UNIVERSIDAD DE BARCELONA FACULTAD DE MEDICINA DESARROLLO DE UNA VACUNA PREVENTIVA CONTRA EL VIH, BASADA EN BCG RECOMBINANTE - PDF

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UNIVERSIDAD DE BARCELONA FACULTAD DE MEDICINA DESARROLLO DE UNA VACUNA PREVENTIVA CONTRA EL VIH, BASADA EN BCG RECOMBINANTE TESIS DOCTORAL: ELIAS B. PEZZAT SAID 21 DE JUNIO DE 2005 IX. MANUSCRITOS EN PREPARACIÓN
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UNIVERSIDAD DE BARCELONA FACULTAD DE MEDICINA DESARROLLO DE UNA VACUNA PREVENTIVA CONTRA EL VIH, BASADA EN BCG RECOMBINANTE TESIS DOCTORAL: ELIAS B. PEZZAT SAID 21 DE JUNIO DE 2005 IX. MANUSCRITOS EN PREPARACIÓN Y COMUNICACIONES ESCRITAS ACEPTADAS Y PRESENTADAS EN CONGRESOS INTERNACIONALES 183 CONFERENCIA INTERNACIONAL SOBRE VACUNAS VIH. Laussane, septiembre 2004 Alpha-antigen promoter from M.tuberculosis and lysine gene complementation prevent the disruption of heterologous HIV-1 gp120 gene expression by genetic rearrangements in BCG strain lysine auxotrophic. A critical issue for recombinant BCG based HIV-1 vaccine development E. Pezzat, G. Virgili-Lopez (*), F. Etcheverry, T. Gallart, M. Plana, C. Gil, JM. Miró, F.García, J. Joseph(**) and JM. Gatell(**). HIV Vaccine Research and Development. AIDS Research Unit. Infectious Diseases Department. Hospital Clinic. August Pi i Sunyer Biomedical Research Institute (IDIBAPS), School of Medicine, University of Barcelona, Barcelona, Spain. * Presenting author; ** Research Directors Background: A promising approach for an HIV vaccine is Mycobacterium bovis Bacillus Calmette Guerin (BCG) as bacterial live recombinant vaccine vehicle. In this study, we have evaluated the disruption of heterologous HIV-1 gp120 gene expression by genetic rearrangements in BCG host Pasteur strain using a replicative (pmv261) vector. We have also employed the replicative (pjh222) vector in BCG host strain lysine auxotrophic complemented with a plasmid carrying a lysine complementing gene from M. smegmatis to compare the HIV- 1gp120 gene expression and plasmid stability in vivo. Methods: The entire DNA coding sequence of HIV-1 gp120 (HXBc2 strain) (from N. Letvin) was amplified by PCR and cloned into different Escherichia coli- Mycobacteria shuttle vectors (from B.Bloom and W.Jacobs) under the regulatory control of BCG hsp60 promoter (pmv261) and M. tuberculosis alpha-antigen promoter (pjh222). The coding sequence was fused to M.tuberculosis 19k-Da lipoprotein signal sequence (pjh222). BCG strains were transformed by electroporation and transformant BCG screened onto 7H10 medium plates containing kanamycin (25 ug/ml). Lysates of recombinant BCG were separated by 10% SDS-PAGE and analyzed for expression by Western blot analysis using monoclonal antibody directed to influenza hemagglutinin epitope. The mycobacterial plasmid DNA was used as template for PCR analysis and DNA sequencing. Results: 12 out of 14 rbcg:hiv-1gp120(261) colonies screened 184 showed a partial deletion in the DNA coding sequence and 2 did show a total deletion. On the other hand, 10 out of 10 rbcg:hiv-1gp120(222) colonies screened kept the fragment DNA intact (DNA sequencing confirmation). The partial deletion (900 bp) corresponds to gp120 core containing 3 glycosilations sites and V3, V4 and V5 regions of HIV-1 env. All rbcg mutants kept the ORF and one of them was analyzed by Western blot detecting a band of 25-KDa (truncated protein) and the expected 67-KDa of the rbcg:hiv-1gp120(222) recombinant protein. Strikingly, the DNA sequence coding for HIV-1 envelope immunodominant CTL epitope (P18 peptide) was also deleted in the rbcg mutants. Conclusion: We have demonstrated that E.coli-Mycobacteria expression vectors containing a weak promoter and lysine complementing gene in BCG host strain lysine auxotrophic do prevent genetic rearrangements involving toxic components and protective B-cell and CTL epitopes from HIV-1 envelope. A critical issue for rbcg based HIV-1 vaccine development. 185 CONFERENCIA INTERNACIONAL SOBRE VACUNAS VIH. Quebec, septiembre 2005 Identification and recruitment of a high risk population for HIV vaccine efficacy trials in Barcelona, Spain. Social and health impact on vulnerable population MF. Etcheverry 1 ; E. Pezzat 1 ; M. Meroño 2 ; M. Montero 3 ; F.García 1 ; E. Fumero 1 ; P.Castro 1 ; J.L. Blanco 1 ; C. Jaques 2 ; A. Capitán 2 ; E. Solf 3 ; JM Miró 3 ; J. Joseph 1* and JM Gatell 1* 1 HIV Vaccine Research and Development Group. AIDS Research Unit. Infectious Diseases Department. Hospital Clinic/IDIBAPS, University of Barcelona, Barcelona, Spain. 2 Ambit Prevenció. NGO for Health and Quality of life, Barcelona, Spain 3 Cruz Roja (Red Cross). Detoxification Unit for drug users, Barcelona, Spain (*) Research Directors Background: Identification and follow-up of HIV-negative high-risk individuals is necessary for the prevention of HIV transmission and for conducting efficacy HIV-1 preventive vaccine human trials. Objectives: i)to identify, recruit and follow-up seronegative individuals at high risk of HIV infection; ii) To estimate the HIV, HBV and HCV prevalence; iv) To evaluate the willingness of this cohort to participate in such trials in Spain. Methods: This multicentric study has involved the Hospital Clínic (HC) and two community based organizations: Red Cross Centre (CR), detoxification Unit for drug users and Ambit Prevenció (AP), day care center for sex-workers on the streets. Epidemiological data, behavior risk factors and willingness were recollected using a confidential questionnaire. HIV positive individuals were referred to a specialized 186 health center. HIV negative individuals were enrolled in this study and followed-up over 1 year. Results: We show our preliminary data on 176 subjects (81 CR,76 from AP and 19 from HC ) followed-up for 12 months in CR and, 6 months in HC and AP. There were 88 men, 82 women and 6 transexual. 52% of them were immigrants, 27% were injecting drug users (IDU), 59% others drugs users and 46% were sex-workers on the streets. The overall HIV seroprevalence was 6% (10/176), 5 of them from CR and 5 from AP. All individuals from HC (stable serodiscordant partners) were seronegative. Also, the hepatitis C virus seroprevalence was 50 % (51/103), 94% were IDUs from CR. Seroprevalence of hepatitis B virus (Anti-HBc) was 18 % (19/105). Among seronegative participants 158/166 (95%) affirmed that they would participate in efficacy HIV vaccine trial. Conclusion: This study provides important data for planning future vaccine efficacy studies in Spain and shows that research involving vulnerable population and interaction between health centers and NGOs is important to implement HIV prevention programs. 187 Alpha-antigen promoter from Mycobacteria spp and lysine gene complementation prevent the disruption of heterologous HIV-1 gp120 gene expression by genetic rearrangements in BCG strain lysine auxotrophic. A critical issue for recombinant BCG based HIV-1 vaccine Elias Pezzat et al. HIV Vaccine Research and Development Group, AIDS Research Unit, Infectious Diseases Department, Hospital Clínic/IDIBAPS), School of Medicine, University of Barcelona, Barcelona, Spain. A promising approach for an HIV vaccine is Mycobacterium bovis Bacillus Calmette Guerin (BCG) as bacterial live recombinant vaccine vehicle. In this study, we have evaluated the disruption of heterologous HIV-1 gp120 gene expression by genetic rearrangements in BCG host Pasteur strain using a replicative (pmv261) vector. We have also employed the replicative (pjh222) vector in BCG host strain lysine auxotrophic complemented with a plasmid carrying a lysine complementing gene from M. smegmatis to compare the HIV-1gp120 gene expression and plasmid stability in vivo. The entire DNA coding sequence of HIV-1 gp120 (HXBc2 strain) was amplified by PCR and cloned into different Escherichia coli- Mycobacteria shuttle vectors under the regulatory control of BCG hsp60 promoter (pmv261) and Mycobacteria spp. alphaantigen promoter (pjh222). 12 out of 14 rbcg:hiv-1gp120(261) colonies screened showed a partial deletion in the DNA coding sequence and 2 did show a total deletion. On the other hand, 10 out of 10 rbcg:hiv-1gp120(222) colonies screened kept the fragment DNA intact. Strikingly, the DNA sequence coding for HIV-1 envelope immunodominant CTL epitope (P18 peptide) was also deleted in the rbcg mutants. We have demonstrated that E.coli-Mycobacteria expression vectors containing a weak promoter and lysine complementing gene in BCG host strain lysine auxotrophic do prevent genetic rearrangements involving toxic components and protective B-cell and CTL epitopes from HIV-1 envelope. A critical issue for rbcg based HIV-1 vaccine. 188 1. Introduction The need for a safe and effective HIV vaccine has never been greater. The UNAIDS report on the global AIDS epidemic updated on december 2004 states that the total number of people living with the human immunodeficiency virus (HIV) has reached its highest level ever: an estimated 39,4 million people are living with HIV and 4,9 million people became newly infected with HIV in In the past year, the global AIDS epidemic killed 3,1 million people, and by 2010, the number of children orphaned by AIDS could be around 25 million [1]. It is estimated that 85% of these new infections occur in developing countries. In addition, ensuring universal access to antiretrovirals in developing countries still presents an enormous challenge [2]. There is strong evidence supporting a role of cytotoxic T lymphocytes (CTLs) in the containment of HIV replication. Several vaccine approaches are being taken to elicit anti-hiv CTL responses. A promising approach is Mycobacterium bovis bacillus Calmette Guérin (BCG) as bacterial live recombinant vaccine vehicle. BCG vaccine has been used to immunize more than two billion individuals against tuberculosis with a long record of safe use in humans and is able also to induce long lasting immunity. CTL induction against HIV-1 and SIV gag or env antigens has been described following immunization of mice or rhesus monkeys with recombinant BCG (rbcg) expressing these antigens. The development and understanding of mycobacterial genetic system has been hindered by the slow growth and special cell wall characteristics of the organism like tendency to aggregate. Many important contributions have provided understanding of this genetic system. Bloom et al. (1987) initially reported the development of E.coli-mycobacterial shuttle plasmids(8). They identified a 1.8-kilobase segment (orim) from plasmid pal5000 isolated from Mycobacterium fortuitum, which supports plasmid replication in M. Smegmatis and BCG. Yamada s group established a foreign antigen secretion system in mycobacteria using α-antigen promoter to express foreign antigens (9). Stover and collegues developed two different systems for propagating foreign DNA in mycobacteria: a multicopy extrachromosomal plasmid and single-copy plasmid, which is integrated into mycobacterial chromosome (10;11). Important contribution has been done by Tyagi et al (2000) on rbcg approach for development of vaccines. They have 189 developed a generic vector system for expression of genes at varying levels in mycobacteria under the control of a battery of mycobacterial promoters of varying strength for elicitation of optimal immune responses(12). Critical issues to be considered in developing rbcg technology include: i) antigen localization;ii) codon optimization;iii) plasmid stability and iv) genetic rearrangements. Genetic rearrangements have been described and occur in eukaryotes and prokaryotes. They may create new sequences, alter functions of existing sequences or place them in new regulatory situations. Have been reported that mycobacteria contains several insertion sequences (IS), and have been described that some of them possess transposition activity in Mycobacterium smegmatis and BCG. The role of such elements has not been explored in heterologous gene expression in mycobacteria. In this study we have evaluated the disruption of heterologous HIV-1 gp120 (HXBc2 strain) gene expression by genetic rearrangements in Mycobacterium Bovis BCG host strain using a replicative ( pmv261::hiv-1gp120 ) vector. We have also employed the replicative (pjh222::hiv-1gp120) and integrative (pjh223::hiv-1gp120) vectors in lysine auxotrophic BCG host strain complemented with a plasmid carrying a wild-type complementing gene, ensuring the maintenance of the recombinant antigen gene to compare the HIV-1gp120 gene expression and plasmid stability in vivo. In case of replicative pmv261::hiv-1gp120 vector, loss of HIV-1gp120 expression was due to a consensus DNA fragment deletion in the HIV-1gp120 gene detected in 10 different BCG clones by PCR. We have not detected any loss of HIV-1gp120 expression in the replicative (pjh222:hiv-1gp120 ) and integrative (pjh223:hiv-1gp120) vectors. 2. Materials and methods 2.1.Bacterial strains and culture methods The bacterial strains used in this study are listed in Table 1. Escheriachia coli cultures were grown in Luria-Bertani (LB) broth or on LB agar plates at 37 0 C. LB was supplemented with kanamycin ( 40 µg/ml). BCG wild type and Lysine auxotroph of BCG strains were transformed by electroporation. Mycobacterial cultures were grown in Middlebrook 7H9 broth (Difco) or on Middlebrook agar 7H10 medium (Difco) supplemented wit albumin-dextrose complex (ADC, Difco) and containing 0.05% Tween 80 and kanamycin ( 25 µg/ml). The L-Lysine Monohydrochloride was obtained 190 from Sigma Chemical, dissolved in destilled water, and used at a concentration of 40 µg/ml. 2.2 Electroporation of mycobacteria For transformation, BCG cultures were grown to an O.D of 0.9 (600nm), sedimented at 3000 rpm, and washed twice by resuspension and centrifugation (3000 rpm) in 10 % glycerol at 4 0 C, and finally resuspended in 1/20th of the original culture volume of cold 10 % glycerol. Then 100 µl of the cold BCG suspension was mixed with plasmid DNA (50-500ng) in a prechilled 0.2 cm electroporation cuvette and transformed using the Biorad Gene Pulser electroporator at 2.5kV, 25 mf, and 1000Ω. After electroporation 1mL 7H9 medium supplemented with ADC and containing Tween 80 and incubated at 37 0 C for 12 hours before plating on Middlebrook agar 7H10 medium (Difco) supplemented with albumin-dextrose complex (ADC, Difco) and containing 0.05% Tween 80 and kanamycin ( 25 µg/ml). 2.3.Construction of Expression vectors The plasmids and the E.coli / mycobacterial shuttle vectors used to express HIV-1 gp120 (SHIV-HXBc2P 3.2) antigen are listed in Table 1 and 2. Plasmid vectors pmv261, pjh222 and pjh223 were used as parental plasmids for all the plasmid constructs described below. Plasmid pmv261 and pjh222 are replicative vectors and pjh223 is an integrative vector. All of them contain a DNA cassette encoding kanamycin resistance, an E.coli origin of replication (orie), and an expression cassette containing a mycobacterial promoter, a multiple cloning site and a transcriptional terminator. The pmv 261 and pjh222 contains a mycobacterial plasmid origin of replication (orim) and the pjh223 plasmid contains a DNA segment carrying the attachment site (attp) and the integrase (int) gene from the mycobacteriophage L5. The entire DNA coding sequence of HIV-1 gp120 was synthesized by PCR, using oligonucleotide primers specific for HIV-1 gp120 gene and cloned into different Escherichia Coli- Mycobacterial shuttle vectors under the regulatory control of BCG hsp60 promoter (pmv261) and Mycobacteria spp. alpha-antigen promoter (pjh222 and pjh223). The coding sequence was fused to Mycobacterium tuberculosis 19kD lipoprotein signal sequence (pjh222 and pjh223). For immunodetection purpose we fused downstream of the HIV-1 gene the influenza peptide (hemagglutinin epitope ) coding sequence and six residues of histidine. The oligonucleotides were designed to 191 incorporate BamHI and HindIII sites at the 5 and 3 termini of the amplified DNA fragment in pmv261 plasmid, and HindIII-HindIII in pjh222 and pjh223 plasmids Plasmid DNA isolation and DNA sequencing The QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany) was used for isolation of plasmid DNA from mycobacteria and from E.coli. For mycobacteria we used the standard QIAprep Spin Miniprep protocol with one change. After addition of buffer P1, lysozyme was added at a concentration of 10 mg/ml and samples were incubated at 37 0 C overnight. The standard protocol was then followed. The mycobacterial plasmid DNA was transformed in E.coli JM109 using 5ul of the isolated plasmid DNA and purified according to the manufacturer s instructions and finally the plasmids pmv261:: HIVgp120 were used as templates for DNA sequencing and for PCR analysis. The nucleotide sequences were determined using the BigDye Terminator v3.1 Cycle Sequencing Kit (applied Biosystems) and ABI PRISM 3100 Genetic Analyzer. DNA sequence alignments were performed using the clustal software Western blot analysis BCG transformants were grown to mid-logarithmic phase in liquid 7H9 (Difco) medium containing kanamycin (25 µg/ml). rbcg cultures were centrifugued at 3000 rpm for 10 minutes at 4 0 C. Pellets were washed twice in PBS plus 0.02 % Tween-80 and resuspended in 1ml of extraction buffer (50mM Tris-HCl ph 7.5, 5mM EDTA, 0.6 % sodium dodecyl sulfate) and 5 µl of 100x protease inhibitor cocktail (1mg/ml aprotinin, 1mg/ml E-64, 1mg/ml leupeptin, 1 mg/ml pepstatin A, 50mg/ml pefabloc SC, and 10 ml DMSO) was added. Cells were sonicated for 4 minutes on ice on Branson sonifier at output control 7, duty cycle 50%. Extracts were centrifugued at rpm for 10 minutes at 4 0 C and supernatants were collected. Proteins were separated on 15 % SDS-polyacrylamide gel. After electroblotting, nitrocellulose membranes were first probed with mouse monoclonal antibody HA.11 directed to influenza hemagglutinin epitope (YPYDVPDYA) and second with HRP-conjugated antibodies. 3. Results 3.1 Cloning of HIV-1gp120 gene into different E.coli-mycobacterial expression vectors 192 The cloning of the HIV-1gp120 gene was confirmed by enzyme restriction analysis. 3.2 DNA fragment deletion detected by PCR and confirmation by restriction enzyme analysis As shown the figure,we detected by BCG colonies PCR the partial deletion using the specific primers that we used for cloning the DNA sequence corresponding to HIV- 1gp120 gene into pmv261 vector. This genetic rearrengement was detected and reproduced also in 12 out of 14 rbcg:hiv-1gp120 (261) colonies by PCR (data not shown). As we can observe in figure, this partial deletion was observed only by BCG colonies PCR when the DNA fragment was inserted into pmv261 vector that contains the hsp60 promoter from BCG. We did not detected by BCG colonies PCR the deletion when the DNA fragment was cloned into pjh222 and pjh223 vectors that contain the alpha antigen promoter. We also did compare the restricton enzyme digestion profile of the plasmid DNA vector pmv261 with and without heterologous DNA insert and pjh222 containing the heterologous DNA insert, pre and post BCG transformation. As shown in figure, the pmv261 plasmid vector without the DNA insert kept the same profile pre and post BCG transformation. Contarily, the pmv261 containing the DNA insert did show a different restriction profile pre and post BCG transformation. On the other hand, the pjh222 plasmid vector containing the DNA insert kept also the same restriction profile pre and post BCG transformation 3.3 Nucleotide sequence analysis and characterization of the HIV-1gp120 gene 12 out of 14 rbcg:hiv-1gp120(261) colonies screened showed a partial deletion in the DNA coding sequence and 2 did show a total deletion. On the other hand, 10 out of 10 rbcg:hiv-1gp120(222) colonies screened kept the fragment DNA intact (DNA sequencing confirmation). The partial deletion (900 bp) corresponds to gp120 core containing 3 glycosilations sites and V3, V4 and V5 regions of HIV-1 env. All rbcg mutants kept the ORF and one of them was analyzed by Western blot detecting a band of 25-KDa (truncated protein) and the expected 67-KDa of the rbcg:hiv-1gp120(222) recombinant protein. Strikingly, the DNA sequence coding for HIV-1 envelope immunodominant CTL epitope (P18 peptide) was also deleted in the rbcg mutants. No deletions were observed with pjh222 and pjh223 plasmids, which
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