The human immunodeficiency virus type-1 (HIV-1) is the leading cause of acquired immunodeficiency syndrome (AIDS) in the world. As there is no cure currently available to treat HIV-1 infections or AIDS, the major focus of drug development efforts has been to target viral replication in an effort to slow down the progression of the infection to AIDS. The aspartyl protease of HIV-1 is an important component in the viral replication cycle and thus, has been an important anti-HIV-1 drug target. Currently there are nine protease inhibitors (PIs) that are being used successfully as a part of highly active antiretroviral therapy (HAART). However, as is with all HIV-1 drug targets, the emergence of drug resistance substitutions within protease is a major obstacle in the use of PIs. Understanding how amino acid substitutions within protease confer drug resistance is key to develop new PIs that are not influenced by resistance mutations. Thus, the primary focus of my dissertation research was to understand the molecular basis for drug resistance caused by some of these resistance substitutions. Until recently, the genetic diversity of the HIV-1 genome was not considered to be important in formulating treatment strategies. However, as the prevalence of HIV-1 continues, the variability of the HIV-1 genome has now been identified as an important factor in how the virus spreads as well as how fast the infection progresses to AIDS. Clinical studies have also revealed that the pathway to protease inhibitor resistance can vary between HIV-1 clades. Therefore, in studying the molecular basis of drug resistance in HIV-1 protease, I have also attempted to understand how genetic variability in HIV-1 protease contributes to PI resistance. In Chapters II, III and Appendix 1, I have examined how clade specific amino acid variations within HIV-1 CRF01_AE and clade C protease affect enzyme structure and activity. Furthermore, I have examined how these sequence variations, which are predominantly outside the active site, contribute to inhibitor resistance in comparison to clade B protease. With the results presented in Chapter II, I was able to show that sequence variations within CRF01_AE protease resulted in structural changes within the protease that might influence enzyme activity. In Chapter III, I focused on how sequence variations in CRF01_AE influence protease activity and inhibitor binding in comparison to clade B protease. Enzyme kinetics data showed that the CRF01-AE had reduced catalytic turnover rates when compared to clade B protease. Binding data also indicated that CRF01_AE protease had an inherent weaker affinity for the PIs nelfinavir (NFV) and darunavir (DRV). In work described in Chapter III, I have also examined the different pathways to NFV resistance seen in CRF01_AE and clade B protease. Using x-ray crystallographic studies I have shown the molecular mechanism by which the two different pathways confer NFV resistance. Furthermore, I provide a rational for why different resistance pathways might emerge in the two clades. In Appendix I, I present results from a parallel study carried out on clade C protease. In Chapter IV, I have examined the role of residue 50 in HIV-1 protease in modulating inhibitor binding. Patients failing amprevavir (APV) and DRV therapy often develop the I50V substitution while the I50L substitution is often observed in patients failing atazanavir (ATV) therapy. This indicates that by making subtle changes at residue 50 the protease is able to confer differential PI resistance. With binding data presented in this chapter I have shown that substitutions at residue 50 change the susceptibility profiles of APV, DRV and ATV. Furthermore, from analyses of protease-inhibitor complexes, I have described structural insights into how substitutions at residue 50 can modulate inhibitor binding. This thesis presents results that reveal mechanistic insights into how a number of resistance substitutions within protease confer drug resistance. The results on non-B clade proteases demonstrate that clade specific sequence variations play a role in modulating enzyme activity and influence the pathway taken to confer PI resistance. Furthermore, the results provide structural insights into how amino acid substitutions outside the active site effectively alter inhibitor binding.

Influenza is a severe disease in humans and animals, causing upwards of 40,000 deaths every year in America alone. Influenza A virus (IAV) also causes periodic pandemics every 10 to 50 years, killing millions of people. Despite this, very few effective therapies are available. All strains of IAV are prone to developing resistance to antibodies due to the high mutation rate in the viral genome. Because of this mutation rate, a yearly vaccine must be generated before every flu season, and efficacy varies year to year. IAV has also mutated to escape several of the clinically-approved small molecule inhibitors. A therapeutic agent that targets a highly conserved region of the virus could bypass resistance and also be effective against multiple strains of IAV. IAV attachment is mediated by many individually weak hemagglutinin–sialic acid interactions that all together make a strong attachment to a host cell. Polymerized sialic acid analogs can recreate these interactions and block infection. However, they are not ideal therapeutics due to solubility issues and in vivo toxicity. We used liposomes as a novel means for delivery of the sialic acid-containing glycan, sialylneolacto-N-tetraose c (LSTc). LSTcbearing decoy liposomes form multivalent, polymer-like interactions with IAV. Decoy liposomes competitively bind IAV in hemagglutination inhibition assays and inhibit infection of target cells in a dose-dependent manner. LSTc decoy liposomes co-localize with IAV, while control liposomes do not. Inhibition is specific, as inhibition of Sendai virus and respiratory syncytial virus is not observed. In contrast, monovalent LSTc does not bind IAV or inhibit infectivity. LSTc decoy liposomes prevent the spread of IAV during multiple rounds of replication in vitro and extend survival of mice challenged with a lethal dose of virus. Considering the conservation of the hemagglutinin binding pocket and the ability of decoy liposomes to form high-avidity interactions with IAV hemagglutinin, our decoy liposomes have potential as a new therapeutic agent against emerging strains.

While memory CD4 T cells are critical for effective immunity to pathogens, the mechanisms underlying their generation are poorly defined. Although extensive work has been done to examine the role of antigen (Ag) in shaping memory formation, most studies focus on the requirements during the first few days of the response known as the priming phase. Little is known about whether or not Ag re-encounter by effector T cells (late Ag) alters CD4 memory T cell formation. Since influenza infection produces a large, heterogeneous, protective CD4 memory T cell population, I used this model to examine the role of late Ag in promoting CD4 memory T cell formation. In the experiments presented in this thesis, I demonstrate that late Ag is required to rescue responding CD4 T cells from default apoptosis and to program the transition to long-lived memory. Responding cells that failed to re-encounter Ag had decreased memory marker expression and failed to produce multiple cytokines upon re-stimulation. Ag recognition is required at a defined stage, as short-term Ag presentation provided 6 days after infection is able to restore canonical memory formation even in the absence of viral infection. Finally, I find that memory CD4 T cell formation following cold-adapted influenza vaccination is boosted when Ag is administered at this stage. These findings imply that persistence of viral Ag presentation into the effector phase is the key factor that determines the efficiency of memory generation. They also suggest that administering Ag during the effector stage may improve vaccine efficacy.

Thesis (MPH.) -- University of Limpopo, 2021 / Introduction: HIV/AIDS remains a disease of public health importance and mother-to-child transmission (MTCT) is one of the major problems. Sub-Saharan Africa is the most severely affected region, accounting for more than 90 percent of paediatric HIV infections. Most of these infections occurred during pregnancy, delivery or breastfeeding making the prevention of mother-to-child transmission (PMTCT) a public health priority. Over the last few years, efforts have been made in Sub-Saharan countries to improve PMTCT and the success of prevention of mother‐to‐child transmission of HIV (PMTCT) is dependent upon high retention of mother‐infant pairs within the PMTCT cascade. Assessing the risk factors for MTCT will help to decrease child morbidity and mortality and strengthen PMTCT programs as there is dearth of evidence regarding factors determining MTCT HIV infection to infants born to HIV positive mothers. The purpose of this study was to investigate the determinants for the human Immunodeficiency Virus positivity rates in the Greater Letaba Municipality. The study objectives were to describe the demographic characteristics of mothers and babies who tested polymerase chain reaction test (PCR)-positive in the Greater Letaba Municipality during the two-year period from 2015 to 2016, in order to determine maternal and neonatal factors associated with high positive PCR; and to determine health system-related factors associated with a high positive PCR result. Methodology The current study followed a quantitative approach in which convenient and purposive sampling was used, focusing on records of infants born from HIV-positive women in all clinics at Greater Letaba Municipality were reviewed. All records of infants who were tested for HIV and the PCR results were positive from birth up to 12 months of age were retrospectively reviewed and for the health care workers, all nurses working as managers of a clinic were interviewed. The Statistical Package for the Social Sciences (SPSS) version 23 computer software and Stata 15 was used. for comparison of categorical variables was done using a Chi-Squared test, whereas continuous variables were compared using a t-test and P-value of <0.05 was considered significant. To determine maternal and neonatal factors associated with high positive PCR, Factor analysis was used with rotated factor loadings done using the Varimax method. Results: A total of 107 records were retrieved and audited. Fisher’s exact test was used to determine the relationship between selected variables, where p<0.05 was set as level of significance. The findings reveal that the number of infants exposed to HIV during pregnancy has steadily increased. The current study further indicates that health system factors such as unskilled or untrained NIM-ART nurses in the facilities is a contributory factor to infant’s positivity rate in Greater Letaba hospital. Equal proportions of both male and female babies were found to be PCR positive at 6 weeks. The study further revealed that the highest proportion of the mothers who gave birth to PCR positive babies for the reporting period were married mothers, in the age group 25-29 years (46.1%). The second largest proportion of mothers who gave birth to PCR positive babies were single mothers in the age group 25-29 years (38.4%). The results show that high PCR positivity can be attributed to about 5 main Factors namely: maternal antenatal history (22% contribution to total variance), maternal HIV care history (18% contribution to total variance), measures of adherence to treatment (17% contribution to total variance), maternal exposure to HIV (14% contribution to total variance) and lastly the ART regimen (12% contribution to total variance). Conclusion: The study findings revealed that there is still vertical transmission of HIV to infants and the prevalence of HIV among infants born from seropositive mothers despite the availability of the latest Prevention of Mother to Child Transmission (PMTCT) Guidelines in all health care facilities. Even though transmission is reduced to the meaningful number (< 5%), there are still appropriate measures that should be taken to reduce the transmission of HIV from mothers to infants. The delayed diagnosis, adherence to ART by mothers, infant ARV prophylaxis at birth and feeding practices contributed the vertical transmission of HIV to infants. Strengthening of the PMTCT of HIV programme, increasing antenatal HIV screening and linking it to care and treatment of HIV positive mothers to obtain zero infant HIV prevalence in the region. Infant prophylaxis and maternal PMTCT interventions should be provided to all exposed infants and mothers based on the guidelines by the health institutions. Nurse-initiated management of antiretroviral treatment (NIM-ART) training of professional nurses is being offered by the Department of Health in South Africa, but it does not yield positive results as far as the PMTCT is concerned. This may be due to shortage of staff, especially trained professional nurses (PN), as well as the workload. Key concepts: Infant and Human immune deficiency virus

Doctorat en Sciences / info:eu-repo/semantics/nonPublished

This dissertation consists of three essays in the field of health economics. The first essay provides the first causal evidence that bargaining power in a relationship shapes pregnancy outcomes and health disparities in the US. A key driver of bargaining power is the availability of potential non incarcerated male partners in the local dating market, which I define at the race by cohort by county level. Because these sex ratios are endogenous, I use a novel instrument that leverages the randomness in sex at birth and the persistence of local demographics to isolate exogenous variation in the relative availability of men. Greater female bargaining power causes better outcomes: fewer out-of-wedlock births, less chlamydia and hypertension among mothers, and fewer infants with APGAR score below the normal level. The marriage market makes a significant contribution to racial disparities in pregnancy health. Specifically, Black women face relatively poor prospects when looking for a partner compared to White women: while there are 102 White men per 100 White women, only 89 Black men are available per 100 Black women. According to my estimates, Black women’s disadvantage accounts for 5-10% of the large racial gap in maternal and neonatal health. The racial difference in male availability is mostly policy-driven, as incarceration accounts for 45% of the gap. A counterfactual policy equalizing county-level incarceration rates for non-violent offenses between Black and White people would prevent 200-700 adverse pregnancy outcomes per year among Black mothers through the bargaining power channel alone. The second essay investigates how reopening hotels and ski facilities in Poland impacted tourism spending, mobility, and COVID-19 outcomes. We used administrative data from a government program that subsidizes travel to show that the policy increased the consumption of tourism services in ski resorts. By leveraging geolocation data from Facebook, we showed that ski resorts experienced a significant influx of tourists, increasing the number of local users by up to 50%. Furthermore, we confirmed an increase in the probability of meetings between pairs of users from distanced locations and users from tourist and non-tourist areas. As the policy impacted travel and gatherings, we then analyzed its effect on the diffusion of COVID-19. We found that counties with ski facilities experienced more infections after the reopening. Moreover, counties strongly connected to the ski resorts during the reopening had more subsequent cases than weakly connected counties. The third essay studies the diffusion of influenza-like illnesses (ILI) through social and economic networks. Using almost two decades of weekly, county-level infection and mortality data from Poland, it studies within and across-counties ILI transmission. Firstly, it evaluates the causal effect of school closures on viral transmission. The results show that closing schools for two weeks decreases the number of within county cases by 30-40%. The decline in infections extends to elderly and pre-school children. In addition, flu-related hospitalizations drop by 7.5%, and mortality related to respiratory diseases among the elderly drops by 3%. Secondly, the paper demonstrates the significant contribution of economic links to diffusion across counties. The disease follows the paths of workers commuting between home and workplace. Together with the structure of the labor mobility networks, these results highlight the central role of regional capitals in sustaining and spreading the virus.

Channel catfish virus disease (CCVD) is the principal viral disease in the United States catfish industry. The CCVD is caused by channel catfish virus (CCV), with mortality reaching up to 100% in fingerlings. CCV is assigned taxonomically to the family Alloherpesviridae, genus Ictalurivirus, species Ictalurid herpesvirus 1 (IcHV-1). To date, virulence, immunogenicity, and genome plasticity of the CCV field isolates have not been investigated. Three genotypes of CCV (IcHV-1A, IcHV-1B, and BCAHV) were identified using restriction fragment length polymorphism (RFLP) analysis. Virulence assessment of three representative isolates of RFLP groups suggests that IcHV-1B (pooled survival [mean ± SE]: 58.3% ± 2.6) showed significantly lower survival than IcHV-1A (68.6% ± 2.4). Re-exposure of the survivors with a representative of IcHV-1A and IcHV-1B isolates indicates a robust cross-protective effect (relative percent survival [RPS]: 80-100%). Antigenic determinants against anti-CCV monoclonal antibody Mab-95 were conserved among IcHV-1A, and IcHV-1B; however, BCAHV possesses antigenically distinct epitopes (Neutralization index [NI] = 0). Although BCAHV and CCV have nearly colinear genomes (except for the absence of ORF16A in CCV), they represent distinct species, given that nucleotide identity is 93.9%. Moreover, infectivity trials indicated that channel and hybrid catfish fingerlings might be refractive to LD50 (1.3×105 TCID50/L) dosage of BCAHV. However, previous exposure to BCAHV has protected the channel and hybrid catfish against the subsequent infection with the ATCC strain of CCV (RPS:100%). Next, two discriminatory duplex probe-based qPCR assays were designed and validated to diagnose latent IcHV-1A and IcHV-1B. Spatio-temporal survey of six Mississippi catfish hatcheries indicated that the prevalence of latent CCV genotypes varied between 25-100%. Lastly, twenty one reference quality genomes of CCV field isolates were assembled, and phylogenomic analyses supported the monophyly of the CCV field isolates with BCAHV as their closest relative. The phylogenomic analyses confirmed the two distinct genotypes (IcHV-1A and IcHV-1B) identified in RFLP analysis and further allowed the segregation of the IcHV-1A genotype into two subgroups, IcHV-1A1 and IcHV-1A2. Results from the current studies lay the foundation for future research and will help formulate efficient management strategies to reduce the economic impact of CCV in the catfish industry.

Despite recent advances in antiretroviral therapy, nearly 37 million people continue to live with human immunodeficiency virus (HIV). Basic and applied research on the assembly of HIV could be enhanced by using a genetically tractable organism, such as yeast, rather than mammalian cells. While previous studies showed that expression of the HIV Gag polyprotein in Saccharomyces cerevisiae spheroplasts resulted in the production of virus-like particles (VLPs), many questions regarding the utility of yeast in HIV assembly remain uninvestigated. Here, we report use of S. cerevisiae for both the production of VLPs with selectively packaged RNA and to evaluate the human Y-box-binding protein 1 (YB-1) in selective RNA packaging into VLPs. Our data reveal: (1) When co-expressed alongside HIV-1 Gag, an RNA mammalian expression cassette is selectively encapsidated and released in VLPs produced from spheroplasts; (2) Inclusion of the 5’UTR-5’Gag RNA upstream of the mammalian expression cassette greatly increased the selectivity to which non-viral RNA was packaged into VLPs; and (3) heterologous expression of the human YB-1 protein in S. cerevisiae did not facilitate the selective packaging of viral RNA into VLPs, likely due to inability to bind upstream elements in the HIV-1 viral RNA. Overall, this research provides a key first step in the use of yeast for the production of viral vectors used in gene therapy, and lays a foundation for further experiments investigating the role of YB-1 and other host proteins in selective RNA packaging.

Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections induce a myriad of disturbances to CD4 T cell functions, including mitochondrial compromise, excessive inflammation, increased telomeric DNA damage and attrition, cellular exhaustion and senescence, and accelerated aging. In this dissertation, the mechanisms underlying metabolic failure, accelerated aging, and cellular dysfunctions were evaluated in CD4 T cells from healthy subjects (HS) treated with a telomere-targeting drug (KML001) or HCV-infected individuals or people living with HIV (PLHIV) compared to HS. We observed that KML001-induced telomere injury resulted in mitochondrial swelling and decreased mitochondrial membrane potential, cellular respiration, mitochondrial DNA (mtDNA) copy number, and ATP production mediated by p53-mediated repression of the master mitochondrial regulators peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and nuclear respiratory factor 1 (NRF-1). We then investigated the mechanisms responsible for T cell dysfunction and metabolic failure during chronic viral infections (HCV, HIV). We observed that chronic HCV infection leads to elevated production of cellular and mitochondrial reactive oxygen species (ROS), impaired mtDNA, and altered levels of proteins responsible for mediating oxidative stress, apoptosis, and mtDNA maintenance, as well as mitochondrial regulators PGC-1α and mitochondrial transcription factor A (mtTFA), contributing to impaired cellular respiration and mtDNA content. Similarly, we demonstrated that latent HIV infection induced disruptions to CD4 T cell homeostasis and increased cellular exhaustion, senescence, and apoptosis and reduced proliferation. We also observed significant repression of mitochondrial respiration, mtDNA content, and mtTFA levels in CD4 T cells from PLHIV, which was reversed via ectopic expression of mtTFA. Finally, we observed elevated cellular and mitochondria ROS production in CD4 T cells from PLHIV, along with significant deregulation of levels of antioxidant defense (superoxide dismutase 1, SOD1) and oxidative stress-induced DNA damage repair (apurinic/apyrimidinic endonuclease 1, APE1) proteins, which were shown to be essential for cellular respiration independently of mtDNA content. Taken together, this research highlights novel multi-leveled mechanisms by which chronic viral infection induces accelerated T cell aging and mitochondrial compromise via deregulating master mitochondrial regulators and provides a diverse collection of novel therapeutic targets that may be applied to various infectious diseases.

Human Immunodeficiency Virus (HIV) infection initiates major metabolic and cell- survival complications. Anti-retroviral therapy (ART) is the current approach to suppress active HIV replication to a level of undetected viral load, but it is not a curative approach. Newer and sophisticated gene editing technologies could indeed be a potent antiviral therapy to achieve a clinical sterilization/cure of HIV infection. Chronic HIV patients, even under a successful ART regimen, exhibit a low-grade inflammation, immune senescence, premature aging, telomeric DNA attrition, T cell apoptosis, and cellular homeostasis. In this dissertation, we investigated CD4 T cell homeostasis, degree of T cell apoptosis, an associated telomeric DNA damage, DNA damage repair signaling, and the apoptotic pathways in CD4 T cells during HIV infection with or without ART treatment. Our data support a DNA damage accumulation, and impaired DNA damage repair in chromosome ends via recruitment of 53BP1 protein to the damaged foci. We found that a key player of DNA damage and repair enzyme, ATM, and its associated checkpoint proteins (CHK1, CKH2) are affected by HIV infection. HIV infection also altered another multifunctional master regulator protein AKT that is crucial in maintaining cellular homeostasis. Curing HIV is the ultimate redemption against HIV-associated complications. To explore the possibility of a functional cure, we investigated the use of a transient and a non-viral CRISPR/Cas9-based gene editing technology targeting the latently incorporated HIV provirus. After performing a nucleofection/electroporation using an in vitro formulated ribonucleoprotein (RNP) constituting a synthetic guide RNA (gRNA) and Cas9 nuclease protein, we demonstrated a significant (maximum 97%) reduction of HIV-mRNA and p24-capsid protein expression, upon stimulation (using PMA) and latency reactivation of latently HIV-infected CD4 T cells and latent-monocytes. Notably, the RNP treatment did not induce any cytotoxic effects, without affecting the abilility of cell proliferation. A sequence specific cleavage of HIV-provirus in two crucial gene locations (targeting vpr/tat genes) showed the most significant suppression of HIV reactivation or latency reversal. We have used DNA sequencing, and T7EI assay to confirm the target-site-specific cleavage of the HIV-proviral genome. Our data confirm the activation of non- homologous end joining (NHEJ) pathway to repair the double-stranded DNA break created by the CRISPR/Cas9 treatment. Taken together, this study provides a new gene therapeutic approach using synthetic gRNA/Cas9 targeting HIV genome, which warrant further in vivo animal and human studies.