Elsevier

Antiviral Research

Volume 90, Issue 1, April 2011, Pages 42-53
Antiviral Research

HIV-1 dual/mixed tropic isolates show different genetic and phenotypic characteristics and response to maraviroc in vitro

https://doi.org/10.1016/j.antiviral.2011.02.005Get rights and content

Abstract

Dual/mixed-tropic HIV-1 strains are predominant in a significative proportion of patients, though few information is available regarding the genetic characteristics, quasispecies composition, and susceptibility against CCR5-antagonists of the primary-isolates. For this reason, we investigated in deep details, both phenotypically and genotypically, the characteristics of 54 HIV-1 primary-isolates obtained from HIV-infected patients. Tropism was assessed by multiple-cycles phenotypic-assay on U87MG-CD4+-CCR5+-/CXCR4+-expressing cells. In vitro selection in PBMCs of X4-tropic viral strains following maraviroc-treatment was also performed.

Phenotypic-assay reported pure R5-tropic viruses in 31 (57.4%) isolates, dual/mixed-tropic viruses in 22 (40.7%), and pure X4-tropic virus in only 1 (1.8%). Among dual/mixed-tropic isolates, 12 showed a remarkably higher replication-efficacy in CCR5-expressing cells (R5+/X4), and 2 in CXCR4-expressing cells (R5/X4+). Genotypic-tropism testing showed a correlation between PSSM-scores, geno2pheno false-positive-rate, and V3-net-charge with both CCR5-usage and syncytium-inducing ability. Moreover, specific gp120- and gp41-mutations were significantly associated with tropism and/or syncytium-inducing ability.

Ultra-deep V3-pyrosequencing showed the presence of a swarm of genetically distinct species with a preference for CCR5-coreceptor not only in all pure R5-isolates, but also in 6/7 R5+/X4-tropic isolates. In both pure-X4 and R5/X4+-isolates, we observed extensive prevalence of X4-using species. In vitro selection-experiments with CCR5-inhibitor maraviroc (up to 2 months) showed no-emergence of X4-tropic variants for all R5- and R5+/X4-isolates tested (while X4-virus remained fully-resistant).

In conclusion, our study shows that dual/mixed-tropic viruses are constituted by different species, whereby those with characteristics R5+/X4 are genotypically and phenotypically similar to the pure-R5 isolates; thus the use of CCR5-antagonists in patients with R5+/X4-tropic viruses may be a therapeutic-option that deserves further investigations.

Introduction

HIV-1 entry into host cells requires gp120-interaction with CD4-receptor and then with either CCR5 or CXCR4 chemokine co-receptor. HIV-1 strains can be phenotypically classified according to virus-ability to use CCR5 and/or CXCR4 co-receptor. Thus, pure R5-tropic and pure X4-tropic virus can use only the CCR5 and CXCR4 co-receptors to enter target-cell, respectively, while dual-tropic virus can use both co-receptors (Berger, 1998, Loftin et al., 2010, Scarlatti et al., 1997, Yi et al., 1999, Yi et al., 2005). In a viral population, the use of both co-receptors can be due either to the presence of dual-tropic clones, to a mixture of pure R5-tropic and X4-tropic clones, or both. This is defined as dual/mixed (D/M) phenotype.

R5-tropic viruses are generally responsible for the establishment of the initial infection, and predominate in most drug-naïve patients (prevalence, >80%) also in advanced stages of the disease (Brumme et al., 2005, Melby et al., 2006, Moyle et al., 2005, Wilkin et al., 2007), while X4-tropic viruses are restricted to advanced stages, and are associated with CD4-cell count decline. In addition, they are rarely observed in either drug naïve (<1%) or treatment-experienced patients (<5%) with preserved immune-function (Brumme et al., 2005, Melby et al., 2006, Moyle et al., 2005, Wilkin et al., 2007, Simon et al., 2010). Conversely, a quite high proportion of patients harbouring dual/mixed-tropic viruses has been observed both in drug-naïve (prevalence ranging 12–15%) and in treatment-experienced patients (prevalence ranging 20–50%) (Church et al., 2008, Huang et al., 2007, Lihana et al., 2009, Moreno et al., 2009, Shepherd et al., 2008).

Preliminary findings recently highlighted the existence of different types of D/M viruses: those that are much more efficient in using the CCR5 co-receptor (R5+/X4), those that use more efficiently the CXCR4 (R5/X4+), and those that can use with the same efficiency both co-receptors (R5/X4) (Loftin et al., 2010, Huang et al., 2007, Symons et al., 2011, Toma et al., 2010). These studies have been conducted using recombinant viruses, while nothing is known about the tropism characteristics of primary HIV-1 isolates, and their pathogenetic potential.

CCR5-antagonists are a new class of anti-HIV-1 drugs that specifically inhibit the entry of CCR5-tropic HIV-1 strains into the target cells by allosteric inhibition of the CCR5 co-receptor (Dorr et al., 2005, Princen and Schols, 2005, Regoes and Bonhoeffer, 2005). Maraviroc is the first approved CCR5 antagonist, which entered in clinical practice in 2007. Although the use of maraviroc is so far recommended only in patients with pure R5-viruses (Department of Health and Human Services, 2009), there is the increasing evidence that the use of this drug may be productively extended also to a subset of patients harbouring D/M-tropic viruses (Symons et al., 2011, Toma et al., 2010, Swenson et al., 2009). Therefore, defining the subset of viruses that can be suppressed by maraviroc is crucial to optimize the use of CCR5 antagonists in clinical practice, also in patients infected with D/M tropic viruses.

In this light, the goal of this study was to phenotypically and genotypically characterize the viral tropism of a large panel of HIV-1 primary isolates obtained from plasma of HIV-1 infected individuals. Particular attention was dedicated to: (a) the definition of R5-, X4-, D/M-populations by the study of viral replication in U87 cell lines expressing CXCR4 or CCR5 coreceptor; (b) full-length gp160 standard population sequencing; (c) V3 ultra-deep pyrosequencing, and evaluation of in vitro selection of X4-tropic viral strains following maraviroc treatment.

Section snippets

Cells

Human astroglioma U87MG-cells expressing CD4-receptor alone or with CXCR4 co-receptor (U87MG-CD4+/CXCR4+) or CCR5 co-receptor (U87MG-CD4+/CCR5+) and their corresponding parental cells were kindly provided by Dr. David Kabat, and were maintained in MEM/EBSS (Gibco) with the addition of 10% fetal bovine serum (FBS–HiClone), 1 mM sodium pyruvate (Euroclone), 0.1 mM non-essential amino acids (Euroclone), 100 U/ml penicillin + 100 μg/ml streptomycin (Euroclone) and 2 mM l-glutamine (Gibco). Medium for

Phenotypic characterization of primary HIV-1 isolates

This study included 71 primary viral-isolates obtained from 71 HAART-treated patients. Their characteristics are reported in Table 1. These isolates were used to infect both U87MG-CD4+/CCR5+ and U87MG-CD4+/CXCR4+cells. Viral infection was evaluated by p24-quantification in culture supernatants. Tropism of 54 out of 71 (76.1%) isolates was successfully characterized in indicator-cells. For the remaining 17 isolates, whose initial TCID50 were very low, viral-replication was not observed either in

Discussion

This study highlights the existence of different types of D/M-tropic viruses, with a preferential co-receptor usage in a considerable rate of clinical isolates, which is largely underestimated by the tropism determination assays currently used in clinical practice. Using primary HIV-1 isolates in multiple cycles phenotypic assay (and not using env pseudoviruses in a single-cycle assay, as generally described in this type of studies), we showed that R5+/X4 viruses (with a preference for CCR5)

Conclusions

Although our results require a clinical validation, they show that among primary HIV-1 isolates, dual-tropic viruses are mostly constituted by a swarm of species with different dual-mixed tropism potential, with a frequently predefined preference for CCR5, and whose replication is well- and long-term controlled by CCR5-antagonists despite their potential ability to enter through CXCR4 co-receptor. Both population-based and ultra-deep sequencing can be used for an optimal prediction of

Transparency declaration

Drs. Stuyver, Vandenbroucke, Van Marck, Mostmans and Aerssens report being employees of Virco BVBA or Tibotec-Virco BVBA, Johnson & Johnson companies, who are owners of the antiretroviral drugs darunavir and etravirine. The other authors declare no competing interests.

Acknowledgements

This work was financially supported by grants from the Italian National Institute of Health, the Ministry of University and Scientific Research, Current and Finalized Research of the Italian Ministry of Health, an unrestricted educational grant from Pfizer, and by the European Commission Framework 7 Programme (CHAIN, the Collaborative HIV and Anti-HIV Drug Resistance Network, Integrated Project no. 223131), and from European AIDS Treatment Network (NEAT, contract number LSHT/CT/2006/037570).

We

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    This work has been presented in part at the 16th Conference on Retroviruses and Opportunistic Infections, Montreal, Canada, 8–11 February 2009, and at the 7th European HIV Drug Resistance Workshop, Stockholm, Sweden, 25–27 March 2009.

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