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June 27, 2007 EUROPEAN JOURNAL OF MEDICAL RESEARCH 127 F.20 (Vortrag) First results of a randomized, controlled phase-IIstudy with a MVA-Nef vaccine in HIV-1 infected patients with CD4 counts > 250/l followed by Structured Treatment Interruption (STI) Harrer T. 1 , Hain J. 2 , Baedecker N. 2 , Vollmar J. 2 , Helm M. 3 , Gorriahn D. 4 , Schneider L. 5 , Jäger H. 6 , Harrer E.G. 1 1 Universitätsklinikum Erlangen, Medizinische Klinik 3, Klinische Infektionsimmunologie, Erlangen, Germany, 2 Bavarian-Nordic GmbH, Martinsried, Germany, 3 Practice Dr. Helm, Nuremberg, Germany, 4 Practice Dr.Gorriahn, Munich, Germany, 5 Practice Dr.Schneider, Fürth, Germany, 6Practice Dr.Jäger, Munich, Germany Introduction: MVA-BN® is a safe viral vector incapable of replicating in human cells. A derived recombinant vaccine, MVA-nef expressing the nef gene from HIV-1-LAI, has previously shown a good safety profile and the capability to induce Nef-specific CD4+ T-cells and CD8+ CTL. To assess the immunogenicity and safety of MVA nef in dependence of dose and in comparison to MVA (IMVAMUNE®) we performed a study in HIV-1 infected patients on HAART. Methods: In this single blind, randomized controlled phase II study 77 patients received 3 s.c. vaccinations of either 1E8 TCID50 IMVAMUNE®, 1E8 TCID50 or 5E8 TCID50 MVAnef (n=26,25,26) at weeks 0, 8, 16. At w20 patients were offered a STI with monitoring at weeks 24/26/28/32/40/52 regarding safety, plasma viral load (VL), CD4 counts and immune response to HIV. Results: Safety: No vaccine related serious adverse reactions were reported. All subjects developed local injection site reactions,mostly mild to moderate. Solicited systemic adverse effects were transient and also mostly mild to moderate, events grade 3 (fever, myalgia, headache, fatigue) occurred only in single cases. MVA-specific antibodies: Both the vaccinia preimmune and the vaccinia naïve patients developed a good antibody response to MVA. STI phase: In total 37 of 77 patients stopped HAART after w20. VL increased in all patients within w0 to w8 after STI followed by a subsequent decrease. There is an indication for a trend that the decrease in VL in patients receiving the MVA-nef vaccine is more pronounced than in the control group with IMVAMUNE® In total 25 patients remained off HAART for at least 32 weeks. Conclusions: MVA-nef proved to be safe and immunogenic in HIV-1-infected patients confirming previous results from clinical studies. Data from the Structured Treatment Interruption indicate a vaccine and dose relationship regarding viral load. Furthermore, the strong induction of an immune response against IMVAMUNE® confirms the potential as a safe and potent smallpox vaccine in HIV-infected subjects. Based on these positive results in HIV infected patients further studies are granted to evaluate the capability of the MVA-vector technology for development of therapeutic HIV vaccines. Acknowledgement: Partially funded by NIAID (N01-AI-40072). F.21 (Poster) Selection of HIV-1 specific scFv antibodies from LTNP derived phage libraries Antoni S. 1 , Hust M. 2 , Dübel S. 2 , Dietrich U. 1 1 Georg-Speyer-Haus, Molekulare Virologie, Frankfurt/Main, Germany, 2 Technische Universität Braunschweig, Institut für Biochemie und Biotechnologie, Braunschweig, Germany Objective: The aim of this study was to generate scFv phage libraries from long-term non-progressors (LTNP) with broadly neutralizing antibodies to select new HIV-1 specific scFv able to neutralize the infection. Methods: cDNA was prepared from B-cells of well characterized LTNPs having broadly neutralizing antibodies against HIV-1. ScFv libraries were generated and screened on different variants of the envelope protein to identify cross-reactive phage. Phage derived scFv sequences were analyzed and grouped according to their homology. ScFv fragments from reactive clones will be analyzed for their neutralizing capacity against HIV-1 by in-vitro neutralization assays. Results: Phage were generated from 2 LTNPs in which we could prove broadly neutralizing antibodies against HIV-1 as the most likely cause for non-progression (Humbert et al., 2007). cDNA was prepared from lymphocyte RNA and used to generate k and l scFv libraries derived from IgG and IgM. First pannings with monomeric forms of Env (SF162, BaL, JR-FL gp120) led to an enrichment of HIV-1 specific phage. Two Env-specific-scFvs were selected so far. Further characterization and new pannings with uncleaved Envs (gp160) and trimeric forms of gp140 are ongoing. Conclusion: ScFv phage generated from LTNP with broadly neutralizing antibodies against HIV-1 could represent a helpful tool to isolate new anti HIV-1 specific scFv fragments for therapeutic or preventive applications. This work is supported by the Deutsche Forschungsgemeinschaft and the BGAG-Walter Hesselbach Stiftung. F.22 (Vortrag) A phase 1 study to evaluate the safety and immunogenicity of a recombinant adeno-associated virus HIV vaccine van Lunzen J. 1 , Mehendale S. 2 , Clumeck N. 3 , Vets E. 4 , Rockstroh J. 5 , Anklesaria P. 6 , Johnson P. 7 , Lehrman J. 8 , Schmidt C. 8 , Excler J.L. 9 , Fast P. 8 , Heald A. 6 1 University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 2 National AIDS Research Institute, Pune, India, 3 St. Pierre University Hostital, Brussels, Belgium, 4 SGS Biopharma, Antwerp, Belgium, 5 University Clinic, Bonn, Germany, 6 Targeted Genetics Corporation, Seattle, United States of America, 7 The Children`s Hospital of Philadelphia, Philadelphia, United States of America, 8 IAVI, New York, United States of America, 9 IAVI, New Dehli, India Background: Delivery of HIV genes within recombinant adeno-associated virus (rAAV) protein capsid is a potent inducer of both HIV-specific antibodies and T-cell responses in animal studies. A rAAV-based vaccine (tgAAC09), consisting of single-stranded DNA from Clade C HIV-1 genes for the gag, protease and part of the reverse transcriptase proteins enclosed within a rAAV2 protein capsid, was developed as a potential component of a HIV vaccine.
128 EUROPEAN JOURNAL OF MEDICAL RESEARCH June 27, 2007 Objective: To evaluate the safety and immunogenicity of tgAAC09 in healthy, HIV-uninfected adult volunteers at low risk for HIV infection. Methods: In this dose-escalation study, 80 healthy, HIV-uninfected volunteers received a single intra-muscular injection of tgAAC09 at different doses: 3x109 DRP (n=16), 3x1010 DRP (n=23), 3x1011 DRP (n=25) or placebo (n=16). In addition, 21 of 50 volunteers received a boost vaccination of tgAAC09 at a dose of 3x1011 DRP or placebo. T-cell responses were assessed by IFN- ELISPOT assay, anti-AAV2 neutralizing titers (NT) by a cell-based assay, and vaccine shedding by PCR. Results: The vaccine was well-tolerated after both initial and boost vaccination. Mild-to-moderate local and systemic reactogenicity was experienced by app. 15-20% of volunteers. No vaccine-related SAE`s were reported. The proportion of reactogenicity and AE`s were evenly distributed among the different dose groups and placebo. There was no evidence of vaccine shedding. At baseline, 48% of European and 97% of Indian volunteers had detectable NT against AAV2. The proportion of volunteers with four-fold or greater rise in anti- AAV2 NT increased with higher doses and after boost vaccination. HIV-specific T-cell responses were detected in 9/64 vaccinees, with 24% (6/25) of volunteers in the highest dose group responding. All responses were to gag epitopes and the magnitude was moderate (38-385 SFC/106 PBMC). No induction of antibody to HIV was observed. Conclusions: Vaccination with tgAAC09 appears to be safe and well-tolerated. Immunogenicity data indicated a moderate response at the highest dose, directed to gag. Future clinical trials will focus on higher doses of tgAAC09, the optimal interval for boost vaccination, the effect of pre-existing immunity to AAV2, and evaluating prime-boost regimens. F.23 (Poster) Induction of neutralising antibodies after immunisation with the transmembrane envelope proteins p15E of gammaretroviruses: Implication for the development of an HIV vaccine Fiebig U. 1 , Langhammer S. 1 , Behrendt R. 1 , Kurth R. 1 , Denner J. 1 1 Robert Koch-Institut, Berlin, Germany Objective: Neutralising antibodies specific for the membrane-proximal external region (MPER) of the transmembrane envelope (TM) protein gp41 of HIV such as 2F5 and 4E10 have been isolated from infected patients. These antibodies are broadly neutralising and decreased the HIV load in clinical trials. However, many attempts to induce such antibodies failed so far. To study the mechanism of induction and the efficacy of neutralising antibodies directed against the retroviral TM proteins, the TM proteins p15E of three different gammaretroviruses (porcine endogenous retrovirus, PERV; feline leukaemia virus, FeLV, and Koala retrovirus, KoRV) were used in immunisation experiments. Methods: Recombinant p15E were produced; rats, goats and cats were immunised, neutralising antibodies were analysed in a real-time PCR based infection assay; ELISAs, Western blots and epitope mappings were performed, cats immunised with FeLVp15E were challenged with infectious FeLV. Results: Antibodies neutralising PERV, FeLV and KoRV were obtained after immunisation with the corresponding p15E. Two epitopes, one located in the N-terminal part near the fusion peptide (designated E1), the other in the C-terminal MPER (E2) of p15E were found in all cases. The localisation of E2 corresponds to the localisation of the epitopes of 2F5/4E10 and despite the evolutionary difference of HIV and gammaretroviruses a limited sequence homology was observed. Neutralising antibodies specific for p15E of FeLV prevented in immunised cats an antigenemia, demonstrating their efficacy in vivo. Conclusion: These data show that the E2 domain represents a highly vulnerable target for neutralisation of retroviruses. Since by us (WO 2005/021574) and others a E1 domain was identified in gp41 of HIV, immunisation with proteins containing these domains may induce broadly neutralising antibodies of the type 2F5 and 4E10. F.24 (Poster) A novel gene therapeutic strategy against HIV-1 infection using the suicide protein tBid Hülsmann P.M. 1 , Hofmann A.D. 1 , Rauch P. 1 , Wolff H. 2 , Berens C. 3 , Metzner K. 1 1 University of Erlangen-Nuremberg, Institute of Clinical and Molecular Virology, Erlangen, Germany, 2GSF-Helmholtz Research Center for Environment and Health, Institute of Molecular Virology, Neuherberg, Germany, 3 University of Erlangen-Nuremberg, Chair of Microbiology, Microbiology, Erlangen, Germany Objective: Specific elimination of human immunodeficiency virus type 1 (HIV-1) infected cells by HIV-1 LTR driven suicide genes presents a promising strategy in gene therapy of HIV-1. Clinical applications of this approach depend on a fast and effective suicide gene which is exclusively expressed in HIV-1 infected cells. These preconditions have not yet been fulfilled and, thus far, success of suicide approaches is limited. It was recently shown that expression of truncated Bid (tBid), a human pro-apoptotic protein, rapidly and efficiently induces apoptosis. Thus, we tested tBid in the context of a novel HIV-1 LTR-based, Tat- and Rev-dependent transgene expression vector as a new potential suicide strategy in the gene therapy of HIV-1. Methods: tBid was cloned into the HIV-1 LTR-based Tatand Rev-dependent expression vector pLRed2xINSR replacing dsRed. The effects of tBid were then analyzed in the presence or absence of the HIV-1 proteins Tat and/or Rev in transiently and stably transfected 293T and HeLa cells. Cell death was determined 24 hours after transfection by PI-staining. The original vector pLRed2xINSR was used as control and dsRed expression determined by FACS and fluorescence-microscopy. Results: tBid, expressed by the strong constitutive CMV promoter, was highly efficient in inducing apoptosis in both 293T and HeLa cells in less than 24 hours showing that tBid is effective in these cell lines. When expression of tBid was driven by the HIV-1 LTR promoter, induction of apoptosis was not observed in both cell lines in the absence of co-factors showing that this system is not leaky. Coexpression of Rev alone did not lead to induction of apoptosis, but coexpression of Tat alone moderately induced apoptosis. This effect was strongly enhanced by the co-expression of Tat and Rev. Cell death was rapidly observed within 24 hours. Equivalent results for dsRed expression/fluorescence were obtained using the control vector pLRed2xINSR.
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