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A Simple Virus Recovery Assay to detect the Replication-Competent HIV-1 Reservoir in Children
Thesis (MScMedSc)--Stellenbosch University, 2021.
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| Format: | Thesis |
| Language: | en_ZA |
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Stellenbosch : Stellenbosch University
2021
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| _version_ | 1867613969686986752 |
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| access_status_str | Open Access |
| author | Van Zyl, Carli |
| author2 | Van Zyl, Gert Uves |
| author_browse | Van Zyl, Carli Van Zyl, Gert Uves |
| author_facet | Van Zyl, Gert Uves Van Zyl, Carli |
| author_sort | Van Zyl, Carli |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MScMedSc)--Stellenbosch University, 2021. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/110125 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:44:35.400Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2021 |
| publishDateRange | 2021 |
| publishDateSort | 2021 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/110125 A Simple Virus Recovery Assay to detect the Replication-Competent HIV-1 Reservoir in Children Van Zyl, Carli Van Zyl, Gert Uves Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Medical Virology. Viral Outgrowth Assay UCTD Antiretroviral agents Human immunodeficiency viruses HIV-positive children RNA Transcript Thesis (MScMedSc)--Stellenbosch University, 2021. Background and Introduction: The early initiation of combined antiretroviral therapy (cART) in children has proved to significantly reduce the morbidity and mortality rate associated with human immunodeficiency virus type 1 (HIV-1) infection. However, even though cART can reduce plasma viral loads to being clinically undetectable, it does not cure HIV-1 infection due to the presence of a latent reservoir where HIV-1 persists as integrated, replication-competent proviruses. For this reason, the search to finding a cure for HIV-1 continues, either by targeting the HIV-1 reservoir directly (eradication approach) or by controlling rebound of viraemia (functional cure approach). To accurately assess the success of such cure strategies, assays to accurately measure the latent reservoir are essential,such as the quantitative viral outgrowth assay (qVOA). In this study, a simple viral outgrowth assay (VOA) was developed that utilises MOLT-4CCR5+ cells for propagation of HIV-1 as well as a sensitive reverse transcriptase real-time polymerase chain reaction (RT-qPCR) assay for detectionof viral outgrowth. Methods Two children (patient 337756 and patient 341622) were selected that were part of the post-Children with HIV Early Antiretroviral Therapy randomised trial (CHER) to be assayed for replication-competent virus,with the VOA,from stored peripheral blood mononuclear cells (PBMCs) formas singleblood collection, which consisted of stimulation to induce viral release followed by co-cultureover28 days. Both children started therapy at two months of age, were ART suppressed for eight years, although both had a recent episode of viraemia (six months before the time point sampled), and both had total HIV-1 deoxyribonucleic acid (DNA) levels of >50copies per million cell equivalents at the time point sampled. Patient 337756 was 11years old and patient 341622 was 10years old at the time point sampled. A total of 29million and 21million PBMCs were assayed for patients 337756 and 341622, respectively. A VOA was implemented and optimised that utilisedMOLT-4CCR5+ cells for HIV-1 expansion and a sensitive, affordable RT-qPCR assay was developed that targets the integrase region ofpolin the HIV-1 genome to detect exponential viral outgrowth. T cells were stimulated by addition of phytohemagglutinin (PHA) and irradiated feeder cells. To determine the sensitivity ofthis RT-qPCR assay, it was compared to the traditional, but more expensive p24 antigen enzyme-linked immunosorbent assay (ELISA). Results Infectious virus was recovered from two out of three VOA wells in one of the two children(patient 337756). The in-house RT-qPCR assay was able to detect virus on day five for this child while the p24 ELISA only had a positive result on day 14. Interestingly, the second VOA well for the same child had low-level exponential outgrowth with the RT-qPCR assay, but the p24 had no positive signals. This proved that the RT-qPCR assay was more sensitive in detecting viral outgrowth compared to the p24 ELISA. The third VOA well for this patient had weak ribonucleic acid (RNA)signal on day 14 as well as DNA signal on day seven. The child who had no infectious virus recovery from stored PBMCs (patient 341622) also had a single instance of weak RNA signal on day 14 in one of the two VOA wells assayed. The single instances of RNA release might have been non-infectious, inducible virus that got released after stimulation. HIV-1 DNA detected in the supernatant were cell derived and may therefore indicate apoptosisor lysis of infected cells. Conclusion: The VOA implemented in this project proved to be more practicable by using MOLT-4CCR5+ cells compared to the gold standard qVOA that makes use of cluster of Differentiation8 (CD8+)-depleted lymphoblasts from different donors.The RT-qPCR assay also proved to be more sensitive than the p24 ELISA. Although, by streamlining and optimising this assay even more, it could be more valuable for future studies in evaluating HIV-1eradication strategies. Nevertheless, this VOA could be a valuable tool in studying the success of latency reversing agents (LRAs) in children on suppressive cART. AFRIKAANSE OPSOMMING: Raadpleeg teks vir opsomming Masters 2021-03-03T13:51:49Z 2021-04-21T14:41:36Z 2021-03-03T13:51:49Z 2021-04-21T14:41:36Z 2021-03 Thesis http://hdl.handle.net/10019.1/110125 en_ZA Stellenbosch University 153 pages application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Viral Outgrowth Assay UCTD Antiretroviral agents Human immunodeficiency viruses HIV-positive children RNA Transcript Van Zyl, Carli A Simple Virus Recovery Assay to detect the Replication-Competent HIV-1 Reservoir in Children |
| title | A Simple Virus Recovery Assay to detect the Replication-Competent HIV-1 Reservoir in Children |
| title_full | A Simple Virus Recovery Assay to detect the Replication-Competent HIV-1 Reservoir in Children |
| title_fullStr | A Simple Virus Recovery Assay to detect the Replication-Competent HIV-1 Reservoir in Children |
| title_full_unstemmed | A Simple Virus Recovery Assay to detect the Replication-Competent HIV-1 Reservoir in Children |
| title_short | A Simple Virus Recovery Assay to detect the Replication-Competent HIV-1 Reservoir in Children |
| title_sort | simple virus recovery assay to detect the replication competent hiv 1 reservoir in children |
| topic | Viral Outgrowth Assay UCTD Antiretroviral agents Human immunodeficiency viruses HIV-positive children RNA Transcript |
| url | http://hdl.handle.net/10019.1/110125 |
| work_keys_str_mv | AT vanzylcarli asimplevirusrecoveryassaytodetectthereplicationcompetenthiv1reservoirinchildren AT vanzylcarli simplevirusrecoveryassaytodetectthereplicationcompetenthiv1reservoirinchildren |