Microbicides as the most effective means to prevent HIV-1 transmission

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esculentum and their topical microbicidal activity

Background on HIV/AIDS It is over 30 years since the Centres for Disease Control and prevention (CDC) first reported acquired immunodeficiency syndrome (AIDS) in 1981 (Fortson, 2011). Since then, UNAIDS (2007a) reported a global total of 2.7 million new infections in 2008. Out of approximately 60 million infections, more than 25 million persons have died of AIDS and currently, more than 33 million persons are HIV positive or living with AIDS (Dieffenbach and Fauci, 2011). Sub-Saharan Africa constitutes 3% of the global population and has an alarming 68% of the world’s individuals living with HIV/AIDS (Lurie and Rosenthal, 2010). According to UNAIDS (2007b), there is no other region with HIV prevalence greater than 1%, yet 5% of adults have been reported to be infected with HIV in sub-Saharan Africa.
Heterosexual intercourse is the predominant mode of HIV type 1 (HIV-1) transmission across the globe which accounts for more than 90% of HIV-1 infections (Kish-Catalone et al., 2006). The World Health Organisation (2009) reported that AIDS is the main cause of death among women of reproductive age across the globe, thus women increasingly bear a disproportionate burden of the pandemic. UNAIDS / WHO (2004) reported that females in the sub-Saharan Africa account just about 57% of the total infected population. These should not raise so many questions because women have become and continue to be victims of sexual violence due to personal, economic, social and cultural issues (Ramjee et al., 2006a). Thus the high HIV infection rates have prompted the UN Joint Programme on HIV/AIDS to call for extra efforts in HIV/AIDS interventions (Sidibe, 2010).

Entry and fusion of the HIV to the host cell

HIV particles have a diameter of 100 nm and are surrounded by viral envelope (or membrane) which is made up of a lipid bilayer (Figure 2) (Fanales-Belasio et al., 2010). Three gp120 envelope glycoproteins that are noncovalently associated to three gp41 transmembrane molecules project from the viral envelope as viral spikes (Pancera et al., 2010). Inside the viral envelope, there is a layer of matrix protein (p17). The capsid (viral core) surrounds two copies of the viral ssRNA, reverse transcriptase, integrase and protease enzymes that are required for HIV replication (reviewed in Gelderblom et al., 1989; Luciw, 1996). A capsid is made up of polymers of protein p24 (Fanales-Belasio et al., 2010).
The virus infects target cells (not only in the genital tract) including dendritic cells, CD4+ lymphocytes and macrophages (Olinger et al., 2000; Clapham and McKnight, 2001; Saphire et al., 2001; Buckheit et al., 2010). Figure 2 also illustrates the structure of HIV subsequent to post-translational modification processes including glycosylation, oligomerisation and cleavage resulting into two subunits, gp120 and gp41 (Zhang et al., 2001). HIV infection is established by attachment of gp120 to the cellular lectins (DC-Sign and related C-type lectins) found on the surface of CD4+ lymphocytes, macrophages and dendritic cells (Figure 3) (Dhawan and Mayer, 2006). Three steps are involved in the entry of the virus thus, (1) attachment to the CD4 receptor; (2) binding to the co- receptor and (3) fusion process. All three steps are mediated by the viral envelope (env) proteins, gp120 and gp41 projecting from the membrane of the virus (Tilton and Doms, 2010). During infection, the gp120 subunits first contact CD4 (Anastassopoulou et al., 2009). The CD4-gp120 complex must also bind to HIV co- receptors, which are 7-transmembrane G-protein-coupled receptors (GPCRs) for chemokines, normally expressed on the cell membrane of various cells including T cells for infection to occur (Jin et al., 2010). The principal HIV co-receptors are CCR5 and CXCR4 (Choi and An, 2011). Binding to the HIV co-receptors causes conformational rearrangement within the trimer that drives gp41 fusion peptide (FP) region to be inserted into the host cell membrane resulting in membrane fusion (Nuttall et al., 2007; Anastassopoulou et al., 2009). FP is situated at the N-terminus of gp41 which is adjacent to two heptad repeats (HR1 and HR2). During fusion, the two heptad repeats interacts together and thereby forming a 6-helix bundle that approximates the HIV envelope and host cell membrane together to form a fusion pore which allows transmission of the viral capsid to the target cell (Matos et al., 2010).

TABLE OF CONTENTS :

• ACKNOWLEDGEMENTS
• LIST OF FIGURES
• LIST OF TABLES
• LIST OF ABBREVIATIONS AND ACRONYMS
• ABSTRACT
• CHAPTER INTRODUCTION AND LITERATURE REVIEW
  • 1.1 Background on HIV/AIDS
  • 1.2 Microbicides as the most effective means to prevent HIV-1 transmission
    • 1.2.1 HIV life cycle
    • 1.2.2 Entry and fusion of the HIV to the host cell
    • 1.2.3 Reverse transcription
    • 1.2.4 Integration
    • 1.2.5 Transcription, viral assembly and budding-off
  • 1.3 HIV prevention
    • 1.3.1 Classes of microbicides and their mode of action
    • 1.3.2 Surfactants / membrane disruptors
    • 1.3.3 Vaginal defense enhancers
    • 1.3.4 Entry / fusion inhibitors
    • 1.3.5 Antiretroviral-based microbicides
    • 1.3.6 Anti-HIV proteins
    • 1.3.7 Chemokines in HIV management
    • 1.3.7.1 Chemokines and their classification
    • 1.3.7.2 Role of chemokines in disease management
    • 1.3.7.3 Chemokine inhibitors
    • 1.3.8 RANTES analogues as microbicide candidates
    • 1.3.8.1 PSC-RANTES
    • 1.3.8.2 5P12-RANTES and 6P4-RANTES
    • 1.3.9 Approaches in production of RANTES analogues
    • 1.3.9.1 Chemical synthesis
    • 1.3.9.2 Microbial fermentation
    • 1.3.9.3 Challenges in production of chemokines
  • 1.4 Plants as alternative production systems
    • 1.4.1 An ideal host for plant protein expression
    • 1.4.1.1. Cereals
    • 1.4.1.2 Fruits and vegetables
    • 1.4.1.3 Tobacco production system
    • 1.4.2 Protein expression systems
    • 1.4.2.1 Transient expression of proteins
    • 1.4.2.2 Modified viral vectors
    • 1.4.2.3 Stable expression of proteins
  • 1.5 Aims of study
• CHAPTER MATERIALS AND METHODS
  • 2.1 Sources of reagents and materials
    • 2.1.1 Chemical and non-chemical reagents
    • 2.1.2 Kits
    • 2.1.3 Plasmids
    • 2.1.4 Computer analysis
    • 2.1.5 Primers
  • 2.2. Microbiological techniques
    • 2.2.1 Bacterial strains
    • 2.2.2 Transformation of E. coli
    • 2.2.3 Transformation of A. tumefaciens
  • 2.3 DNA preparation and analysis
    • 2.3.1 Nucleic acid manipulation
    • 2.3.2 Preparation of plasmid DNA
    • 2.3.3 Polymerase Chain Reaction (PCR)
    • 2.3.5 Cloning into expression vectors
  • 2.4 Preparation of plant biomass
    • 2.4.1 General plant husbandry (Nicotiana benthamiana)
    • 2.4.2 Agroinfiltration of N. benthamiana with MagnICON constructs
    • 2.4.3 Agroinfiltration of N. benthamiana with pTRA constructs
    • 2.4.4 Agroinfection of Lycopersicon esculentum with MagnICON constructs
  • 2.5 Protein analysis
    • 2.5.1 Crude protein extraction from N. benthamiana leaves
    • 2.5.2 Crude protein extraction from L. esculentum fruits
    • 2.5.3 Protein determination
    • 2.5.4 Measuring pH of the lyophilised tomato fruit material
    • 2.5.5 Size-exclusion chromatography
    • 2.5.6 Protein purification by Protino Ni-IDA
    • 2.5.7 Protein concentration by trichloroacetic acid (TCA) method
    • 2.5.8 SDS-PAGE for Coomassie staining and western blot
    • 2.5.9 High resolution by Tris-tricine gel
    • 2.5.10 Western blot analysis
    • 2.5.11 Enzyme-linked immunosorbent assay (ELISA)
  • 2.6 Assays
  • 2.6.1 Cytotoxicity assay
  • 2.6.2 HIV-1 pseudovirus neutralisation assay
• CHAPTER EVALUATION OF RANTES ANALOGUES EXPRESSION IN NICOTIANA BENTHAMIANA
  • 3.1 Motivation
  • 3.2 Results and discussion
  • 3.2.1 Sequence alignment, vector design and construction
  • 3.2.2 Effect of subcellular targeting of 5P12 on accumulation and cell viability
    • 3.2.2.1 Phenotypes of infiltrated leaves
    • 3.2.2.2 Effect of infiltration method on protein yield
    • 3.2.2.3 Needle injection
  • 3.2.2.4 Vacuum infiltration
  • 3.2.3 SDS-PAGE and immunoblot analysis of plant-made 5P
  • 3.2.4 In vitro cytotoxicity effects of plant-made 5P
  • 3.2.5 In vitro efficacy effects of plant-made 5P
  • 3.2.6 Specificity of plant-made 5P
  • 3.3 Summary and conclusion
• CHAPTER EVALUATION OF 5P12-RANTES ANALOGUE EXPRESSION IN LYCOPERSICON ESCULENTUM
  • 4.1 Motivation
  • 4.2 Results and discussion
  • 4.2.1 Phenotypes of tomato fruits agroinjected with a GFP construct
  • 4.2.2 Phenotypes of whole tomato fruits agroinjected with 5P12 construct
  • 4.2.3 Total soluble protein (TSP) in tomato fruits
  • 4.2.4 Total 5P12 expression in L. esculentum
  • 4.2.5 Coomassie stained SDS-PAGE analysis
  • 4.2.6 Western blot analysis
  • 4.3 Conclusion
• CHAPTER  DISCUSSION AND CONCLUSION
  • 5.1 Agroinfiltration methods
  • 5.2 MagnICON constructs
  • 5.3 pTRA constructs
  • 5.4 Efficacy testing and specificity of the plant-made 5P
  • 5.5 Conclusion
  • REFERENCES

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