Genotypic and phenotypic characterization of HIV-1 from cerebrospinal fluid and blood compartments in patients with cryptococcal meningitis.

Doctor of Philosophy in the School in Virology. University of KwaZulu-Natal. Durban, 2017.The combination of HIV and cryptococcal meningitis (CM) is a major cause of morbidity and mortality in sub-Saharan Africa. The phylogenetic relatedness of HIV-1 subtype C (HIV-1C) variants in peripheral blood and the central nervous system (CNS) compartments of individuals with CM is unknown. Additionally, the major and alternative coreceptor usage of HIV-1C in those compartments are not clear and these have implications for the pathogenesis of the virus, and the use of coreceptor blocking therapies. For genotypic studies, 16 antiretroviral therapy naïve individuals with CM were randomly selected. We conducted single-genome, or bulk PCR amplification and sequencing of full-length HIV-1 env genes from plasma and/or cerebrospinal fluid (CSF) of the participants. Additionally, we estimated the prevalence of CXCR4-using variants in our cohort using coreceptor usage prediction algorithms (CPAs). Next, we evaluated the usage of CCR3, CCR5 and CXCR4 expressed on NP2/U87-CD4 cells by HIV-1C Envs derived from the plasma and/or CSF of 14 of the 16 participants. CCR3 alone, or in combination with the other receptors. Overall, our results have improved the understanding of HIV-1C pathogenesis in the peripheral blood and CNS compartments of individuals with end-stage infection and CM, and provides clinically relevant information for therapies including coreceptor antagonists in this setting

Novel CD4-immunoglobulin fusion proteins as HIV-1 immunotherapeutics

The initial step of HIV entry into host cells involves the specific binding of gp120 envelope glycoprotein to the cell-surface CD4 protein, expressed by antigen-presenting cells. Extensive systemic infection of host cells by HIV leads to immune dysfunction and ultimately the demise of infected individuals due to AIDS. The gp120-CD4 interaction (KD of approximately 2 X 10-9 M) is of primary importance to the infection process of HIV and poses a validated therapeutic target. Mutational analyses of CD4 show that residues in domains 1 and 2 of CD4 contribute to gp120 binding. Soluble CD4 protein, composed of domains 1 and 2 or 1 to 4 only, has been shown to compete with cell-surface CD4 for gp120 binding and inhibit host cell infection. Soluble CD4 achieves viral inhibition by uncoating HIV of its envelope proteins, reducing the amount of cell-surface CD4 recognition by gp120 and preventing the cell-to-cell spread of HIV by syncytium formation. Due to the short serum-lifetime of soluble CD4 in the blood, modified forms of it have been generated which contain life-time extending immunoglobulin portions attached to them. The attachment of soluble CD4 in place of immunoglobulin variable regions may increase the valence of CD4 available for gp120 binding and may contribute extra immune effector functions depending on the Fc portion type. PRO 542 (CD4-IgG2), composed of four two-domain CD4 molecules replacing the variable regions of antibody IgG2, was found to have greater valence and effector functions than soluble CD4. Like soluble CD4, PRO 542 was deemed safe at all concentrations tested during clinical trials; however tests were halted as it could not significantly reduce viral load in patients. In this study, we designed constructs which produced a PRO 542-like protein (kCD4-IgG2wt) in a novel way. Constructs encoding a mutant S60C PRO 542-like molecule (kCD4-IgG2S60C) were also generated to produce a heterotetramer intended to bind gp120 with even higher affinity by virtue of intermolecular redox-disulphide exchange. The uniquely produced heterotetrameric kCD4-IgG2wt and kCD4-IgG2S60C proteins were expressed in Human Embryonic Kidney 293T cells and collected in culture supernatant. Purification of kCD4-IgG2wt and kCD4-IgG2S60C was of limited success; however both proteins could bind Protein-A agarose beads like NIH PRO 542 implying that their Fc portions were fully folded. Immunoprecipitation tests showed the ability of kCD4-IgG2wt to bind gp120 from HIV-1Bal like PRO 542 confirming the high structural and functional integrity of kCD4-IgG2wt. Finally, surface plasmon resonance studies (SPR) were conducted to evaluate the ability of supernatant-contained kCD4-IgG2wt and kCD4-IgG2S60C to bind six different gp120 molecules from genetically diverse viruses. SPR studies included purified PRO 542 obtained from the National Institutes of Health (NIH), two-domain CD4 (2dCD4wt) and an S60C mutant two-domain CD4 (2dCD4S60C) as positive controls. The culture supernatant from mock transfections was used as a negative control. According to SPR data, supernatant-containing kCD4-IgG2wt binds specifically to gp120 of HIV-1CN54, HIV-1SF162, HIV-1IN25925, HIV-1ZA706010164, HIV-196ZM651 and HIV-1Bal like NIH PRO 542, 2dsCD4wt and 2dsCD4S60C. Monoclonal antibody 17b, which binds the co-receptor binding site of gp120 was observed to bind gp120 from HIV-1SF162, HIV-1IN25925, HIV-1ZA706010164 and HIV-1Bal, after the gp120s co-receptor binding site was exposed by supernatant-contained kCD4-IgG2wt, NIH PRO 542, 2dsCD4wt and 2dsCD4S60C. Low concentrations of expressed kCD4-IgGS60C did not permit the observation of kCD4-IgG2S60C binding to any of the tested gp120s. Results obtained from this study showed that the novel strategies employed to produce heterotetrameric kCD4-IgG2wt, which was identical in structure and function to NIH PRO 542, were successful. Novel and functional CD4-based molecules with activity against HIV gp120 may be generated using the strategies described in this study

AIDS-related mycoses: the way forward.

The contribution of fungal infections to the morbidity and mortality of HIV-infected individuals is largely unrecognized. A recent meeting highlighted several priorities that need to be urgently addressed, including improved epidemiological surveillance, increased availability of existing diagnostics and drugs, more training in the field of medical mycology, and better funding for research and provision of treatment, particularly in developing countries