Strappe, Padraig Michael; (2001) Quantitation of proviral load and the distribution of drug resistant mutations in human immunodeficiency virus infection. Doctoral thesis (Ph.D.), University College London.

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Abstract

Infection with Human Immunodeficiency virus type 1 (HIV-1) induces a chronic and progressive disease process with a broad spectrum of clinical manifestations from acute primary infection to AIDS which is often associated with opportunistic infections and AIDS. The course of the disease is also associated with increasing levels of viral replication and destruction of the immune system. Treatment of HIV-1 infection with single antiretroviral drugs provides a limited duration of benefit due to the emergence of drug resistant strains of the virus during monotherapy. The aim of this study was to examine levels of HIV-1 proviral DNA load and the distribution of HIV-1 zidovudine resistance mutations in a group of 79 patients who were receiving zidovudine monotherapy and also in multiple post-mortem tissues samples from patients who died of AIDS. A quantitative polymerase chain reaction (QPCR) assay was firstly developed to accurately measure HIV-1 proviral DNA load. The QPCR assay was based on coamplification of known copy number of a control sequence with patient HIV-1 DNA which allowed accurate quantitation. Measurement of proviral DNA load in post-mortem tissues from 11 patients who died of AIDS revealed high levels in lymph node and spleen samples, significant levels were also found in frontal lobe brain tissue. The sequence of the V3 loop of HIV-1 gp120 was obtained by direct sequencing of a selection of the tissues and 15 of the 20 sequences analysed showed a macrophage tropic, non syncytium inducing (NSI) phenotype. Mutations in HIV-1 reverse transcriptase which confer resistance to zidovudine were examined by a point mutation assay (PMA) which showed differences in levels of resistant genotypes between different tissues in individual patients. Separate evolution of drug resistant virus in different anatomical sites may reflect the efficiency of zidovudine in different tissues or the selective tissue tropism of HIV-1. The profiles of resistance at the five codons of HIV-1 RT examined were also different between patients and the presence of mutation at codon 41 and 215 was indicative of long term zidovudine monotherapy. Monitoring the emergence of drug resistant HIV-1 plays an important role in successful antiretroviral therapy and rapid PCR based assays for resistant genotypes have been demonstrated. In this thesis a Line Probe assay (LiPA) was evaluated for the detection of mutations at a number of codons of HIV-1 conferring Zidovudine resistance. Detection of resistant mutations by the LIPA test in PCR amplified DNA from a selection of PM tissues and whole blood samples showed 100% agreement with the sequence of HIV-1 RT obtained the samples. The LiPA test was both a rapid and sensitive assay for drug resistant mutations and wild type sequences. HIV-1 proviral DNA load and the presence of zidovudine resistant mutation at codons 41 and 215 was examined in 79 individual patients who had received zidovudine monotherapy. Expressing proviral DNA load per milliliter (ml) of whole blood or per unit of CD4 cells demonstrated how measurement of proviral load levels can be confounded by variation in cell numbers during the course of infection. Expression of proviral load per ml of blood showed a slight increase over time from baseline, whereas proviral load expressed per 103 CD4 cells showed a more significant change over time which mirrored the change in CD4 levels over time. Kaplan Meier analysis of proviral DNA load demonstrated that individuals with levels of > 4.0 log lo DNA copies had a shorter time for progression to a 50% CD4 cell decrease and also a shorter time to death. Further analysis of proviral DNA load as a marker for disease progression using Cox proportional hazard models showed a strong relationship between proviral load expressed per 10 CD4 cells and progression to death. The relationship of zidovudine resistance to proviral load, CD4+ T-cell count and disease progression was also examined. A significant correlation was found between presence of resistance mutation and a low CD4 cell count, resistance mutation was also strongly associated with AIDS and with a progression to death in a Cox proportional hazard model. The results presented in this thesis of measurements of proviral DNA load and drug resistance mutations combined with an in-depth statistical analysis provide an important insight into HIV-1 disease progression in a group of patients receiving zidovudine monotherapy.

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