M.Sc. / It is the widely accepted view that the human immunodeficiency virus (HIV) is the causative agent of acquired immunodeficiency syndrome (AIDS) and that South Africa harbors mainly HIV type 1 subtype C (HIV-1 C). Extensively characterized biological isolates (especially of HIV-1 subtype C) for use in HIV/AIDS vaccine and drug development are not readily available. This study evaluated three different protocols for the expansion of virus from infected PBMC's of 68 HIV/AIDS patients (designated HJ1 — 22 and INN1 — 97). Factors influencing the success of a protocol for the expansion of HIV-1 were 1) the amount and time of addition of IL-2 and PHA to the culture media; 2) the fact that freshly isolated clean PBMC's (treated with PHA prior to co-cultivation) was necessary while infected PBMC's could be used fresh or frozen; 3) whether the absence or presence of polybrene as a tissue culture additive had any effect. The I-11V-status of patients could be confirmed with rapid tests and/or NASBA assays, while successful expansion of the virus could be confirmed or refuted by determining p24 levels of sera or culture supematant (with values ranging from <7.8pg/ml to about 280pg/ml). Less sensitive assays like the reverse transcriptase (RT) and gpl 20 ELISA's give much lower absorbance values when compared to the p24 ELISA. Using expanded virus to infect PBMC's and T-cell lines (PM1, U87.CD4-CCR5, U87.CD4-CXCR4 and CEM.NKR-CCR5) and then measuring p24 levels showed that the final protocol chosen was capable of producing high titre, biologically active virus. To further test the biological activity of the isolates, the virus was used in assays evaluating the potential inhibitory ability of natural products and neutralizing antibodies. PCR using universal primers (SK22/SK38/SK39) was not consistently successful in amplifying out the correct sized region of gag (for SK22/SK39 a fragment of 600bp and a 115bp fragment for SK38/SK39 was obtained but not for all the samples). Primers (Cgag189(+/-)) were designed during this project to specifically amplify an 189bp region of gag from subtype C, which proved (in some instances) to be more successful. PCR amplification of the proviral DNA fragments was confirmed by sequencing of selected PCR products. Virus titre was determined by calculating TCID50 (login: 1.054). By modifying expansion and detection protocols it is possible to standardize the process to suit a particular isolate and/or circumstance. This production of large volumes of high titre, biologically active isolates has filled a desperate need for reagents to aid HIV researchers in the development of an effective vaccine or other drug therapy.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:uj/uj:2562 |
| Date | 16 August 2012 |
| Creators | Hill, Emma. |
| Source Sets | South African National ETD Portal |
| Detected Language | English |
| Type | Thesis |
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