Low affinity Fcγ receptors (FcγR) mediate key immune effector mechanisms through the engagement of the Fc portion of immunoglobulin G (IgG). These receptors are involved in multiple biological processes, including clearance of antigen/antibody immune complexes, enhancement of antigen presentation, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, regulation of antibody production, and activation of inflammatory cells. FcγR phenotypic variability modulates these processes through altering receptor IgG subclass binding affinity (FcγRIIa-H131R and FcγRIIIa-F158V), subcellular localization (FcγRIIb-I232T), post-translational modification (FcγRIIIb-HNA1a/b/c), expression of an otherwise pseudogene (FcγRIIc), and receptor surface density (gene copy number variability and promoter haplotypes). Accumulating data suggest that FcγR-mediated effector functions play a significant role in HIV-1 protective immunity, which is substantiated by the association of FcγR phenotypic variants with HIV-1 disease outcome. This study set out to characterize FcγR functional variability in the South African population, and to investigate the potential role thereof in HIV-1 transmission and disease progression in South African Black individuals. Since the only known determinant of FcγRIIIa surface density – FCGR3A gene copy number – is rare, this study investigated novel genetic determinants of FcγRIIIa expression by flow cytometry and nucleotide sequencing. FcγRIIIa expression on peripheral blood mononuclear cells was characterized for 32 South African Caucasian individuals and 22 South African Black individuals (Chapter 3). Significant differences in the proportion of FcγRIIIa-positive monocytes and FcγRIIIa expression levels on natural killer (NK) cells were observed between the population groups. A novel four-variant FCGR3A intragenic haplotype that associated with increased surface expression of FcγRIIIa on NK cells was detectable in Caucasian individuals, but not Black individuals and may account for the observed population differences. Further exploration of genetic diversity at the low affinity FCGR gene locus was extended to include all currently known functional variants of FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb using a commercial multiplex ligation-dependent probe amplification assay (Chapter 4). Thirty-two South African Caucasian individuals and 131 South African Black

Thesis (M.Sc. (Med.))--University of the Witwatersrand, Faculty of Health Sciences, 2013. / Human immune deficiency virus (HIV) infection contributes to child mortality rates in South Africa. Investigations of newer technologies for improving early infant diagnosis of HIV in the South African setting could reduce child mortality as life saving treatment can be accessed early in life. This study investigated three technologies: a fourth generation rapid HIV test and two viral load (VL) platforms. Determine Combo (DC) is a qualitative fourth generation rapid test that is able to detect HIV antibodies and p24 antigen simultaneously. The performance of DC was evaluated in the field on samples from pregnant and postpartum women; in the laboratory, on stored samples from children and with the addition of heat denaturation. In the maternal DC study 90 (8 .8%) of 1019 women tested HIV positive of whom 59 (17.1% prevalence) were pregnant and 31 % (4.6% prevalence) were postpartum. The sensitivity and specificity of the antibody component of DC on plasma was 100%(Confidence Interval (CI): 95.9- 100%) and 99.8%(CI: 99.2-99.9%) respectively. Three postpartum patients tested false positive for HIV antibodies (n=2) and p24 antigen (n=1). No true positive p24 antigen was detected DC was performed on stored samples from 182 (90%) HIV-exposed and 20 (10%) HIV-unexposed children aged from birth to six years. The DC HIV antibody component returned false negative results in 2 HIV-infected children; one clinically symptomatic and one asymptomatic aged 7 and 23 months respectively. The sensitivity of DC HIV antibody was 100% (CI :94.3-100%) in infants aged 6 months and younger with a specificity of 100% (CI:81.6-100%) for all ages. Of the 61 HIV infected infants tested , the DC p24 antigen was reactive in only one clinically symptomatic infant resulting in a sensitivity for detection of HIV infection of 1.7% (CI 0.3-8.9%). A heat denaturation technique designed to improve p24 antigen detection was applied to HIVinfected samples but failed to enhance p24 antigen detection on DC. HIV viral load (VL) molecular assays are used to confirm an HIV-infected diagnosis and for VL monitoring. In South Africa, plasma is the gold standard sample for VL monitoring in infants even though dried blood spots (OBS) are the preferred specimen type in resource-constrained settings and for early infant diagnosis. The use of OBS specimens for HIV VL monitoring would convenience resource limited settings. The OBS matrix therefore requires validation to determine accuracy (for establishing diagnosis) and precision (for VL monitoring) compared to plasma VL. This study investigated the accuracy and precision limits of OBS VL on the Roche Cobas AmpliPrep-Cobas TaqMan HIV-1 v2.0 assay (CAP/CTM) and the Abbott RealTime HIV-1 assay (m2000) platforms on samples from HIV-infected adults and children. The CAP/CTM was investigated on OBS containing 751J1 blood and the m2000 was investigated using one (50IJI) and two (2x501J1) OBS. Compared to plasma VL, OBS VL from adults and children were higher in the lower range «310g,<1000copies/ml) and lower values in the higher range (>510g, >185,000copies/ml) on the CAP/CTM in the study of OBS VL accuracy. Additionally, OBS VL values were >log1.0 higher in 42/100 (42%) of adult and 16/49 (33%) of measurements from children, which will have clinical significance. On the m2000 platform, the differences between plasma and OBS VL were lower in the range >5 log and higher in the range 2 log copies/ml (100 copies/ml) to 4 log copies/ml (10000 copies/ml). Compared to plasma VL, OBS VL values were >log1.0 higher in 20/82 (24%) adult and 7/43 (16%) of measurements from children. Both platforms demonstrated 100% specificity in testing stored OBS from HIV-uninfected infants who were diagnosed negative on HIV DNA PCR. Acceptable limits for plasma VL precision is a coefficient of variation (CV) <35% and standard deviation (SO) :50.19 log. Where plasma VL :5510g, OBS VL demonstrated poor precision with CV>40% in 8/10 patients and total SO>0.30 log in 4/10 patients on the CAP/CTM. The m2000 total SO was >210g between adult plasma and OBS VLs under the 4 log copies/ml cut-off, irrespective of the number of DBS used. DBS VLs were unreliable when using precision limits used on plasma VLs on both platforms. In conclusion the DC test does not offer any advantage over currently available rapid tests in diagnosing new infection in women and children. The two VL platforms can be used to establish an HIV status in treatment naive patients in view of the 100% specificity. HIV-infected patients on treatment with undetectable plasma VL will always have detectable DBS VL on CAP/CTM, but equally undetectable DBS VL on the m2000. With DBS, the CAP/CTM assay generates higher VL values in the lower VL range than on plasma likely due to amplification of proviral DNA. Both platforms display poor intra- and inter- assay precision, using plasma VL based criteria and the variances would potentially affect clinical decision making. The acceptable limits for plasma VL precision cannot be applied to DBS VL on either platform.

HIV-1 is a global pandemic with about 39 million people infected. In India, 2.9 million people are infected and about 2 lakh new infections have been reported last year. To date, there is no cure for HIV/AIDS. Current treatment, which is associated with serious side effects, only delays the onset of AIDS and death. Thus, HIV/AIDS is responsible for a global health concern imposing significant healthcare costs, especially in low- and middle-income regions such as India and Africa, and a marked loss of quality of life to infected individuals. Understanding factors impacting vaccine design and drug development via mathematical modelling of HIV-1 transmission, evolution and pathogenesis and discerning the subtype and region specific differences are a crucial part of the overall strategy of reducing the burden of HIV/AIDS. The strain dominant in India is HIV-1 subtype C (HIV-1C). Treatment guidelines have largely been based on studies on HIV-1 subtype B (HIV-1B), dominant in the west. In this thesis, we have attempted to understand the dynamics of the spread of HIV-1C, leading to new guidelines and intervention strategies applicable to India. We have for the first time estimated the basic reproductive ratio, R0, of HIV-1 subtype C (HIV-1C), a proxy for its fitness and virulence, using clinical data of infected patients from India. We employed measurements of viral load decay dynamics during treatment and estimated R0, and the critical efficacy, εc, for successful treatment of HIV-1C infection. Clinical data showed that the viral load in patients in India was significantly higher than in the west. Yet, in 6 months following the start of treatment, 87.5% had undetectable viral load, indicating an excellent response to ART, comparable to the west. We analyzed the clinical data using a mathematical model and estimated the median R0 to be 5.3. The corresponding εc was ∼0.8. These estimates of R0 and εc are smaller than current estimates for HIV-1B, suggesting that HIV-1C exhibits lower in vivo fitness compared to HIV-1B, which allows successful treatment despite high baseline viral loads. New treatment guidelines thus emerge that are less stringent than in the west. HIV-1C is far more prevalent globally than HIV-1B. This is surprising in light of our findings above of a lower fitness of HIV-1C than HIV-1B. To understand this observation, we next developed a mechanistic paradigm of HIV-1 transmission. HIV-1 has been hypothesized to optimize its transmission potential (TP) in an infected population by modulating its steady state viral load (VSS), a robust marker of virulence. The mechanism of this optimization is paradoxical and poorly understood given that HIV-1 mutates rapidly in vivo in response to selection pressure by the host immune system. We hypothesize that the HIV-1 TP is not solely a function of VSS as proposed earlier, but a function of two variables - VSS and R0, which function such that R0 is optimized within an infected individual in response to the immune system while VSS is optimized across individuals such that transmission is optimized. On this TP(VSS, R0) landscape, we find that HIV-1C lies closer to the optimum than HIV-1B, suggesting an explanation for the global spread of HIV-1C. This leads to the intriguing implication that the lower virulence of HIV-1C may be because it has evolved more along the TP(VSS, R0) landscape than HIV-1B. Lastly, we examined the role of recombination on HIV-1 adaptation. Following transmission, HIV-1 adapts in the new host by acquiring mutations that allow it to escape from the host immune response at multiple epitopes. It also reverts mutations associated with epitopes targeted in the transmitting host but not in the new host. Moreover, escape mutations are often associated with additional compensatory mutations that partially recover fitness costs. It is unclear whether recombination expedites this process of multi-locus adaptation. To elucidate the role of recombination, we constructed a detailed population dynamics model that integrates viral dynamics, host immune response at multiple epitopes through cytotoxic T lymphocytes, and viral evolution driven by mutation, recombination, and selection. Using this model, we computed the expected waiting time until the emergence of the strain that has gained escape and compensatory mutations against the new host’s immune response, and reverted these mutations at epitopes no longer targeted. We found that depending on the underlying fitness landscape, shaped by both costs and benefits of mutations, adaptation proceeds via distinct dominant pathways with different effects of recombination, in particular distinguishing escape and reversion. Specifically, recombination tends to delay adaptation when a purely uphill fitness landscape is accessible at each epitope, and accelerate it when a fitness valley is associated with each epitope. Our study points to the importance of recombination in shaping the adaptation of HIV-1 following its transmission to new hosts, a process central to T cell-based vaccine strategies.

Objectif : Étudier l'association entre l’utilisation de contraceptifs hormonaux et le risque d'acquisition du VIH-1 chez les femmes au Malawi, en Afrique du Sud, en Zambie et au Zimbabwe. Devis : Analyses secondaires de 2887 femmes âgées de 17-55 ans ayant participé à l’étude HPTN 035, une étude de phase II/IIb sur l’efficacité de deux gels microbicides pour prévenir la transmission du VIH chez les femmes à risque. Méthodes : L'association entre l'utilisation de contraceptifs hormonaux et le risque d'acquisition du VIH-1 a été évaluée en utilisant des modèles de Cox. Des risques relatifs sont estimés où le groupe de référence est celui des femmes qui n’utilisent pas de contraceptifs hormonaux. De plus, un modèle multivarié de Cox est utilisé afin de contrôler pour les facteurs potentiellement confondants. Résultats : Les contraceptifs injectables ont été utilisés par 52,1% des femmes, alors que les contraceptifs oraux ont été utilisés par 20,7% de celles-ci. Pendant l'étude, il y a eu 192 séroconversions. L'incidence observée du VIH était de 2,28; 4,19 et 4,69 pour 100 personne-années pour les contraceptifs oraux, injectables et non hormonaux, respectivement. Lors de l’analyse multivariée, nous n'avons trouvé aucune association significative entre l’usage des contraceptifs hormonaux et l’acquisition du VIH-1. Le risque relatif ajusté (RRa) pour les contraceptifs oraux est de 0,573 (IC de 95% : [0,31-1,06]) et 0,981 (IC de 95% : [0,69 ; 1,39]) pour les contraceptifs injectables. Conclusions : Bien que cette étude ne démontre pas d’association entre l’usage des contraceptifs hormonaux et le VIH-1, nous concluons toutefois que ces méthodes de contraception ne protègent pas contre le VIH-1, et il est ainsi recommandé aux femmes utilisant des hormones contraceptives de toujours utiliser le condom pour prévenir l'infection au VIH-1. / Objective: To investigate the association between the use of hormonal contraceptive and the risk of acquiring HIV-1 infection in women from Malawi, South Africa, Zambia and Zimbabwe. Design: Secondary analyses of 2887 women aged 17-55 years who participated in the HPTN 035 trial, a Phase II/IIb trial on the efficacy of two microbicide gels to prevent HIV transmission in women at risk in Africa. Methods: The association between the use of hormonal contraceptive and the risk of acquiring HIV-1 was evaluated using Cox proportionnal models. Relative risks of exposed women were estimated using as a reference group the women who do not use hormonal contraceptives. In addition, a multivariate Cox model was used to control for potentially confounding factors. Results: Injectable contraceptives were used by 52.1 % of women, while oral contraceptives were used by 20.7% of them. During the study, there were 192 seroconversions. The observed HIV-1 incidence was 2.28, 4.19 and 4.69 per 100 woman-years for oral, injectable and non-hormonal contraceptive users, respectively. In multivariate analysis, we found no significant association between the use of hormonal contraceptives and HIV-1 acquisition. The adjusted relative risk (aRR) for oral contraceptives was 0.573 (95% CI: [0.31 to 1.06]) and 0.981 (95% CI: [0.69, 1.39]) for injectable contraceptives. Conclusions: Although this study did not demonstrate an association between hormonal contraceptive use and the risk of HIV-1 infection, we conclude, however, that these methods of contraception do not protect against HIV-1, and it is thus recommended that women using contraceptive hormones always use condoms to prevent HIV-1.