The evolution of immune escape during HIV infection is well documented, yet some CD8+ T-cell epitopes remain conserved even in the presence of strong cognate responses. This report investigates the frequency of sequence variability within the HLA-A2 restricted immunodominant epitope SLYNTVATL (SL9). Sequencing results, from 15 HIV+ HLA-A2+ individuals, support a very high degree of conservation of this epitope sequence with only focused, conservative changes evident in proviral quasispecies. These observations suggest that changes to the SL9 sequence may have deleterious effects on viral replication and are therefore limited in vivo. To investigate the sequence constraints imposed on the SL9 epitope by HIV replicative fitness, HIV plasmid clones incorporating synonymous and non-synonymous mutations throughout the SL9 epitope region were constructed. Growth of the resultant congenic viruses in mono-infection assays indicated a wide diversity of SL9 variant viruses that grew efficiently. Furthermore, dual-infection viral competition assays demonstrated that SL9 variants known to arise in vivo were equally fit to consensus SL9 virus and several novel epitope variants were also able to compete effectively. Collectively, the data reported in this thesis reveal that the viral pressures acting to conserve the SL9 epitope sequence are less than previously assumed and highlight the lack of understanding of protective CD8+ T-cell action during HIV infection.

90% of Human Immunodeficiency Virus (HIV) infections are acquired across the genital or gastrointestinal mucosa, and infection leads to profound depletion of CD4+ lymphocytes. Antiretroviral therapy can restore blood CD4+ T cells. However, immune dysfunction and defects in mucosal antimicrobial defence persist. Some CD4+ T-subsets, particularly antimicrobial Th17 cells, show enhanced susceptibility to HIV infection and are also preferentially depleted in the course of HIV infection; the latter may allow microbial translocation into the bloodstream. Genital infections have been shown to have direct mucosal immune effects and to increase susceptibility to HIV; however, the effect of systemic infections, such as Malaria (which is holo-endemic in some HIV prevalent regions) is unknown. Understanding the relationship between HIV, highly susceptible immune cells, immune activation and malaria infection on mucosal tissues has been the main focus of my thesis. In HIV-infected individuals, I explored whether HIV antiretroviral therapy restores gut Th17 populations and improves gut antimicrobial defences. Therapy restored gut Th17 populations in some, but not all individuals, but antimicrobial defence remained impaired. I then piloted a novel mucosal-optimized PCR assay to measure cervical immune gene responses, as standard mucosal assays are inadequate. I succeeded in measuring mitogen-induced, but not HIV-specific, cervical immune responses in HIV-infected individuals. Next, using this PCR platform I examined mitogen-induced cervical immune responses in individuals demonstrating reduced susceptibility to HIV, and found that they had reduced production of both Th17-associated and pro-inflammatory cytokines from cervical cells. Finally, in a murine model I found that malaria caused genital and gastrointestinal mucosal immune activation, and increased both the expression of mucosal HIV susceptibility immune markers, and mucosal T cell immune activation. In summary, insufficient gastrointestinal Th17 cells restoration does not underlie persistent mucosal immune activation and microbial translocation in HIV-infected people on therapy. A reduced frequency of highly susceptible Th17 cells in the cervix of HIV-exposed but uninfected individuals was identified as a correlate of reduced HIV susceptibility. Malaria, a common systemic infection in HIV-endemic countries, may enhance susceptibility to HIV through increasing putative immune markers of HIV susceptibility and immune activation in potential mucosal sites of HIV exposure.

The evolution of immune escape during HIV infection is well documented, yet some CD8+ T-cell epitopes remain conserved even in the presence of strong cognate responses. This report investigates the frequency of sequence variability within the HLA-A2 restricted immunodominant epitope SLYNTVATL (SL9). Sequencing results, from 15 HIV+ HLA-A2+ individuals, support a very high degree of conservation of this epitope sequence with only focused, conservative changes evident in proviral quasispecies. These observations suggest that changes to the SL9 sequence may have deleterious effects on viral replication and are therefore limited in vivo. To investigate the sequence constraints imposed on the SL9 epitope by HIV replicative fitness, HIV plasmid clones incorporating synonymous and non-synonymous mutations throughout the SL9 epitope region were constructed. Growth of the resultant congenic viruses in mono-infection assays indicated a wide diversity of SL9 variant viruses that grew efficiently. Furthermore, dual-infection viral competition assays demonstrated that SL9 variants known to arise in vivo were equally fit to consensus SL9 virus and several novel epitope variants were also able to compete effectively. Collectively, the data reported in this thesis reveal that the viral pressures acting to conserve the SL9 epitope sequence are less than previously assumed and highlight the lack of understanding of protective CD8+ T-cell action during HIV infection.

There are currently more than 33 million people worldwide who are infected with HIV-1 despite development of novel treatments and knowledge of prevention strategies. Within the Pumwani area of Nairobi, Kenya there is a group of commercial sex workers who are highly exposed to HIV-1. A small subset of these women have been classified as resistant to HIV-1 infection as they remain HIV un-infected despite as many as 60 unprotected sexual exposures to HIV each year. A better understanding of such a natural model of HIV resistance would be invaluable to inform the development of a protective HIV vaccine or microbicide. Globally, heterosexual transmission of HIV across mucosal surfaces is responsible for the bulk of new infections and thus it is important to examine both the macro and the micro environments of the vaginal mucosa in efforts to determine what enhances and what thwarts HIV-infection. Previous studies have shown elevated levels of RANTES, a natural ligand for the dominant HIV co-receptor CCR5, in cervicovaginal secretions of HIV-resistant women. Additionally, a novel HIV-inhibitor, Trappin-2 was previously shown to be elevated in cervicovaginal secretions of HIV-resistant women. To test the hypothesis that RANTES and Trappin-2 in cervicovaginal fluid are important mediators of HIV resistance we will: 1) measure RANTES in a much larger group of women from the Pumwani cohort, and 2) measure Trappin-2 levels in samples taken at different time points, and 3) correlate Trappin-2 levels in cervicovaginal fluid with biological confounding variables, and 4) investigate whether SDF-1 plays a role in HIV-disease progression in HIV-positive women. / February 2009

HIV/AIDS is a global health problem of immense magnitude, with 33 million people living with HIV and 2 million AIDS-related deaths per year. As the development of drug resistance undermines treatment efficacy, the long-term success of anti-retroviral therapy depends upon the introduction of novel drugs aimed at additional targets essential for the viral life cycle. With a critical role in many viral diseases including the proteolytic maturation of the HIV-1 envelope glycoprotein gp160, the secretory pathway proprotein convertases (PCs) represent a potential anti-viral target. Our laboratory has reported the identification of Spn4A, a potent naturally occurring secretory pathway serine protease inhibitor directed at the prototype PC member, furin. Because of the requirement for the PCs in the production of infectious HIV-1, we hypothesized that strategic manipulation of PC activity by Spn4A and Spn4A-engineered variants would provide a means of effectively limiting HIV-1 infection. This thesis details the investigation of the anti-proteolytic activities and anti-HIV-1 properties of recombinant adenoviruses expressing Spn4A and Spn4A bio-engineered variants, including a secreted recombinant Spn4A (Spn4A S). Our data shows that the expression of Spn4A S in MAGI-CCR5 cells and furin-deficient LoVo cells inhibited the PC-dependent processing of the HIV-1 envelope precursor gp160. Furthermore, inhibition of processing resulted in a nearly complete reduction of productive HIV-1 infection as determined by HIV-1 Tat-driven β-galactosidase activity and multinuclear activation of a galactosidase indicator (MAGI) assays. Complementing the previously described anti-furin activity of Spn4A, our studies indicate that Spn4A S inhibits additional PCs involved in gp160 maturation, and that PC inhibition can serve as an effective means of limiting HIV-1 infection. With the central role of the PCs in the replication and pathogenesis of numerous infectious agents, the identification of Spn4A S as an efficacious HIV inhibitor establishes Spn4A as a prospective broad-based agent for the inhibition of PC-related diseases.

Confidentiality is considered an integral component of medical practise, yet there has been debate within the medical community as to whether there should be exceptions to the obligation to protect patient’s confidences. In the cases involving medical patients with deadly sexually transmittable disease like HIV/AIDS, physicians feel caught between two basic principles – keeping of medical confidentiality and public safety. Bioethicists would favour breaking of confidentiality when the public safety and the life of someone are endangered. However, considering the complexities and discrimination in connection with HIV/AIDS in Nigerian context, many would be tempted to discourage the notification of partners who risk being infected, through the moral obligation of 'duty to warn', but some others would argue that not notifying people of such threat to life would only help in spreading the virus to ignorant partners of an index patient. I argued that there is an overridden utilitarian principle to save others from harm, but some others cite the negative effects the breaking of medical confidentiality would have on the healthcare system as a reason not to favour partner notification. Nevertheless, people would appreciate the value of breaching confidentiality in HIV/AIDS related cases when various forms of discrimination and stigmatisations are criminalised and policies to protect the fundamental rights of people living with HIV/AIDS (PLWHA) are strictly adhered to.

Chemokines are important mediators of leukocyte migration. Chemokines bind to G protein–coupled receptors (GPCR) and cause conformational changes that trigger intracellular signaling pathways involved in inflammation, injury healing, cancer, metastasis, and HIV infections. No direct structural information about any chemokine receptor is available, but the structure of chemokines has been well studied. Structural studies of chemokines coupled with cell-biological investigations may lead to a better understanding of the mechanisms of chemokine-receptor interactions. In this Ph.D. project, I studied the structural and functional relationship between chemokines and chemokine receptors using NMR, X-ray crystallography, and mutagenesis approaches, coupled with several different cell-biology assays. We found that the conserved “chemokine fold” can support different dimerization types in the chemokines family, although changing the dimers from CC- to CXC-type fold is not readily accomplished. I also used an engineered covalently-bound dimer of the MIP-1β mutant, MIP-1β-A10C, to study the relationship between dimerization of chemokines and their interaction with the CCR5 receptor. My results suggest that MIP-1β dimer neither bind nor activate the CCR5 receptor. I also studied the biophysical properties of one N-terminal awkward mutant of P2-RANTES, which was originally selected by others from a phage display using CCR5-expressing cells. Although the NMR and X-ray crystal studies revealed that the wild type RANTES is a tight homodimer, analytical ultracentrifugation reveals that P2-RANTES is a monomer in solution, the 1.7 Å resolution X-ray crystal structure of P2-RANTES was found to be a packed tetramer. The mutated N-terminal residues play a very important role in the tetramerization in the X-ray crystal structure. Finally I used the HIV-1 env mediated cell-cell fusion assay to study the combination of chemokines or chemokine variants with anti-HIV peptides C37 or/and T-20. A surprisingly synergistic effect was found between P2-RANTES and C37 or T-20. This combination stratagem may lead to further useful drug combinations or drug delivery for more potent anti-HIV treatments.

Thèse de doctorat : psychologie : Metz : 2005. / Thèse soutenue sur ensemble de travaux. Bibliogr. f. 262-275. Annexes.

Thèse de doctorat : Mathématiques appliquées : Metz : 2009. Thèse de doctorat : Mathématiques appliquées : Yaoundé 1 : 2009. / Thèse soutenue en co-tutelle. Titre provenant de l'écran-titre. Notes bibliogr.

Zugl.: Düsseldorf, Univ., Diss., 2009