymphocyte recognition due to genetic variations in the main immunogenic region of the human immunodeficiency virus 1 NEF protein.

Human immunodeficiency virus (HIV) induces strong responses from human histocompatibility leukocyte antigen (HLA) class I-restricted cytotoxic T lymphocytes (CTL). In a previous report we identified an immunodominant region (amino acids 73-144) in the NEF protein that was recognized by CD8+ class I-restricted CTL of most asymptomatic individuals. Analysis of the 73-144 region by peptide sensitization, experiments using overlapping peptides corresponding to the LAI isolate identified the peptide sequences located between residues 73 and 82 or 84 and 92 and the peptide sequence between residues 134 and 144 as cognate peptides for HLA-A11- and HLA-B18-restricted epitopes, respectively. This report describes the variable demonstrable reactivities of CTL obtained from HLA-A11 or HLA-B18 seropositive, asymptomatic patients who all had a response to the virus NEF protein, but who did not always recognize appropriate cognate peptides. The high mutation rate of HIV probably facilitates the selection of mutants that can avoid the cellular immune response. We therefore analyzed the variability of these epitopes restricted by HLA-A11 and HLA-B18. We sequenced several viral isolates from HLA-A11 and HLA-B18 donors who recognized certain HLA-peptide complexes and from those who did not. A CTL sensitization assay was used to show that some mutations led to a great reduction in CTL activity in vitro. This might be due to failure of the mutated epitope to bind major histocompatibility complex class I molecule. A simple assay was used to detect peptides that promoted the assembly of class I molecules. Some of these mutations at major anchor positions prevented HLA-A11/peptide binding, and consequently impaired recognition of the HLA-peptide complex by the T cell receptor.

T cell receptor gene usage in the response to lambda repressor cI protein. An apparent bias in the usage of a V alpha gene element.

The T cell response to the lambda repressor cI protein is directed to the same region of the protein (residues 12-26) in both BALB/c and A/J mice. A panel of T cell hybridomas specific for P12-26 in the context of either I-Ek or I-Ad have been isolated To further understand the molecular interaction between the TCR and the Ia-P12-26 complex, the primary structures of the TCR of five T cell hybridomas have been determined. Southern and Northern analyses indicate that two members of the V alpha 3 gene family are used by 13 out of 14 I-Ek-restricted T cells. Four different V beta genes are used by these T cell hybridomas, while the majority (8 out of 13) express V beta 1 in combination with the J beta 2.1 element. No clear correlation can be seen in this system between gene usage and MHC restriction. In addition, the fine specificity of I-Ek-restricted T cells to a single amino acid substitution [Phe22/His22]P12-26 is not attributed to the usage of particular V alpha and V beta elements. The V alpha 3 family gene is also used by a few I-Ad-restricted T cells. Interestingly, these I-Ad T cells share a reactivity pattern more similar to that of I-Ek-restricted T cells than other I-Ad-restricted T cells. The nonrandom selection V alpha 3 is also demonstrated by the fact that V alpha 3 is used by P12-26-specific, but not by cytochrome c- or staphylococcal nucleus-specific, I-Ek-restricted T cells. This suggests that although antigen specificity may not be accounted for by either chain of the TCR, the members of V alpha 3 genes may be selected by the antigen (P12-26).

Human papillomavirus 16-specific T cell responses in classic HPV-related vulvar intra-epithelial neoplasia. Determination of strongly immunogenic regions from E6 and E7 proteins.

Cell-mediated immunity directed against human papillomavirus 16 (HPV-16) antigens was studied in 16 patients affected with classic vulvar intra-epithelial neoplasia (VIN), also known as bowenoid papulosis (BP). Ten patients had blood lymphocyte proliferative T cell responses directed against E6/2 (14-34) and/or E6/4 (45-68) peptides, which were identified in the present study as immunodominant among HPV-16 E6 and E7 large peptides. Ex vivo enzyme-linked immunospot-interferon (IFN)-gamma assay was positive in three patients who had proliferative responses. Twelve months later, proliferative T cell responses remained detectable in only six women and the immunodominant antigens remained the E6/2 (14-34) and E6/4 (45-68) peptides. The latter large fragments of peptides contained many epitopes able to bind to at least seven human leucocyte antigen (HLA) class I molecules and were strong binders to seven HLA-DR class II molecules. In order to build a therapeutic anti-HPV-16 vaccine, E6/2 (14-34) and E6/4 (45-68) fragments thus appear to be good candidates to increase HPV-specific effector T lymphocyte responses and clear classic VIN (BP) disease lesions.

Immunological self, nonself discrimination.

The ability of immunodominant peptides derived from several antigen systems to compete with each other for T cell activation was studied. Only peptides restricted by a given transplantation antigen are mutually competitive. There is a correlation between haplotype restriction, ability to bind to the appropriate transplantation antigen, and ability to inhibit activation of other T cells restricted by the same transplantation antigen. An exception was noted in that a peptide derived from an antigen, bacteriophage lambda cI repressor, binds to the I-Ed molecule in a specific way, yet is not I-Ed-restricted. Comparison of the sequence of the repressor peptide with that of other peptides able to bind to (and be restricted by) I-Ed and a polymorphic region of the I-Ed molecule itself revealed a significant degree of homology. Thus, peptides restricted by a given class II molecule appear to be homologous to a portion of the class II molecule itself. The repressor-derived peptide is identical at several polymorphic residues at this site, and this may account for the failure of I-Ed to act as a restriction element. Comparison of antigenic peptide sequences with transplantation antigen sequences suggests a model that provides a basis for explaining self, nonself discrimination as well as alloreactivity.

Cytoplasmic domain heterogeneity and functions of IgG Fc receptors in B lymphocytes.

B lymphocytes and macrophages express closely related immunoglobulin G (IgG) Fc receptors (Fc gamma RII) that differ only in the structures of their cytoplasmic domains. Because of cell type-specific alternative messenger RNA splicing, B-cell Fc gamma RII contains an insertion of 47 amino acids that participates in determining receptor function in these cells. Transfection of an Fc gamma RII-negative B-cell line with complementary DNA's encoding the two splice products and various receptor mutants indicated that the insertion was responsible for preventing both Fc gamma RII-mediated endocytosis and Fc gamma RII-mediated antigen presentation. The insertion was not required for Fc gamma RII to modulate surface immunoglobulin-triggered B-cell activation. Instead, regulation of activation involved a region of the cytoplasmic domain common to both the lymphocyte and macrophage receptor isoforms. In contrast, the insertion did contribute to the formation of caps in response to receptor cross-linking, consistent with suggestions that the lymphocyte but not macrophage form of the receptor can associate with the detergent-insoluble cytoskeleton.

[Designing an effective AIDS vaccine: strategies and current status]. / Mise au point d'un vaccin prophylactique contre l'infection par le VIH. Où en est la recherche clinique?

PURPOSE: The human immunodeficiency virus (HIV) and the acquired immunodeficiency syndrome induce account for over 40 million deaths in the past 20 years. Given that the currently available treatments to prevent HIV transmission and disease are not effective in eradicating the virus, vaccination likely represents the only efficacious adapted response to the global impact of this infection. This paper reviews the challenges encountered in the development of an HIV vaccine as well as the different vaccine approaches and main HIV vaccine candidates evaluated in clinical trials. CURRENT KNOWLEDGE AND KEY POINTS: In spite the tremendous progress in HIV research, the major challenges that are encountered in the development of an HIV vaccine remain of scientific order and include viral specificities, absence of correlates of immune protection and limitations of existing animal models. Over 30 vaccine candidates have been evaluated in clinical trials. These vaccine approaches include the use of recombinant envelope proteins, DNA vaccines, live-vectored recombinant vaccines, subunit vaccines and prime-boost regimens combining various vaccine candidates. Although the protective efficacy of these candidate vaccines has yet to be demonstrated, some vaccination regiments appear to dampen initial viremia and prolong disease-free survival. FUTURE PROSPECTS AND PROJECTS: Faced with the challenges in developing an HIV vaccine, international consortia and new methodologies have been proposed in order to accelerate the development and screening process of new candidate HIV vaccines. Moreover, in the absence of a protective vaccine, the impact of a vaccine that confers partial protection needs to be seriously considered.

Weak anti-HIV CD8(+) T-cell effector activity in HIV primary infection.

HIV-specific CD8(+) T cells play a major role in the control of virus during HIV primary infection (PI) but do not completely prevent viral replication. We used IFN-gamma enzyme-linked immunospot assay and intracellular staining to characterize the ex vivo CD8(+) T-cell responses to a large variety of HIV epitopic peptides in 24 subjects with early HIV PI. We observed HIV-specific responses in 71% of subjects. Gag and Nef peptides were more frequently recognized than Env and Pol peptides. The number of peptides recognized was low (median 2, range 0-6). In contrast, a much broader response was observed in 30 asymptomatic subjects with chronic infection: all were responders with a median of 5 peptides recognized (range 1-13). The frequency of HIV-specific CD8(+) T cells among PBMC for a given peptide was of the same order of magnitude in both groups. The proportion of HIV-specific CD8(+)CD28(-) terminally differentiated T cells was much lower in PI than at the chronic stage of infection. The weakness of the immune response during HIV PI could partially account for the failure to control HIV. These findings have potential importance for defining immunotherapeutic strategies and establishing the goals for effective vaccination.

Mapping of a functional autoimmune epitope on the beta 1-adrenergic receptor in patients with idiopathic dilated cardiomyopathy.

The presence and properties of serum autoantibodies against beta-adrenergic receptors in patients with idiopathic dilated cardiomyopathy were studied using synthetic peptides derived from the predicted sequences of the human beta-adrenergic receptors. Peptides corresponding to the sequences of the second extracellular loop of the human beta 1- and beta 2-adrenergic receptors were used as antigens in an enzyme immunoassay to screen sera from patients with dilated cardiomyopathy (n = 42), ischemic heart disease (n = 17), or healthy blood donors (n = 34). The sera of thirteen dilated cardiomyopathy patients, none of the ischemic heart disease patients, and four of the healthy controls monospecifically recognized the beta 1-peptide. Only affinity-purified antibodies of these patients had a inhibitory effect on radioligand binding to the beta 1 receptor of C6 rat glioma cells. They recognized the receptor protein by immunoblot and bound in situ to human myocardial tissue. We conclude that a subgroup of patients with idiopathic dilated cardiomyopathy have in their sera autoantibodies specifically directed against the second extracellular loop of the beta 1-adrenergic receptor. These antibodies could serve as a marker of an autoimmune response with physiological and/or pathological implications.

Phase I trial of recombinant adenovirus gene transfer in lung cancer. Longitudinal study of the immune responses to transgene and viral products.

Animal studies indicate that the use of replication-deficient adenovirus for human gene therapy is limited by host antivector immune responses that result in transient recombinant protein expression and blocking of gene transfer when rechallenged. Therefore, we have examined immune responses to an adenoviral vector and to the beta-galactosidase protein in four patients with lung cancer given a single intratumor injection of 10(9) plaque-forming units of recombinant adenovirus. The beta-galactosidase protein was expressed in day-8 tumor biopsies from all patients at variable levels. Recombinant virus DNA was detected by PCR in day-30 and day-60 tumor biopsies from all patients except patient 1. A high level of neutralizing antiadenovirus antibodies was detected in patient 1 before Ad-beta-gal injection whereas it was low (patient 3) or undetectable in the other two patients. All patients developed potent CD4 type 1 helper T cell (Th1) responses to adenoviral particles which increased gradually over time after injection. Antiadenovirus cytotoxic T lymphocyte responses were consistently boosted in the two patients examined (patients 3 and 4). Sustained production of anti-beta-galactosidase IgG was observed in all patients except patient 1. Consistent with anti-beta-gal antibody production, all patients except patient 1 developed intense, dose-dependent Th1 responses to soluble beta-galactosidase which increased over time. Strong beta-galactosidase-specific cytotoxic T lymphocyte responses were detected in patients 2, 3, and 4. Our results clearly show that despite the intensity of antiadenovirus responses, transgene protein expression was sufficient to induce strong and prolonged immunity in three patients. Recombinant adenovirus injected directly into the tumor is a highly efficient vector for immunizing patients against the transgene protein.

Heterogeneity of HLA-G gene transcription and protein expression in malignant melanoma biopsies.

Nonclassical MHC class I HLA-G antigen expression is tissue specific and is thought to play a role in tolerance of the semiallogeneic fetus by the maternal immune system. Ectopic expression of HLA-G by tumor cells provides them with an additional mechanism of escape from immunosurveillance by host cytotoxic effector mechanisms. The aim of this study was to assess the expression of nonclassical HLA-G antigens in ex vivo human melanoma biopsies. HLA-G mRNA levels corresponding to both membrane-bound and soluble protein isoforms were analyzed in tumor specimens obtained from primary or metastatic melanomas of 23 patients. High levels of HLA-G transcription were detected in tumor specimens in 5 of 23 patients and found to be comparable in both lymph node and skin metastases. HLA-G mRNA transcript levels at tumor sites in 18 of these patients were compared with those in samples of their own healthy skin and were higher in the tumor tissue in 12 patients. Differential expression of mRNA transcripts corresponding to soluble and membrane-bound HLA-G was also observed in some tumor biopsies. HLA-G protein expression was detected in tumors that exhibited high levels of HLA-G transcription by immunofluorescence of frozen sections and Western blot analysis of both tumor and healthy skin biopsies, using anti-HLA-G-specific monoclonal antibodies. This work provides evidence that HLA-G gene transcription and protein expression can be up-regulated ex vivo in melanoma. Our finding that several of the tumors studied expressed high levels of HLA-G provides additional clues as to how a tumor can be selected in vivo to escape from cytotoxic antitumor responses, constituting a new parameter to be considered in the design of therapeutic approaches aimed at enhancing antitumor immune responses.

HLA class I binding regions of HIV-1 proteins.

To identify HIV peptides containing HLA class I binding regions, different studies have been performed. These include the detection of interactions between HIV peptides and purified HLA molecules in solid-phase assays, the measurement of HLA molecule assembly in the presence of peptide added to cell lysates, and the detection of inhibition of CTL-mediated cytolysis by competition between peptides on target cells. To date, the HIV epitopes recognized by anti-HIV CTL are from the Env, Gag, Nef, and Pol proteins and they are identified using synthetic peptides of 12 to 20 amino acids. The search for a correlation between known HIV CTL epitopes and the results of HLA/peptide interaction assays reveals that: (1) most of the peptides that are positive in the assembly assay contain a HLA-A2 peptide motif but the correlation between these positive peptides and the CTL epitopes is not obvious; (2) a high proportion of HIV epitopes are included in the peptides positive in solid-phase binding and in inhibition of cytolysis assays, although these tests do not allow us to predict HLA restriction; (3) the HLA-A2 peptide motif is not systematically included in HLA-A2-restricted CTL epitopes, this observation raising the possibility that other sequences are involved in HLA binding.

CDR3 size analysis of T cell receptor V beta transcripts: follow-up study in a patient with T cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is generally considered to be a clonal disorder arising as an expansion of committed lymphoid precursors. The generation of functional T cell receptor (TCR) genes involves DNA rearrangement processes. This predisposes immature lymphoid cells to abnormal rearrangements. Previous analysis of the TCR genes strongly suggested the clonal origin of human T-ALL. We present a sensitive clonal analysis of bone marrow TCR V beta transcripts in a case of T-ALL. This study allows the analysis not only of TCR V beta transcripts in tumor cells but also the temporal modification of the global TCR repertoire in the follow-up of clinical specimens from bone marrow aspirates. Moreover, we used clone-specific junctional region oligonucleotide probes corresponding to the clonal leukemic population for the molecular monitoring of the malignant clone throughout the clinical course of the disease. This molecular fingerprint appears to be a sensitive method to detect minimal residual disease. It shows that junctional regions of rearranged TCR beta genes corresponding to the tumor cells can also be detected at the time of the complete remission.

Direct detection of peptide-dependent HLA variability by surface plasmon resonance.

Cytotoxic T lymphocytes (CTL) recognize antigens as peptides associated with molecules of the major histocompatibility complex (MHC). The accurate characterization of antigenic peptides requires knowledge of how peptides bind to MHC molecules, and hence the conformational changes they can induce. Several reports have indicated that the conformation of the MHC class I molecule plays a role in T cell recognition. We therefore studied the interaction of a series of viral epitopes with HLA-A2, -A3, -B7 and -B8 molecules to determine how peptides could induce conformational changes in HLA molecules. This was done either directly with class I heavy chains in lysates of peptide-loading deficient T2 cells, or with purified material from B-EBV transformed cell lines. The peptide-induced HLA conformations were assessed using monoclonal anti-HLA antibodies (mAbs) and detected by surface plasmon resonance (SPR). Antigenic peptides specifically bound to the HLA molecule, even when assembly occurred in a mixed solution of HLA molecules. Distinct patterns of reactivity to a given peptide-bound class I molecule were obtained with monomorphic and allele-specific anti-HLA mAbs.

Physicochemical studies on the antigen-antibody complexes of two monoclonal antibodies against rabbit thymocytes.

We have characterized two monoclonal antibodies directed against rabbit thymocyte antigens. Using internally labelled MD3 cells (a transformed T-cell line), ART-F immunoprecipitated a membrane glycoprotein of 95,000 mol. wt, while ART-A immunoprecipitated two major glycoproteins, one of 95,000 mol. wt, the other of 105,000 mol. wt as analysed on reduced SDS-PAGE. ART-A recognizes 650,000 sites both on rabbit thymocytes and MD3 cells with an association constant of 1.5 X 10(5)/M. ART-F shows a biphasic binding curve with a high affinity constant of 3.3 and 0.6 X 10(8)/M and a low affinity constant of 2.2 and 6.9 X 10(5)/M for thymocytes and MD3 cells respectively. The numbers of high affinity and low affinity antigens are 200,000 for the former and 800,000 for the latter. On the basis of the kinetic data, the difference between low and high affinity is attributed to the difference in association-rate constants. The affinity constants calculated from the reaction-rate constants are in good agreement with those obtained from equilibrium measurements. While ART-F prevents the binding of ART-A, ART-A does not inhibit the affinity binding of ART-F. The analysis of the equilibrium, inhibition and kinetic data allow us to present a model for the structural relation of the epitopes recognized on the T-cells by the two monoclonal antibodies.

Immune recognition of a molecule naturally presented as a monomeric or an oligomeric structure: the model of the human chorionic gonadotropin alpha subunit.

The immune recognition of a molecule naturally presented as a monomeric or an oligomeric structure is analyzed using the human chorionic gonadotropin alpha subunit (hCG-alpha) as a model. Indeed, hCG-alpha circulates as either a free subunit or combined to the beta subunit (hCG-beta) to form the dimeric hCG hormone. A T cell study was performed in BALB/c (H-2d) mice which were found to be high responders to hCG-alpha. Mice were immunized with the free hCG-alpha or the dimeric hCG alpha/beta, and their lymph node cells were challenged in vitro with either alpha subunits from different species, hCG or peptides spanning the entire primary structure of hCG-alpha. Proliferation and IL-2 assays demonstrated that hCG-alpha-primed lymph node cells responded equally well to hCG-alpha and hCG alpha/beta, suggesting that both the free and combined hCG-alpha subunits are processed in a similar way. Among the various synthetic peptides used, only those mimicking the hCG-alpha(59-92) C-terminus portion were able to stimulate hCG-alpha-primed lymph node cells, demonstrating that this region contains immunodominant T cell recognition site(s). The hCG-alpha(23-43) and (32-59) peptides, although incapable of stimulating T cells primed with hCG-alpha, elicited a T cell response when used as immunogens. These regions encompassed cryptic epitopes which were not generated during hCG-alpha processing in H-2d mice. The T cell epitopes of hCG-alpha above described as immunodominant or cryptic on the free alpha subunit, had similar characteristics when the alpha/beta dimer was used as the immunogen. In contrast, T cells primed with peptides mimicking immunodominant sites recognized differently the hCG-alpha and the hCG alpha/beta antigens. Moreover, the analysis of the B cell response to all the immunogenic hCG-alpha peptides indicated that they bear B and T cell epitopes as well. Antibodies elicited against the hCG-alpha(59-92) or (32-59) peptide were capable of recognizing the alpha subunit in its free form but not in the alpha/beta hCG dimer. Such study deserves attention for the comprehensive mechanisms of the immune response to hCG as well as for the design of anti-hCG vaccines.

Localisation of three epitopes of the env protein of feline immunodeficiency virus.

The envelope protein of the feline immunodeficiency virus (FIV) was analyzed using several epitope prediction programs based on profiles of hydrophilicity, antigenicity, and probability of residues to lie on the protein surface. Tentative homologies with the immunodominant epitope sites in simian virus (SIV) or human immunodeficiency virus (HIV) such as the V3 loop, the site of cleavage between surface envelope protein (SU) and transmembrane envelope protein (TM), and sites of N-glycosylation were thus identified. Five peptides corresponding to potential epitopes were synthesized. Four out of five peptides (P99, P100, P101, P103) were from the FIV surface envelope protein (SU). The last one (P102) was from the FIV transmembrane envelope protein TM. Three of these peptides (P99, P100, and P102) were recognized in ELISA by almost all the sera from infected cats. The peptide from TM (102) was recognized by sera from both naturally infected and inoculated cats, whereas peptides P99 and P100 (from SU) were recognized mainly by sera from naturally infected cats. On the basis of these results we propose that peptides P99, and P100 from SU and P102 from TM constitute epitopes on the FIV env protein.

Different patterns of HIV-1-specific cytotoxic T-lymphocyte activity after primary infection.

OBJECTIVE: To analyse the HIV-1-specific cytotoxic T-lymphocyte (CTL) responses of nine HIV-seropositive subjects in relation with primary infection. METHODS: Anti-HIV CTL were generated by in vitro stimulation of peripheral mononuclear cells obtained from HIV-seropositive donors at various times after primary infection. They were tested against several structural or regulatory HIV-1 proteins, using autologous target cells infected with recombinant vaccinia viruses expressing one of the HIV-1LAI proteins. RESULTS: An important CTL activity was found during the first month following seroconversion only in those donors who showed clinical symptoms during primary infection. The temporal evolution of this response differed for each subject; one remained a non-responder even 30 months after seroconversion. The structural proteins were recognized particularly early, while the antigenicity of regulatory proteins appeared later. CONCLUSION: Different patterns of HIV-specific CTL response can be observed after primary infection. The evolution of infection in these different HIV-seropositive subjects will be particularly interesting to analyse.

Lipopeptides induce cell-mediated anti-HIV immune responses in seronegative volunteers.

OBJECTIVE: Test the efficacy of a mixture of six NEF (N1, N2, N3), GAG (G1, G2) and ENV (E) lipopeptides in the induction of B- and T-cell anti-HIV responses. DESIGN: A randomized phase I open-label dose-finding trial. Twenty-eight healthy seronegative volunteers received the lipopeptides, with or without the adjuvant QS21. METHODS: Anti-HIV-peptide antibodies were detected by enzyme-linked immunosorbent assay and Western blotting. Induction of cellulary responses was assessed by proliferative test and (51)Cr-release assay. RESULTS: Local and systemic adverse reactions were always mild or moderate. After three injections an antibody response was detected in 25 out of 28 volunteers (89%). T cells from 19 (79%) of the 24 volunteers proliferated in response to at least one peptide. The majority of the volunteers had induced a multispecific proliferative response; that is, cells from volunteers proliferated to two (five of 19), three (five of 19), four (three of 19) or five peptides (one of 19). Cytotoxic responses by anti-HIV CD8+ lymphocytes could be tested in 24 volunteers, 13 (54%) of whom had clear and reproducible responses, with strong activity in the remaining 12 (> 20% of specific lysis), and polyepitopic responses were detected in at least seven of the 13 responders. Cytotoxic responses were found against the whole NEF protein (clade B LAI) in three of four tested volunteers and cross-reactions with the proteins of clade B (MN) and clade A (Bangui) HIV-1 strains, and also HIV-2 ROD, were detected in one of two tested volunteers. CONCLUSIONS: Lipopeptides are promising immunogens for an AIDS vaccine.

Persistent alterations in T-cell repertoire, cytokine and chemokine receptor gene expression after 1 year of highly active antiretroviral therapy.

OBJECTIVES: To examine T-cell repertoire modifications, the evolution of T-helper (TH)1/TH2 cytokine imbalance and modifications in chemokine receptor expression when the viral load is decreased by 2-3 log10 copies/ml under highly active antiretroviral therapy (HAART). DESIGN: Sixteen patients previously treated with zidovudine and lamivudine, with CD4 cells below 300 x 10(6)/l and viraemia above 30000 copies/ml were treated by saquinavir and ritonavir together with both reverse transcriptase (RT) inhibitors (ANRS 069 trial). T-cell repertoire, chemokine receptor and lymphokine expression were studied from peripheral blood mononuclear cells sampled at weeks 0, 24 and 48. METHODS: T-cell repertoire study was carried out using the Immunoscope method. Interleukin (IL)-12 receptor beta2, CC-chemokine receptor (CCR)-3, CXC-chemokine receptor-4 and CCR-5 expression in CD4+ cells was measured by kinetic quantitative PCR and IL-2, IL-4, IL-10, IL-13, interferon (IFN)-gamma were measured using a quantitative RT-PCR assay with homologous internal standards. RESULTS: Repertoire alterations were more frequent in CD4- than in CD4+ cells and persisted despite undetectable viraemia. Increased CCR-3 expression and spontaneous IFN-gamma as well as mitogenic induced IL-13 were observed at baseline and decreased slightly under HAART. CONCLUSION: The CD8+ cell repertoire alterations were profound, whereas the CD4+ cell alterations were moderate and both persisted unchanged under HAART. The TH1/TH2 imbalance was more related to TH2 over-expression than to TH1 deficiency and persisted for at least 1 year under HAART.

Transient expression of genes transferred in vivo into heart using first-generation adenoviral vectors: role of the immune response.

Gene therapy for heart diseases requires availability of an efficient vector for gene transfer into myocardium. Recombinant adenovirus expressing the Escherichia coli beta-galactosidase (beta-Gal) gene was shown to infect rat cardiocytes efficiently in vivo. However, a time course of gene expression showed that transgene expression was maximal during the first week following injection, then declined and disappeared by day 21. An immunosuppressive treatment prolonged beta-Gal expression for at least 21 days. On the contrary, a preimmunization of the animals by two intraperitoneal injections of the vector led to a decreased transgene expression 48 hr after intramyocardial injection and to a barely detectable expression at the sixth day. Appearance of adenovirus neutralizing antibodies in preimmunized animals could have contributed to such a refractoriness to further adenoviral infection. Finally, a neonatal intrathymic injection of the vector was able to induce long-term LacZ expression for more than 2 months after heart injection, although neutralizing as well as anti-beta-Gal antibodies were detected in sera of the animals. These results indicate that an immune response against first-generation replication-defective adenoviral vectors is a major cause of transient transgene expression, a cellular response being most probably responsible for ablation of transgene expression in immunocompetent animals.