Construction, stability and immunogenicity of recombinant BCG expressing HIV-1 subtype C gag under the control of MtrA promoter, with or without the leader sequences

XIV CHAPTER 1: INTRODUCTION . 1 1.1. GLOBAL IMPACT OF HIV AND AIDS . 1 1.2. THE VALUE OF INDUCTION OF BOTH HUMORAL AND CELLULAR IMMUNE RESPONSES IN HIV VACCINE DEVELOPMENT . 2 1.3. PHASE III TRIALS AND THEIR IMPLICATIONS IN HIV VACCINE DEVELOPMENT . 3 1.4. ELITE CONTROLLERS AND THEIR INFORMATION ON CONTROL OF HIV INFECTION . 7 1.5. HIV VACCINE VECTORS AND VACCINATION STRATEGIES . 8 1.5.1. Viral vectors . 9 1.5. 2. Virus like particles . 9 1.5. 3. Envelope-based vaccines . 10 1.5. 4. DNA vaccines . 10 1.5. 5. Viral sequences used in HIV vaccines . 10 1.5. 6. Prime-boost regimes . 11 1.6. DEVELOPMENT OF RBCG AS AN HIV VACCINE VECTOR. 14 1.6.1. Disadvantages of using rBCG as a vaccine . 16 1.6.2. Stability of rBCG vaccines . 17 1.6. 3. Plasmid shuttle vector features . 17 1.6. 4. Protein translocation systems in mycobacteria . 17 1.6. 4.1. Sec-mediated system . 18 1.6. 4.2. TAT system . 18 1.6. 4.3. Snm/ESX-1 system . 19 1.6. 5. Characteristics and use of signal peptides . 20 1.6. 6. Examples of signal peptides . 21 1.6. 6.1. The 19kDa signal sequence . 21 U ni ve rs ity o f C ap e To w n ii 1.6. 6.2. The α-antigen signal sequence . 22 1.6. 7. Use of leader peptides to determine the types of immune responses elicited . 23 1.6. 8. Mycobacterial promoters . 23 1.6. 9. The hsp60 promoter . 25 1.6.10. The mtrA promoter . 25 1.7. CODON OPTIMIZATION OF FOREIGN GENES . 26 1.8. AIM . 26 CHAPTER 2: MATERIALS AND METHODS . 28 2.1 STUDY DESIGN . 28 2.2 MATERIALS AND GENERAL METHODS FOR THE GROWTH AND CULTURING OF ESCHERICHIA COLI . 28 2.2.1 E. coli strains, growth media and culture conditions . 28 2.2.2 Preparation of competent E. coli cells . 28 2.2.3 Transformation of E. coli with shuttle plasmid vectors . 29 2.2.4 Enzymatic reactions . 29 2.2.4.1 Agarose gel electrophoresis of DNA . 29 2.2.4.2 Restriction endonuclease digestion, DNA extraction and purification . 30 2.2.4.3 Blunting of restriction sites by T4 DNA polymerase . 30 2.2.4.4 DNA ligation . 30 2.2.5 Small scale plasmid DNA isolation . 31 2.2.6 Large scale plasmid isolation . 31 2.3 CONSTRUCTION OF E. COLI/MYCOBACTERIAL SHUTTLE VECTORS . 32 2.3.1 Construction of 1 st phase plasmids . 34 2.3.2 Construction of 2 nd phase plasmids . 35 2.4 MATERIALS AND METHODS FOR THE GROWTH AND CULTURING OF MYCOBACTERIA . 35 2.4.1 Mycobacterial strains, growth media and culture conditions . 35 2.4.3 Preparation of electro-competent mycobacterial cells . 36 2.4.4 Electro-transformation of shuttle plasmid vectors into mycobacteria . 37 2.4. 5. Total protein extraction . 38 2.4. 5.1 Bead beating method . 38 2.4. 6 Quantification of total protein from mycobacterial cultures . 38 2.5 EVALUATION OF VECTOR FITNESS . 39 2.5.1 Indication of vector fitness by growth rates on solid medium . 39 2.5. 2 Indication of vector fitness by growth rates in liquid medium . 40 2.6 EVALUATION OF PLASMID STABILITY . 40 2.7. EVALUATION OF BASAL LEVEL EXPRESSION OF HIV-1 GAG BY P24 CAPTURE ELISA ASSAY . 40 2.8. PREPARATION OF RBCG VACCINE STOCKS . 41 U ni ve rs ity o f C ap e To w n iii 2.9 EVALUATION OF RBCG VACCINE STABILITY BY RESTRICTION ENDONUCLEASE MAPPING OF ISOLATED PLASMID DNA . 41 2.10 VACCINATION OF BALB/C MICE WITH THE RBCG VACCINES AND SAAVI-MVA-C . 42 2.10. 1 Preparation of Mouse splenocytes . 43 2.10. 2 Fluorescence-activated cell sorting (FACS) analysis to determine cell population in mouse spleens . 44 2.10. 3 Assessment of CD8 and CD4 T-cell responses in spleens . 44 2.10. 4 Mouse IFN- and IL-2 ELISPOT assays . 44 2.10. 5 Validation of vaccination dose and rBCG recovery from mouse spleens . 46 CHAPTER 3: RESULTS . 47 3.1 SUMMARY OF RESULTS . 47 3.2 DEVELOPMENT OF STABLE VACCINE SHUTTLE VECTORS . 49 3.2.1 Selection of optimal shuttle vector . 49 3.2.2 Construction of plasmid shuttle vectors for rBCG-HIV vaccines . 51 3.2.2.1 Construction of pEMGag2(V3SV5), pEM19Gag2(V3SV5) and pEM Gag2(V3SV5) . 52 3.2.2.2 Construction of pEM(V3SV5), pEM19(V3SV5) and pEM(V3SV5) . 56 3.2.3 Determination of vector fitness by growth rates . 60 3.2.3.1. Indication of fitness by growth rates on solid medium .