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Table of contents
Chapter 1- Review of Literature
1.1 Lentiviruses of Human and Animals
1.1.1 Retroviruses
1.1.2 Lentiviruses
1.1.3 Timeline of Lentivirus discovery
1.2 Natural History of HIV-1
1.2.1 Discovery of AIDS and the causative agent HIV
1.2.2 Epidemiology and Geographic distribution
1.2.3 HIV-1 Transmission
1.2.4 Transmitted Founder (T/F) virus
1.2.5 Tropism / Target cells
1.2.6 Pathogenesis
1.3 Structure and genetic organization of HIV-1
1.3.1 Structure / Morphology of HIV virions
1.3.2 Genomic organization and proteins
1.3.3 Replication and virus life cycle
1.3.4 Cell factors interfering with HIV-1 replication
1.4 HIV Immunology and Vaccination
1.4.1 Overview of HIV-1 Immunology 1.4.1.1 HIV vs Innate immunity
1.4.1.2 The adaptive T cell response to HIV-1
1.4.1.3 The antibody response
1.4.1.4 Obstacles to an efficient vaccine
1.4.1.5 Goals of a successful vaccine
1.4.2 Strategies used to develop HIV Vaccines
1.4.2.1 Live attenuated vaccines
1.4.2.2 Subunit Vaccine
1.4.2.2.1 Recombinant Protein subunit vaccines Virus like particle (VLPs)
1.4.2.2.2 Synthetic Peptide
1.4.2.3 DNA vaccine
1.4.2.4 Recombinant viral vector vaccines
1.4.3 Clinical Trials phase IIb and phase III trials
1.4.3.1 VAX003/VAX004
1.4.3.2 MrK/ STEP -HVTN502/ & HVTN503 –Phambili study
1.4.3.3 RV144
1.4.3.4 HVTN505
1.4.4 Development of innovative HIV vaccines in our lab
1.4.5 Cytokines as adjuvants
1.5 SIV and SHIV as NHP models for HIV.
1.5.1 SIVs as challenge viruses
1.5.2 SHIVs as challenge viruses
1.5.3 The importance of a CCR5 tropic challenge virus
1.5.4 The need of novel development of a T/F CCR5 clade C envelop in a challenge virus
1.6 Antiretroviral drugs and HIV-1 Latency
1.6.1 ART- Antiretroviral drugs
1.6.2 Latency and Persistence
1.6.2.1 Pre-integration latency:
1.6.2.2 Post-integration latency:
1.6.2.3 Persistence
1.6.3 Mechanism of post integration latency
1.6.3.1 Integration site and orientation
1.6.3.2 Chromatin Remodeling
1.6.3.3 Host transcription factors and viral latency
1.6.3.4 Elongation factors and viral latency
1.6.3.5 Tat feedback and viral latency
1.6.3.6 Post transcriptional mechanism of latency-the micro RNA (miRNA).
1.6.4 Reservoirs, types of cells and anatomical reservoirs
1.6.5 Current models to study latency
1.6.5.1 Primary cell models of latency
1.6.5.2 Humanized mouse models
1.6.5.3 Non-human primate (NHP) model
1.6.5.4 Experimental approach to detect the latently infected cells in vivo
1.6.5.4.1 Detecting the integrated HIV-1 DNA and Cell associated RNA
1.6.5.4.2 Detecting cells carrying the replication- competent virus/ Assay to measure the latent reservoir
1.6.6 Prospects for treatment of latency
1.6.6.1 ART intensification
1.6.6.2 Using transcriptional inhibitors to control the HIV-1 progression
1.6.6.3 Strategies to reactivate latent provirus from reservoirs (“Flushing out”/ “Shock and Kill”)
1.6.6.4 Strategies based on the increasing apoptosis susceptibility
1.6.6.5 Other therapies targeting latency
1.6.6.6 Challenges in treatment of latency
1.7 Natural History of CAEV
1.7.1 Discovery and Epidemiology
1.7.2 Genomic Organization
1.7.3 Tropism & Target cells
1.7.4 Pathogenesis
Chapter 2- Materials and Methods
2.1 Materials
2.1.2 Plasmids
2.1.2.1 Cloning Vector
2.1.2.2 Infectious molecular plasmid genomes
2.1.3 Chemicals Buffers and Reagents.
2.1.3.1 Microbiology reagents
2.1.3.2 Bacterial Growth Media
2.1.3.3 LB Agar Plates
2.1.3.4 SOC Medium
2.1.3.5 Molecular Biology Reagents
2.1.3.5.1 Reagents for restriction enzyme digestions
2.1.3.5.2 Reagents for DNA modifications
2.1.3.5.3 Reagents for PCR
2.1.3.7 Buffers for Plasmid DNA isolation
2.2 Microbiology Methods.
2.2.1 Growth and storage of bacterial strains.
2.2.2 Transformation by heat shock method
2.2.2.1 Transformation protocol for JM109 and Stable -2 competent cells.
2.2.2.2 Transformation protocol for dam-/dcm- Competent E. coli cells.
2.2.3 Isolation of Plasmid DNA
2.2.3.1 Mini preparation of plasmid DNA (10-15 μg)
2.2.3.2 Midi preparation of plasmid DNA (200-500 μg)
2.2.4. DNA Quantification
2.2.6 Extraction and Purification of PCR products / DNA from agarose gel
2.3 Molecular Biology and Cloning
2.3.1 Polymerase Chain Reaction (PCR)
2.3.2 Restriction enzyme digestion
2.3.3. DNA Modifications
2.3.3.1 Dephosphorylation of DNA 5’
2.3.3.2 Klenow
2.3.3.3 T4 DNA polymerase
2.3.4 Ligation
2.3.5 DNA precipitation by ethanol
2.3.6 DNA Sequencing
2.3.7 Oligonucleotides -Primers Used
2.3.7.1 Oligonucleotides used for PCR
2.3.7.2 Oligonucleotides for site directed mutation
2.3.7.3 Primers for whole genome sequencing of the CAL-SHIV-IN+
2.3.7.4 Primer pairs used for introducing restriction sites for cloning.
2.4 Cell Culture
2.4.1 Media and Reagents
2.4.1.1 The media were purchased from Eurobio or Thermo Fisher Scientific.
2.4.1.2 The antibiotics and supplements used were purchased from EuroBio.
2.4.1.3 Transfection reagents:
2.4.1.4 X-gal staining solution:
2.4.1.5 PFA stock solution 20%
2.4.1.6 Giemsa-May-Grünwald staining reagents
2.4.2 Cell lines
2.4.3 Transfection of plasmid DNA into HEK-293T cells or TZM-bl cells.
2.4.3.1 Liposome mediated transfection:
2.4.3.2 Calcium Phosphate mediated transfection
2.4.4 Infection of cell lines
2.4.5 TZM-bl and MGG stainings
2.4.6 Adaptation of viruses to cell lines (cell free and cell associated)
2.4.7 Isolation; activation and infecting the PBMCs
2.4.8 Titration of viral stocks
2.4.9 Genomic DNA isolation
2.4.10 Quantitative analysis of SIV Gag p27 by ELISA
2.4.11 Virus harvest and storage
2.5 Cloning strategies used for Molecular virology
2.5.1 Construction of CAL-SHIV-IN+
2.5.1.1 Cloning of synthetic vif gene
2.5.1.2 Site directed mutagenesis to exchange the mutated vif
2.5.1.3 Introducing the polypurine tract (PPT) of CAEV
2.5.2 Construction of the SHIV-YCC
2.5.3 Construction of the CAL-SHIV-IN-
2.5.3.1 Construction of the CSH-DIN and introduction of an AgeI site and “Kozak” re-initiation spaced sequence
2.5.3.2 Construction steps to introduce functional vif and env
2.5.3.3 Construction of CSH-DIN-eGFP
2.5.3.4 Construction steps to introduce missing 60bp of RNaseH
2.5.3.5 Construction of CSH-DIN with the IRES eGFP (under construction)
2.5.4 Construction of CSH-DIN with Transmitted/Founder (T/F) Env & WARO Env
2.5.5 Construction of SHIV-AD8EO with Transmitted/Founder Envelopes
2.5.6 Construction of the SHIV-KU2 with WARO Envelope using Gibson Assembly Thesis Objectives
CHAPTER 3- RESULTS
PART I- The CAL-SHIV-IN- Vaccine Construct and derivatives
3.1 The CAL-SHIV-IN- vaccine prototype
3.1.1 Construction of the CSH-DIN
3.1.1.1 Strategies and molecular construction of CSH-DIN vaccine
3.1.2 Construction of the CSH-DIN expressing eGFP
3.1.2.1 Introduction of eGFP coding sequences at the AgeI site
3.1.3 CSH-DIN constructs with T/F envelopes
3.1.3.1 Molecular biology and construction of the CSH-DIN with T/F envelops
3.1.3.2 In cellulo studies of the CSH-DIN with T/F envelopes
3.1.3.2.1 The CSH-DIN-T/F-31, CSH-DIN-T/F-18, CSH-DIN-T/F-78, CSH-DIN-WARO vaccine prototypes produce single-cycle viruses
3.1.3.2.2 Tropism CSH-DIN-T/F-31, CSH-DIN-T/F-18, CSH-DIN-T/F-78, CSH-DIN-WARO studies on different cell lines
3.1.3.3 Quantification of Viral protein production (Gag p27 ELISA)
PART II- Novel replication-competent constructs
3.2. The replication competent chimeric lentiviruses with CAEV LTRs
Strategy 1: Construction and analysis of CAL-SHIV-IN+ & CSH-INP constructs
Strategy 2: Construction and analysis of the SHIV-YCC construct
3.2.1. Strategy 1-Molecular biology and construction of the CAL-SHIV-IN+ and CSH-INP
3.2.1.1 Strategy to develop the CAL-SHIV-IN+ construct
a) The CAL-SHIV-IN+
b) CSH-INP
3.2.1.2 Checking of the CAL-SHIV-IN+
a) The CAL-SHIV-IN+
3.2.1.3 Site directed mutagenesis to obtain a correct vif gene
3.2.2 In cellulo studies of the replication competency of the CAL-SHIV-IN+ and CSH-INP vectors .
3.2.3- Adaptation on different cell lines
3.2.4- Adaptation on monkey PBMCs
3.3- Strategy 2- Construction of the SHIV-YCC replication competent virus
3.3.1 In cellulo studies of the SHIV-YCC
3.3.1.1 Production of viruses and infection
3.3.1.2 Adaptation to different cell lines
3.3.1.3 Adaptation to monkey PBMCs
3.3.1.4. Quantification of Viral production
b) Real-Time Quantitative Reverse Transcription PCR
PART III- Novel SHIVs containing primary envelopes
3.4 SHIV-WARO
3.4.1 Constructions of the SHIV-KU2/WARO
3.4.2 Quantification of Gag p27 viral production
3.5 SHIV-AD8EO with T/F Clade C envelope & SHIV-KU2 with WARO envelope
3.5.1 Constructions of the SHIV-AD8EO-T/F
3.5.2 In cellulo studies of the SHIV-T/F
3.5.2.1 Transfection and infection
3.5.2.2 Tropism studies on different cell lines
Chapter 4- Discussion and Future Prospects
4.1 The Single Cycle Replication Competent Vaccine
4.1.1 Prospects for a Single Cycle Replication Competent Vaccine
4.2 The replication competent constructs to study host/virus interactions involved in latency and persistence the development of a SHIV for experimental infection studies
a) CAL-SHIV-IN+
b) CSH-INP
c) SHIV-YCC
4.2.1 Prospects of the CSH-INP and SHIV-YCC replication competent constructs
4.3 SHIV-AD8EO with T/F Envelops
Prospects of the SHIV-AD8EO T/F
Conclusion
