Phenotypic and genotypic drug resistance assays are used routinely for management of treatment experienced, HIV-1 infected patients in North America and Western Europe. As anti-retroviral therapy becomes a reality in resource-poor countries, the need for resistance testing to monitor the emergence of drug resistance and to guide subsequent regimen selection will increase. In many of these countries, subtype B is not the predominant subtype of HIV-1 that is present. Current recombinant virus-based, commercially available phenotypic assays use a subtype B-based vector into which patient-derived protease (PR) and/or reverse transcriptase (RT) sequences are transferred. Preliminary results indicate the existence of subtype-specific differences in RC and susceptibility to certain drugs, but it is not known whether these are artifacts of inter-subtype incompatibility between patient insert and test vector, or reflect inherent differences in viral fitness or susceptibility among subtypes. The goal of the proposed research is to develop a phenotypic susceptibility assay for PR and RT inhibitors and RC using a subtype-specific reporter gene-containing vector that matches the subtype of the patient virus. To determine whether the mismatch between the patient virus and the testing vector influences assay results, we propose to construct a test vector from subtype C, and use this vector to generate a comparative data set from subtype B and C patient virus sequences inserted into matched or mismatched vectors. This subtype C-specific resistance assay (""""""""PhenoSenseHIV-C"""""""") could then be used to support international trials of antiretroviral therapy in Africa and Asia, as well as in North America and Western Europe for patients infected with subtype C HIV-1. Future experiments will depend on the results of this phase I project, which is anticipated to last 2 years. If the data indicates that significantly different results are obtained for subtype C samples in the IGW-B backbone, construction of other subtype IGW (e.g. from subtype A) will be undertaken in phase II. However, if the results indicate that there are no influences of backbone-insert on resulting Phenotypic and Replication Capacity data, ViroLogic's currently commercialized PhenoSense HIV- assay will be further validated for use with subtype C viruses.
