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.

Conceptual structure and the structure of concepts: a distributed account of category-specific deficits.

We present a new account of the fine-grained structure of semantic categories derived from neuropsychological, behavioral, and developmental data. The account places theoretical emphasis on the functions of the referents of concepts. We claim (i) that the distinctiveness of functional features correlated with perceptual features varies across semantic domains; and (ii) that category structure emerges from the complex interaction of these variables. The representational assumptions that follow from these claims make strong predictions about what types of semantic information are preserved in patients showing category-specific deficits following brain damage. These claims are illustrated with a connectionist simulation which, when damaged, shows patterns of preservation of distinctive and shared functional and perceptual information which varies across semantic domains. The data model both dissociations between knowledge for artifacts and for living things and recent neuropsychological evidence concerning the robustness of functional information in the representation of concepts.

Identification in humans of HPV-16 E6 and E7 protein epitopes recognized by cytolytic T lymphocytes in association with HLA-B18 and determination of the HLA-B18-specific binding motif.

Human papilloma virus type 16 (HPV-16) is the HPV most frequently associated with cervical carcinoma in humans. For the prevention or treatment of cervical carcinoma, the E6 and E7 oncoproteins appear to be good targets for vaccine-induced cytotoxic T lymphocytes (CTL). Lipopeptide vaccination is an efficient way of stimulating cellular responses. However, to synthesize effective lipopeptides, it is necessary to define which epitopes are immunogenic. In this study we first determined that peptide 80 - 88 of the E6 protein was recognized by CTL from a healthy donor in association with the HLA-B18 molecule. We then defined the HLA-B18 anchoring peptide motif by testing the binding of various short peptides with the HLA-B18 molecule and showed that it was related to the HLA-A1-specific peptide motif. Furthermore, in analyzing the potential E7 epitopes susceptible to associating with HLA-B18, we demonstrated that peptide E7 44 - 52 gave the strongest binding. It could also be recognized by CTL from peripheral blood mononuclear cells (PBMC) of the same healthy donor. Finally, with PBMC from a patient with a cervical intraepithelial neoplasia grade 3, we found CTL which recognized the E6 80 - 88 epitope. We have hence identified two peptides encoded by the E6 and E7 proteins which are presented by the HLA-B18 molecule and could be included in a vaccine against HPV-16.

Is AIDS vaccine research at a turning point?

Although initial anti-ENV vaccines have failed, better results may be obtained with "non-ENV" vaccines capable of inducing cell-mediated responses and perhaps mucosal reactions. A turning point in AIDS vaccine research has been reached in that there are three precise questions to answer: 1. Are the protections obtained in monkeys significant and reproducible and could they be made more frequent? 2. Can more frequent, polyepitopic, and long-lasting cell-mediated responses in human subjects be obtained? 3. Is it possible to induce significant mucosal responses? The decision as to whether phase III trials must be launched will depend on answers to these questions. It may be possible to organize international AIDS vaccine research so that these answers could be obtained in 3 to 4 years, but this would require a frank acceleration of the present endeavors.

Multiepitopic B- and T-cell responses induced in humans by a human immunodeficiency virus type 1 lipopeptide vaccine.

We have attempted to develop an anti-human immunodeficiency virus (HIV) lipopeptide vaccine with several HIV-specific long peptides modified by C-terminal addition of a single palmitoyl chain. A mixture of six lipopeptides derived from regulatory or structural HIV-1 proteins (Nef, Gag, and Env) was prepared. A phase I study was conducted to evaluate immunogenicity and tolerance in lipopeptide vaccination of HIV-1-seronegative volunteers given three injections of either 100, 250, or 500 microg of each lipopeptide, with or without immunoadjuvant (QS21). This report analyzes in detail B- and T-cell responses induced by vaccination. The lipopeptide vaccine elicited strong and multiepitopic B- and T-cell responses. Vaccinated subjects produced specific immunoglobulin G antibodies that recognized the Nef and Gag proteins. After the third injection, helper CD4(+)-T-cell responses as well as specific cytotoxic CD8(+) T cells were also obtained. These CD8(+) T cells were able to recognize naturally processed viral proteins. Finally, specific gamma interferon-secreting CD8(+) T cells were also detected ex vivo.

Murine retroviral vector producer cells survival and toxicity in the peritoneal cavity of dogs.

Retroviral vector producer cells (VPC) can effectively transfer genes in vivo. To develop a safe method to target gene delivery into intraperitoneal tumors, we have examined the toxicity of intraperitoneal (i.p.) infusion of retroviral VPC in a xenogeneic canine model. Mongrel dogs were injected intraperitoneally (i.p.) with 2 x 10(9) murine LTKOSN.2 VPC. The animals did not demonstrate acute toxicity and tolerated the i.p. infusion of the cells without difficulty. Starting 7 days after i.p. injection, the dogs received intravenous injections of ganciclovir (GCV) twice daily (5 mg/kg) for 7 days. The treatment dogs underwent peritoneal washings on days 3, 7 and 14 after their initial infusion of cells to study the persistence of the VPC. GCV treatment did not cause significant toxicities. Dogs underwent serial blood tests to evaluate bone marrow, renal, liver and immunological function. Complete blood counts, electrolytes and renal function remained normal throughout the study. Although, transient mild elevations occurred of serum alkaline phosphate, the remaining hepatic enzymes remained normal. Histologic examination of tissues from animals sacrificed after the i.p. administration of the VPC revealed no tissue destruction of the normal peritoneal lining. The dogs mounted an antibody response to the murine VPC that was first observed 7 days post injection. PCR analysis of selected tissues after GCV administration did not reveal persistent vector sequences. These results demonstrated that the injection of xenogeneic VPC is not accompanied by significant adverse effects over a 1 month period following administration into the canine peritoneal cavity. These data support the potential clinical application of the VPC in Phase I clinical trials in humans.

Murine retroviral vector producer cells survival and toxicity in the dog liver.

To develop a safe method to target gene delivery into intrahepatic tumors, we examined the toxicity of intrahepatic (IH) injection of retroviral vector producer cells (VPC) into the canine liver. VPC have been demonstrated to effectively transfer genes in vivo. To evaluate for adverse effects form xenogeneic cell transplantation, mongrel dogs were injected IH with 1 x 10(9) murine LTKOSN.2 VPC divided into three aliquots. The animals were then monitored for acute toxicity induced by the VPC. The intraoperative IH injections of the cells were tolerated without difficulty. Starting 7 days after IH injection, the dogs then received intravenous ganciclovir (GCV) twice daily (5 mg/kg) for 7 days. GCV treatment did not cause significant toxicities. Dogs underwent serial blood tests to evaluate bone marrow, renal, liver and immunological function. Complete blood counts, electrolytes, liver function and renal function tests remained normal except for mild elevations of alkaline phosphatase. Histologic examination of liver tissues from the IH injection site revealed no apparent normal tissue destruction induced by the VPC. Two of the four treated dogs underwent liver biopsy on day 3. These biopsy specimens were cultured and persistent, viable VPC were recovered. The dogs mounted an antibody response to the murine VPC that was first demonstrated 5 days post injection. PCR analysis demonstrated low level gene transfer into dog liver tissue. Overall, our results demonstrate that IH xenogeneic VPC injections are not accompanied by significant adverse effects over a 1 month period following administration into the canine liver. These data support the safety aspects of using murine VPC in Phase I clinical trials.

Characterization of a multi-lipopeptides mixture used as an HIV-1 vaccine candidate.

A multi-component vaccine has been defined, which contains six different synthetic 24- to 32-amino acid lipopeptides derived from the sequence of HIV-1 proteins. The physicochemical properties of the lipopeptide components were compatible with multi-dimensional analysis, using RP-HPLC, Edman sequencing, electrospray mass spectrometry, and 2D-NMR. Detailed analysis of the impurity profiles led to the detection and evaluation of the relative proportions of most by-products: several contaminants resulted from the formation of acetylated fragments, transpeptidation reactions with succinimide or piperidide formation, or methionine and/or tryptophan mono-oxidations. The first batch to be produced underwent extensive pharmacotoxicological testings to confirm its safety; this vaccine candidate has now been used in phase I clinical trials. Despite the complexity of such multi-lipopeptide vaccines, our findings suggest the possibility of preparing a clear and precise assignment of by-products to toxicologically qualified impurities in the eventuality of a future production of several successive batches.

Preliminary evaluation of the new hematology analyzer COULTER GEN-S in a university hospital.

The COULTER GEN-S system (COULTER Corp, Miami, USA) is an automated hematology instrument that is designed to provide a complete hematological profile including white blood cells (WBC), complete blood count (CBC) differential count (diff) and the reticulocyte parameters. It was evaluated in our laboratory over a one month period. A preliminary study was performed using the GEN-S software revision 1D. The evaluation had two purposes: 1) evaluation of the GEN-S specifications; 2) comparison of its analytical performance with the hematology analyzer currently used in our laboratory. The first part of the evaluation showed that the COULTER GEN-S is reproducible, has linearity beyond the specifications given by the manufacturer and produces stable results up to 48 hours after blood collection. The evaluation of analytical performance included: 1) a comparison between the GEN-S CBC and diff numerical results and the COULTER STKS (COULTER Corp, Miami, USA). These comparisons showed that the results given by the two methods are similar and suggested that the COULTER GEN-S could replace the current hematology instrument in use in our laboratory; 2) a performance analysis, to measure the system's ability to detect morphologic abnormalities, when compared to the reference blood smear examination. This part of the evaluation was performed on both normal and abnormal samples. The results of this analysis showed that the sensitivity of the GEN-S was excellent, especially regarding blast cells, immature granulocytes, nucleated red blood cells (NRBCs) and platelet clumps when using the complete suspect flagging system of the instrument. In addition, the use of the review criteria in our laboratory allowed to detect all hematological diseases. The overall false negative rate was 0.9%. Thus we consider that the COULTER GEN-S system is suited for use in medium-to large hospital laboratories which perform more than 100 CBC/day. Overall, the instrument had excellent performance, with a throughput of more than 100 samples/h. Its user friendly workstation has complete patient data management, which is in compliance with good laboratory practices in France.

Eliciting hyperacute xenograft response to treat human cancer: alpha(1,3) galactosyltransferase gene therapy.

Xenograft hyperacute rejection in humans occurs as a secondary response to a cellular glycosylation incompatibility with most non-human mammalian species. A key component of hyperacute rejection, alpha(1,3)galactosyl (agal) epitopes present on the surface of most non-human mammal cells, is bound by host anti-agal IgG antibodies leading to the activation of complement and, cellular lysis (1). The enzyme causing specific glycosylation patterns, alpha(1,3)galactosyltransferase [alpha(1,3)GT], directs the addition of agal to N-acetyl glucosamine residues in the trans Golgi apparatus in most mammalian species including Mus musculus, but not old world primates, apes or humans. In this report, we cloned both a truncated and full length murine alpha(1,3)GT gene into a retroviral vector backbone in order to transfer alpha(1,3)galactosyl epitopes into human A375 melanoma cells. Expression of agal epitopes on A375 cells after alpha(1,3)GT gene transfer was demonstrated using FITC-labeled ligand and FACS analysis. These cells were exposed to human serum for 30 minutes and > 90% of the agal expressing cells were killed by this treatment. These pretreated cells failed to establish tumors after implantation into athymic nude mice. This is the first report of retroviral vector transfer of the alpha(1,3)GT gene into human tumor cells in an attempt to elicit hyperacute rejection as a novel anti-cancer gene therapy strategy.

Evolution of cytotoxic T lymphocyte responses to human immunodeficiency virus type 1 in patients with symptomatic primary infection receiving antiretroviral triple therapy.

The impact of highly active antiretroviral treatment (HAART) on anti-human immunodeficiency virus (HIV) cytotoxic T lymphocytes (CTL) was studied in 17 patients with recent symptomatic HIV-1 primary infection receiving triple combination therapy. Anti-HIV CTL were initially detected in 15 patients. In 6, CTL disappeared rapidly and persistently after initiation of therapy. Most of them had a rapid and sustained decrease in plasma HIV RNA to undetectable levels. Conversely, in 6 other patients, CTL remained detectable, which was associated with a less efficient control of viral replication. In 3 others, CTL disappeared only transiently, without clear correlation with the virologic profile. Altogether, despite individual variations, there was a positive correlation between viral replication and anti-HIV-1 cytotoxicity in most subjects, suggesting that the persistence of viral antigens is the main determinant for the maintenance of CTL activity. This raises the question of the potential benefit of anti-HIV CTL induction by immunotherapy in acute seroconverters treated by HAART.

Cross-reactions between the cytotoxic T-lymphocyte responses of human immunodeficiency virus-infected African and European patients.

The great variability of protein sequences from human immunodeficiency virus (HIV) type 1 (HIV-1) isolates represents a major obstacle to the development of an effective vaccine against this virus. The surface protein (Env), which is the predominant target of neutralizing antibodies, is particularly variable. Here we examine the impact of variability among different HIV-1 subtypes (clades) on cytotoxic T-lymphocyte (CTL) activities, the other major component of the antiviral immune response. CTLs are produced not only against Env but also against other structural proteins, as well as some regulatory proteins. The genetic subtypes of HIV-1 were determined for Env and Gag from several patients infected either in France or in Africa. The cross-reactivities of the CTLs were tested with target cells expressing selected proteins from HIV-1 isolates of clade A or B or from HIV type 2 isolates. All African patients were infected with viruses belonging to clade A for Env and for Gag, except for one patient who was infected with a clade A Env-clade G Gag recombinant virus. All patients infected in France were infected with clade B viruses. The CTL responses obtained from all the African and all the French individuals tested showed frequent cross-reactions with proteins of the heterologous clade. Epitopes conserved between the viruses of clades A and B appeared especially frequent in Gag p24, Gag p18, integrase, and the central region of Nef. Cross-reactivity also existed among Gag epitopes of clades A, B, and G, as shown by the results for the patient infected with the clade A Env-clade G Gag recombinant virus. These results show that CTLs raised against viral antigens from different clades are able to cross-react, emphasizing the possibility of obtaining cross-immunizations for this part of the immune response in vaccinated individuals.

Adoptive immunotherapy for leukemia: donor lymphocytes transduced with the herpes simplex thymidine kinase gene for remission induction. HGTRI 0103.

This study will evaluate the safety and efficacy of allogenic donor lymphocyte infusions in patients who have relapsed hematologic malignancies after allogeneic bone marrow transplantation (BMT). Donor lymphocyte transfusions have resulted in the cure of some patients with relapsed leukemia or lymphoproliferative disorder after allogeneic BMT, but has been complicated by the development of graft versus host disease (GvHD). We hypothesize that a retroviral vector containing the Herpes simplex thymidine kinase (HStk) gene will allow for retention of the anti-leukemia response of transfused donor lymphocytes while allowing for the adverse effects of GVHD to be mitigated. Patients with relapsed hematologic malignancies after allogeneic BMT will be infused with ex vivo gene modified donor lymphocytes. The Herpes Simplex thymidine kinase (HStk) gene will be transduced into the cells ex vivo using LTKOSN. 1 vector supernate. Insertion of the HStk gene into lymphocytes confers a sensitivity to the anti-herpes drug ganciclovir (GCV). This selective destruction of donor lymphocytes in situ will be used to abrogate the effect of graft versus host disease, if it develops.

Positive role of macaque cytotoxic T lymphocytes during SIV infection: decrease of cellular viremia and increase of asymptomatic clinical period.

We have measured cellular viremia and observed clinical outcome of macaques from two cohorts, the first including 12 macaques infected by SIVmac251 and the second including 12 macaques immunized by lipopeptides and then challenged by SIVmac251. In the first cohort (SIV-infected macaques), 3 patterns of CTL responders were determined: high, low and non-responders. In the macaques belonging to pattern of low and non-responders, cellular viremia, measured by growing the virus from PBMC, was continuously high during the first 6 months after infection, and five macaques developed AIDS within 14.4 +/- 7.7 months. Conversely, in the six high-responder macaques, cellular viremia was constantly low and only one macaque developed AIDS at 19 months, the five others being alive at 24 months. After immunization with lipopeptides, 7/12 macaques showed CTL responses and among these, after SIV challenge, cellular viremia was continually low, and no disease was observed at 22 months of follow-up. Conversely, the five non-responder macaques displayed persistent high viremia and macaques developed AIDS within 12.6 +/- 2.9 months after SIV challenge. These data strongly suggest that the presence of cytotoxic responses is inversely correlated with cellular viremia and correlated with overall survival and thus in an important component of the immune response in vaccinated individuals. It supports the idea that a strengthening of the CTL responses, if possible, might be beneficial in HIV-infected human beings.

Herpes simplex thymidine kinase gene transfer is required for complete regression of murine colon adenocarcinoma.

The herpes simplex thymidine kinase (HStk) gene induces regression of epithelial tumors after ganciclovir (GCV) administration. This observation has been attributed to both gene transfer and metabolic cooperation between cells (the bystander effect). This study evaluates the relative roles of the bystander effect and gene delivery of the HStk gene by the LTKOSN.2 vector. MC38 colon adenocarcinoma cells, syngeneic for C57/B16 mice, were used. Whereas in vitro proliferation assays demonstrated a bystander effect, significantly greater inhibition of proliferation occurred with HStk gene transfer. In mixtures containing 75 per cent MC38 cells with no vector (MC38 NV) and 25 per cent MC38 pretransduced with LTKOSN.2 (MC38 TK), proliferation was inhibited by 62 +/- 5 per cent. In mixtures containing 75 per cent MC38 NV with 25 per cent HStk vector-producing cells (LTKOSN.2 VPC), proliferation was inhibited by 97 +/- 1 per cent. In vivo subcutaneous mixture experiments utilized MC38 NV cells inoculated at a 1:1 ratio with various treatment cell groups followed by administration of GCV. Tumor volumes (mean +/- standard error) at 30 days were: 264 +/- 66 mm3 for MC38 TK, 0 for LTKOSN.2 VPC, 1009 +/- 335 mm3 for lacZ VPC (beta-galactosidase VPC), and 1012 +/- 212 mm3 for NIH3T3 (nontransduced cells). These data suggest that in vivo, the bystander effect alone causes tumor inhibition, but gene transfer is necessary for complete tumor elimination in immunocompetent mice.

Gene therapy for colon cancer with the herpes simplex thymidine kinase gene.

BACKGROUND: The suicide gene and prodrug, herpes simplex thymidine kinase (HStk) and ganciclovir (GCV), are now in clinical trials for recurrent malignancies. METHODS: We evaluated in vitro and in vivo efficacy of HStk gene transfer and GCV treatment of colonic adenocarcinoma in a syngeneic murine model. RESULTS: In vitro analysis demonstrated that CT-26 adenocarcinoma cells transduced with LTKOSN.2 retroviral vector inhibited the proliferation of wild-type CT-26 (nontransduced) cells after GCV exposure. Cooperative killing with HStk gene therapy was shown in vivo, mixtures of HStk CT-26 transduced cells (CT-26 TK), and nontransduced (CT-26 NV) cells and tumors containing only 9% CT-26 TK cells demonstrated complete regression after GCV (100 mg/kg). CONCLUSIONS: This in vitro and in vivo demonstration suggests that metabolic cooperation permits destruction of tumors even when gene transfer is effective only to a relatively small portion of the tumor. These important results suggest new avenues can be developed for the treatment of this lethal malignancy.