Quantitative evaluation of the recombinant HIV-1 phenotype to protease inhibitors by a single-step strategy

Objective: To develop and optimize a fast and quantitative recombinant stra tegy for evaluating the HIV-1 phenotype to protease inhibitors (PI). Design and methods:A non-replicative HIV-1 molecular vector (designated p D elta pro Delta env) capable of expressing exogenous HIV-l protease-encoding sequences was developed in this study. The HIV-1 protease sequences were a mplified from either viral isolates or plasma samples (both from 21 HIV-l-i nfected individuals, 19 of whom were failing different anti-HIV-l combinati on treatments) and cloned in the p Delta pro Delta env backbone. The HIV-1 recombinant phenotype to PI was determined directly after transfection of v iral chimeric clones by measuring protease activity and calculating a perce ntage sensitivity index (SI%; the ratio between the results from each clone and those from a PI-sensitive reference strain). Results: The SI% values obtained from the recombinant clones paralleled the IC50 results of the viral isolates and documented different degrees of res istance and cross-resistance to PI, compatible, with few exceptions, with t he respective genotype. Interestingly, an inverse correlation between SI% v alues and the presence of primary mutations for resistance to PI (P = 0.003 8 and P = 0.0414, for indinavir and ritonavir, respectively) and a differen ce in SI% between samples harbouring an increasing number of mutations (ind inavir, P = 0.022; ritonavir, P = 0.0466) were observed. Conclusion: The data substantiate the reliability of the novel strategy for a fast (5 day) quantitative evaluation of HIV-1 phenotype to PI, and indic ate that this method may contribute to the understanding of mechanisms of v irus resistance to PI. (C) 2000 Lippincott Williams & Wilkins.