HIV-1 integrase inhibitor mutations: analysis of structural and biochemical effects.

Doctoral Degree. University of KwaZulu-Natal, Durban.Introduction. Combination antiretroviral therapy (cART), composed of drugs from different drug classes, is an effective HIV-1 treatment strategy. As part of cART, integrase strand transfer inhibitors (INSTIs) have become essential drugs and are now recommended for use in first-line, second-line, and subsequent HIV-1 treatment regimens. Though highly potent, the use of first-generation INSTIs Raltegravir and Elvitegravir still resulted in the development of integrase drug resistance mutations. Second-generation INSTIs Dolutegravir, Bictegravir, and Cabotegravir were developed to combat the emerging resistant virus strains to first-generation INSTIs and are considered some of the best antiretroviral drugs in HIV-1 treatment. Despite the fundamental changes and improved performance in second-generation INSTIs, they are not immune to drug resistance. This highlights the need to understand the molecular mechanisms of resistance to INSTIs. This thesis, through a combination of structural and biochemical methods, seeks to understand resistance development in South African HIV-1 subtype C (HIV-1C) viruses and the effect of resistance mutations on enzyme-substrate binding, DNA binding, and 3’ processing. Methods. A total of 48 HIV-1C sequences were analyzed in this study, of which 7 had a virologic failure (i.e. plasma viral loads >1000 copies/mL) and 41 were INSTI naïve isolates (32 treatment-naïve South African HIV-1C integrase sequences downloaded from GenBank and 9 INSTI-naïve isolates amplified in our laboratory). Virologic failures were receiving at least 6 months of INSTI-based cART and presented at the King Edward VIII hospital, a 3rd line regimen referral hospital in Durban, South Africa. Viral RNA was extracted, and the integrase region was amplified and sequenced using Sanger sequencing. To investigate the effect of mutations on the integrase structure, wild-type and representative mutant isolates were modeled on the SWISS model online server and visualized in Chimera v1.13.1. Raltegravir, Elvitegravir, and Dolutegravir were docked into each of the structures using the AutoDock-Vina Plugin available on Chimera, and molecular dynamics simulations were conducted using the AMBER 18 package. Integrase biochemical assays were carried out using a wild-type protein and the 3 mutant recombinant proteins that were expressed and purified. Integrase - LTR binding and 3’ processing assays were then performed. Results. Only one of the 7 (14,28%) INSTI-treated isolates had major mutations (i.e., G140A and Q148R). In addition, this isolate harboured the E157Q minor mutation and previously identified polymorphisms. Interestingly, S119T & V151I, located near the integrase active site, were only found in INSTI failures. Structural analysis results showed a reduced binding affinity for the mutants, which was supported by their weaker hydrogen-bond interaction compared to the wild-type. Our findings showed that the G140A+Q148R double mutant had the strongest effect on the HIV-1C protein structure and binding of EVG and RAL with binding free energies of -12.49 and -11.45 kcal/mol for EVG and RAL, respectively, which are approximately three times lower than the wild-type binding energy. Biochemical assays performed with purified integrase showed a decrease in integrase-LTR binding for all mutants. The 3’ processing activity was slightly decreased in the mutants compared to the wild-type protein; however, no appreciable differences were observed across the mutant isolates. Conclusions Changes near the highly conserved active site residues in HIV-1C integrase core domain and mutations in the 140’s loop have a negative effect on in vitro integrase activity, suggesting that these changes impact viral integration. While they are still few reports of INSTI resistance-associated mutations (RAMs) in South Africa , identification of the G140A+Q148R double mutant for the first time in South African HIV-1 clinical samples, and the identification of S119T and V151I in INSTI-treated patients warrants further investigation. This data broadens the understanding of HIV-1C resistance against INSTIs and adds to the available knowledge of drug resistance mutations that guide therapeutic decisions

Rapid epidemic expansion of the SARS-CoV-2 Omicron variant in southern Africa

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) epidemic in southern Africa has been characterised by three distinct waves. The first was associated with a mix of SARS-CoV-2 lineages, whilst the second and third waves were driven by the Beta and Delta variants, respectively1-3. In November 2021, genomic surveillance teams in South Africa and Botswana detected a new SARS-CoV-2 variant associated with a rapid resurgence of infections in Gauteng Province, South Africa. Within three days of the first genome being uploaded, it was designated a variant of concern (Omicron) by the World Health Organization and, within three weeks, had been identified in 87 countries. The Omicron variant is exceptional for carrying over 30 mutations in the spike glycoprotein, predicted to influence antibody neutralization and spike function4. Here, we describe the genomic profile and early transmission dynamics of Omicron, highlighting the rapid spread in regions with high levels of population immunity

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance.

Investment in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing in Africa over the past year has led to a major increase in the number of sequences that have been generated and used to track the pandemic on the continent, a number that now exceeds 100,000 genomes. Our results show an increase in the number of African countries that are able to sequence domestically and highlight that local sequencing enables faster turnaround times and more-regular routine surveillance. Despite limitations of low testing proportions, findings from this genomic surveillance study underscore the heterogeneous nature of the pandemic and illuminate the distinct dispersal dynamics of variants of concern-particularly Alpha, Beta, Delta, and Omicron-on the continent. Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve while the continent faces many emerging and reemerging infectious disease threats. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Role of angiopoietin-2, endoglin, and placental growth factor in HIV-associated preeclampsia

Objective: The factors angiopoeitin-2 (Ang-2), endoglin (Eng), and placental growth factor (PlGF) have been implicated in the pathophysiology of preeclampsia (PE). This study assessed their serum levels in HIV-negative and HIV-positive pregnant normotensive and pre-eclamptic women. Methods: Participants were recruited at the antenatal clinic, serum samples were evaluated using the Bioplex Human Cancer Biomarker (panel 2). Results: Ang-2 and Eng levels were higher, whilst PlGF levels were lower in the PE compared with the normotensive group. Pregnancy type had no significant effect on Ang-2 and showed a significant interaction with Eng (p < 0.0001) and PlGF (p = 0.0033). HIV status had no significant effect on angiopoeitin-2 (p = 0.4), Eng (p = 0.4), and PlGF (p = 0.7) but the levels were slightly higher in the HIV-negative cohort. Conclusions: This study demonstrates an elevation of Ang-2 and Eng in pre-eclamptic compared with normotensive pregnant women implicating their role in its pathogenesis

Emergence of SARS-CoV-2 Omicron lineages BA.4 and BA.5 in South Africa

DATA AVAILABILITY : All of the SARS-CoV-2 genomes generated and presented in this article are publicly accessible through the GISAID platform (https://www.gisaid.org/). The GISAID accession identifiers of the sequences analyzed in this study are provided as part of Supplementary Table 1. Other raw data for this study are provided as a supplementary dataset at https://github.com/krisp-kwazulu-natal/SARSCoV2_South_Africa_Omicron_BA4_BA5. The reference SARS-CoV-2 genome (MN908947.3) was downloaded from the National Center for Biotechnology Information database (https://www.ncbi.nlm.nih.gov/).CODE AVAILABILITY : All custom scripts to reproduce the analyses and figures presented in this article are available at https://github.com/krisp-kwazulu-natal/ SARSCoV2_South_Africa_Omicron_BA4_BA5.Three lineages (BA.1, BA.2 and BA.3) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant of concern predominantly drove South Africa’s fourth Coronavirus Disease 2019 (COVID-19) wave. We have now identified two new lineages, BA.4 and BA.5, responsible for a fifth wave of infections. The spike proteins of BA.4 and BA.5 are identical, and similar to BA.2 except for the addition of 69–70 deletion (present in the Alpha variant and the BA.1 lineage), L452R (present in the Delta variant), F486V and the wild-type amino acid at Q493. The two lineages differ only outside of the spike region. The 69–70 deletion in spike allows these lineages to be identified by the proxy marker of S-gene target failure, on the background of variants not possessing this feature. BA.4 and BA.5 have rapidly replaced BA.2, reaching more than 50% of sequenced cases in South Africa by the first week of April 2022. Using a multinomial logistic regression model, we estimated growth advantages for BA.4 and BA.5 of 0.08 (95% confidence interval (CI): 0.08–0.09) and 0.10 (95% CI: 0.09–0.11) per day, respectively, over BA.2 in South Africa. The continued discovery of genetically diverse Omicron lineages points to the hypothesis that a discrete reservoir, such as human chronic infections and/or animal hosts, is potentially contributing to further evolution and dispersal of the virus.The South African Medical Research Council (SAMRC) with funds received from the National Department of Health. Sequencing activities for the National Institute for Communicable Diseases (NICD) are supported by a conditional grant from the South African National Department of Health as part of the emergency COVID-19 response; a cooperative agreement between the NICD of the NHLS and the US Centers for Disease Control and Prevention (CDC) (U01IP001048 and 1 NU51IP000930); the African Society of Laboratory Medicine (ASLM) and Africa Centers for Disease Control and Prevention through a sub-award from the Bill and Melinda Gates Foundation (grant number INV-018978); the UK Foreign, Commonwealth and Development Office and Wellcome (221003/Z/20/Z); and the UK Department of Health and Social Care, managed by the Fleming Fund and performed under the auspices of the SEQAFRICA project. This research was also supported by the Coronavirus Aid, Relief, and Economic Security Act (CARES ACT) through the CDC and COVID International Task Force (ITF) funds through the CDC under the terms of a subcontract with the African Field Epidemiology Network (AFENET) (AF-NICD-001/2021). Sequencing activities at KRISP and the Centre for Epidemic Response and Innovation are supported, in part, by grants from the World Health Organization, the Rockefeller Foundation (HTH 017), the Abbott Pandemic Defense Coalition (APDC), the US National Institutes of Health (U01 AI151698) for the United World Antivirus Research Network (UWARN) and the INFORM Africa project through IHVN (U54 TW012041) and the South African Department of Science and Innovation (SA DSI) and the SAMRC under the BRICS JAF (2020/049). Sequencing at the Botswana Harvard AIDS Institute Partnership was supported by funding from the Bill and Melinda Gates Foundation, the Foundation for Innovation in Diagnostics, the National Institutes of Health Fogarty International Centre (3D43TW009610-09S1) and the HHS/NIH/ National Institute of Allergy and Infectious Diseases (NIAID) (5K24AI131928-04 and 5K24AI131924-04).http://www.nature.com/naturemedicineam2023Medical VirologySDG-03:Good heatlh and well-bein