Detection and significance of drug- resistant HIV-1 minor populations

Abstract: The human immunodeficiency virus (HIV) is the cause of the HIV/AIDS pandemic affecting millions of people around the world. Treatment with antiretroviral drugs, if used properly, leads to a dramatic decrease in virus production in the patient and prevents the onset of AIDS. However, treatment is not always successful, mainly due to the extraordinary ability of the virus to change and become resistant to the antiretroviral drugs. The general aim of this thesis was to study the development of HIV-1 drug resistance, specifically the detection and significance of small populations of drug-resistant virus in infected individuals that can go undetected by the standard resistance tests used in the clinics today. In study I, we developed and evaluated an assay called selective real-time PCR (SPCR), which can detect and display the proportions of different populations of drug-resistant virus carrying the clinically relevant M184I/V mutations in the HIV-1 reverse transcriptase enzyme. These mutations confer high-level resistance to one of the most commonly used drugs today, lamivudine (3TC). The SPCR assay was then applied to several different clinical cohorts in the subsequent papers. We found SPCR to be a well-functioning, sensitive and reliable method to study the kinetics of antiretroviral resistance development in HIV-1, which was able to detect resistance mutations not seen by conventional genotyping assays. In study II, we evaluated the presence of M184I/V-containing minor viral populations and their potential role in treatment failure in didanosine (ddI)-treated patients experienced with multiple nucleoside reverse transcriptase inhibitors. We found that the survival advantage of virus strains having the M184I/V mutations were limited during ddI treatment, suggesting that the presence of these mutations should not preclude the use of ddI in nucleoside-experienced patients. In study III, we investigated to which extent unique drug resistance patterns appear in cerebrospinal fluid as compared to blood in patients who had failed 3TC-containing therapy. We found differences in drug resistance patterns in both minor and major populations, which were likely to be related to differences in the selective pressure of antiretroviral drugs within the two compartments rather than unique evolutionary pathways. Minor populations were not seen to play a major role in resistance development in cerebrospinal fluid in the patients we tested. In study IV, we studied to which extent selection of M184I/V mutations occurs in treatment-naïve patients during the first months after initiation of antiretroviral therapy containing two, three or four drugs. We found the selection of drug-resistant variants to be rare in the three- and four-drug cohorts, but common in the two-drug cohort, suggesting that current treatment regimens initiated in treatment-naïve patients should be sufficient to stop resistance development during the first phase of viral decay, as long as the patients are adherent. The clinical significance of minority populations in the development of resistance to antiretroviral drugs is an important topic to be defined. SPCR and similar assays will provide valuable tools to further expand the current knowledge in this field.