Novel Recombinant Virus Assay for Measuring Susceptibility of Human Immunodeficiency Virus Type 1 Group M Subtypes To Clinically Approved Drugs

Abstract

, and CRF12-13) and displaying a viral load range of 200 to >500,000 RNA copies/ml was 97%. The drug susceptibility measurements, based on discrimination between infected and noninfected cells on a single-cell level by flow cytometry, were reproducible, with coefficients of variation for resistance ranging from 7% to 31%, and were consistent with scientific literature in terms of magnitude and specificity.Despite continued improvements in treatment of human immunodeficiency virus type 1 (HIV-1)-infected patients, therapy failure still occurs, often leading to drug resistance development, which necessitates a change in regimen. At this moment, clinicians have at their disposal 22 drugs, targeting the viral protease (PR), the reverse transcriptase (RT), the integrase (IN), the transmembrane glycoprotein (gp41), or the interaction between the surface glycoprotein (gp120) and the cellular coreceptor CCR5. The choice of which drugs to include in the next regimen is based upon the likelihood of the drugs being active against the mutant virus present in the patient with incomplete suppression of replication (11).Currently, genotypic drug resistance testing is used more frequently within the clinical setting of patient follow-up due to practical reasons, such as a shorter turnaround time, easier implementation within laboratories, and lower cost, but also due to the fact that no clinical trial has yet provided sufficient evidence for supporting phenotypic over genotypic drug resistance testing. Nevertheless, the interpretation of genotypic drug resistance testing can be very complex, which makes phenotypic drug resistance testing in certain settings very useful and even necessary (48).Increasing numbers of drug resistance mutations have been identified within gag, pol, and env, reflecting the genetic flexibility of HIV-1. These mutations can directly boost the ability of the virus to specifically replicate in the presence of the drug (major mutations) or indirectly aid the virus by increasing its replication capacity in general, whether in the presence or absence of the drug (minor or compensatory mutations). In general, the major mutations are found at the drug binding sites within the targeted protein (or the viral protein that interacts with the cellular target), whereas the others can be found at distinct sites within the target protein or even other proteins. In this respect, PR inhibitor (PI) mutations have been observed not only within the PR but also at several sites within its Gag substrate (13,28).The use of genotypic data to determine if certain drugs are still active against patient-derived virus is further complicated by the presence of natural polymorphisms in non-B strains and by baseline subtype-dependent combinations of mutations that occur during treatment, leading to discordances between different interpretation algorithms (43,51). Several studies have revealed novel mutations or differences in the prevalence of known mutations in non-B subtypes (1, 3). Others have demonstrated the impact of the genet...

Novel Method for Simultaneous Quantification of Phenotypic Resistance to Maturation, Protease, Reverse Transcriptase, and Integrase HIV Inhibitors Based on 3′Gag(p2/p7/p1/p6)/PR/RT/INT-Recombinant Viruses: a Useful Tool in the Multitarget Era of Antiretroviral Therapy

“…Multiple commercial (28,51,57,71) or in-house (3,7,9,21,30,31,33,42,47,61,62,67,77) phenotypic assays are currently available to quantify recombinant virus susceptibility to different drug classes; however, none has been able to simultaneously evaluate resistance to antiretroviral drugs targeting gag, protease, reverse transcriptase, and integrase coding regions. One of the main advantages of the ViralARTS HIV system is the ability to construct and test recombinant viruses carrying larger HIV-derived fragments.…”

“…Unlike previous approaches that utilize homologous recombination in mammalian cells (4,9,28,33,57,77,80) or ligation-based cloning techniques (21,51,67) to create recombinant viruses, here we used a yeast-based recombination/gap repair method to introduce patient-derived HIV sequences into a vector, with the final goal of producing replication-competent chimeric viruses (15). As described above, a large HIV genomic region from the Gag protein p2 to the integrase coding region was RT-PCR amplified as a large fragment (3,428 nt) or two overlapping fragments (1,657 nt and 2,002 nt) from plasma samples or HIV-1 isolates (Fig.…”

“…Homologous recombination in mammalian cells of PCR-derived HIV sequences into vectors devoid of the corresponding sequence was one of the first and, thus, far more common, methods used (9,28,33,80). Another frequent technique takes advantage of intrinsic or engineered restriction sites to clone patient-derived PCR products into a vector using restriction digestion and ligation (7,21,47,51,67).…”

“…Historically, phenotypic drug susceptibility assays have used HIV-1 isolates (30) or replication-competent recombinant viruses (9,21,28,33,42,61,67,71) derived from patient samples by cocultivation or PCR amplification, respectively. The use of clinical HIV-1 isolates, in addition to being time-consuming and not amenable for high-throughput process, requires a period of virus propagation that usually alters the original in vivo viral quasispecies distribution, affecting the proportion of viruses which may or may not be harboring drug resistance mutations (34,44).…”

“…Susceptibility of the recombinant viruses to various HIV inhibitors can be quantified by indirectly monitoring cytopathic effects caused by the replicating virus (28,33,61) or by directly measuring full virus production via viral protein levels in the cellfree supernatant, e.g., reverse transcriptase activity (11) or p24 antigen (42,47). The inclusion or a reporter gene in the viral genome (i.e., firefly luciferase [51,77], Renilla luciferase [21,77], and fluorescent proteins [9,80]) or a virus-induced reporter gene within the target cells (7,67) provides a measure of virus infection at the step of HIV-1 transcription and is commonly employed with replication-competent or pseudotyped viruses.…”