Impact of frequent natural polymorphisms at the protease gene on the in vitro susceptibility to protease inhibitors in HIV-1 non-B subtypes

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Abstract

Background: Naturally-occurring polymorphisms at the human immunodeficiency virus type 1 (HIV-1) protease which have been associated to resistance to protease inhibitors (PIs) in clade B viruses are frequently found in non-B subtypes, with unknown clinical significance. Objective: To assess the susceptibility of non-B viruses to different PIs. Study design: Plasma samples from 58 drug-naive individuals infected with HIV-1 non-B subtypes (2A, 22C, 2D, 1F, 29G and 2J) defined by phylogenetic analyses of the protease gene were tested using a phenotypic assay (PhenoSense, ViroLogic, South San Francisco, CA, USA). Twenty of them were further analyzed with another assay (Antivirogram™, Virco, Mechelen, Belgium). All 58 non-B viruses harbored amino acid substitutions associated with reduced PI susceptibility in clade B (positions 10, 20, 36, 63, 70, 77 and 82). Results: Using PhenoSense-HIV™ assay, all but two individuals harbored viruses completely susceptible to all six PIs tested (indinavir (IDV), ritonavir (RTV), saquinavir (SQV), nelfinavir (NFV), amprenavir (APV), lopinavir (LPV)). The two viruses with reduced susceptibility belonged to clade G. The first virus, which had K20I, M36I and V82I, showed 2.9-fold decreased susceptibility to APV, while the second virus showed 3.9-fold decreased susceptibility to both NFV and RTV, with amino acid substitutions K20I, M36I, L63P and V82I. Of note, several other viruses displayed the same constellation of mutations but without showing any reduced susceptibility, suggesting that these polymorphisms per se do not affect PI susceptibility. Conclusion: PI susceptibility in HIV-1 non-B viruses seems to be preserved despite the presence of polymorphic changes which have been associated to PI resistance in clade B viruses.

Introduction

Human immunodeficiency virus type 1 (HIV-1) mutates rapidly, and nucleotide substitutions, deletions, insertions, and rearrangements are common during the course of the infection (Coffin, 1995). Methods based on nucleotide sequence analyses allow us to recognize the phylogenetic relationships between different virus sequences. So far, HIV-1 can be divided into three distinct and highly divergent groups: M (major), O (outlier), and N (non-M, non-O). Several genetic variants can be recognized within HIV-1 group M, including 9 subtypes (A–D, F–H, J and K) and 15 major circulating recombinant forms (CRFs) (Kuiken et al., 2000, Robertson et al., 2000). The occurrence of recombination events contribute to expand the degree of genetic heterogeneity of HIV-1 in vivo (Peeters, 2000).

The global distribution of different forms of HIV-1 is a dynamic process (Paraskevis and Hatzakis, 1999). Tracking different HIV-1 subtypes and CRFs is of crucial importance for understanding trends in the AIDS pandemic. In North America and West Europe, HIV-1 subtype B is the most prevalent clade, although a rapid spreading of other variants has been highlighted in recent years (Alaeus et al., 1997a, Alaeus et al., 1997b; Barlow et al., 2001, Boni et al., 1999, Fleury et al., 2003; Holguin et al., 2000a, Holguin et al., 2000b, Holguin et al., 2002a, Holguin et al., 2002b; Op de Coul et al., 2001). The diversity of HIV-1 may affect diagnostic tests (Apetrei et al., 1996, Baldrich-Rubio et al., 2001, Candotti et al., 2000), including those used for viral load measurements (Alaeus et al., 1997a, Alaeus et al., 1997b; Holguin et al., 1999, Jenny-Avital and Beatrice, 2001).

Drug resistance testing has become an important tool in the management of HIV-infected individuals undergoing antiretroviral therapy (Youree and D’Aquila, 2002). Both genotyping and phenotyping methods appear to be equally useful for determining the susceptibility of HIV-1 to antiretroviral drugs (Dunne et al., 2001). While the genotype identifies genetic positions at the HIV genome associated with drug resistance, the phenotype exams in vitro the relative susceptibility of virus to different drug concentrations. Drug susceptibility tests have been designed for and performed mainly on subtype B strains. However, the increasing global spread of non-B subtypes highlights the need to determine the performance of all commercial drug resistance assays testing HIV-1 subtypes other than B.

Naturally-occurring genetic polymorphisms at the pol gene encoding the targets for anti-HIV drugs, such as the reverse transcriptase (RT) and protease, result from the high rate of HIV-1 replication and the low fidelity of the RT enzyme. Such changes have been found among drug-naive individuals infected with non-B subtypes (Becker-Pergola et al., 2000, Cane et al., 2001, Grossman et al., 2001, Holguin et al., 2002a, Pieniazek et al., 2000, Shafer et al., 1997, Vergne et al., 2000). Secondary or accessory protease mutations K20I, M36I and V82A are significantly more common among non-B viruses (review in Holguı́n and Soriano, 2002). Some of these changes are associated to protease inhibitors (PI) resistance (Hirsch et al., 2003; update November–December 2003 in http://www.iasusa.org). Overall, protease change M36I may be considered as a genetic marker for HIV-1 group M non-B subtypes (Holguı́n et al., 2002a), and the K20I substitution appears almost exclusively in protease sequences belonging to subtype G viruses (Holguı́n et al., 2002a).

The clinical relevance of these baseline polymorphisms in non-B viruses has not been determined yet. Although the response to antiretroviral therapy may be independent of clade and baseline polymorphisms (Frater et al., 2001, Hermans et al., 2002), the presence of a high number of substitutions at positions associated with drug resistance might influence in some extent the risk of treatment failure (Loemba et al., 2002, Lorenzi et al., 1999, Perez et al., 2001, Perno et al., 2001).

Section snippets

Material and methods

To assess the susceptibility of non-B viruses to different PIs, plasma samples collected from 58 drug-naive individuals infected with non-B subtypes were phenotyped using PhenoSense-HIV™ (ViroLogic, South San Francisco, CA, USA). Twenty of them were additionally analyzed with Antivirogram™ (Virco, Mechelen, Belgium). Both commercial assays use HIV-1 genomes generated by recombination between PCR amplified pol products derived from patient’s viruses and a subtype B proviral clone having a

Results

The 58 clinical specimens included in the study belonged to non-B HIV-1 subtypes and were classified as 2A, 22C, 2D, 1F, 29G and 2J. The origin of these individuals, viral subtype, mutations at the protease associated to PI resistance, as well as the corresponding sequence accession numbers are shown in Table 1. Thirty-seven specimens were collected in four different HIV clinics in Spain since 1997–2002, and had been previously characterized as non-B strains (Holguin et al., 2000a, Holguin et

Discussion

The results of this study suggest that current phenotypic assays can be used confidently for testing the susceptibility to antiretroviral drugs in HIV-1 non-B subtypes. Despite a higher rate of polymorphisms at positions associated with PI resistance in non-B drug-naive subjects was found, a compromised PI susceptibility was not recognized using two different phenotypic tests. The recognition of a slightly reduction in PI susceptibility in two clade G viruses (nos. 25 and 27) could be the

Acknowledgements

This work was supported in part by grants from Asociación Investigación y Educación en SIDA (AIES) and Red de Investigación en SIDA (RIS). We would like to thank Dr. Marı́a José Pena (Hospital Dr. Negrı́n, Las Palmas de Gran Canaria), Dr. Jorge del Romero (Centro Sandoval, Madrid), and Dr. Belén Aracil (Hospital de Móstoles, Madrid) for providing some of the clinical specimens.

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