Abstract
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.