HIV-2 infections are mainly restricted to West Africa; some European countries are also concerned by HIV-2 infection, mainly Portugal and France [1,2]. HIV-2 presents differential response to some antiretroviral drugs, as observed with the natural resistance of HIV-2 to nonnucleoside reverse transcriptase inhibitors, to fusion inhibitor and the decreased susceptibility to some of the protease inhibitors [3–6]. HIV-2 susceptibility to maraviroc, a C-C chemokine receptor type 5 (CCR5) inhibitor, has now been demonstrated by several in-vitro phenotypic studies and in-vivo case reports [7–12].
CCR5 inhibitors’ use requires tropism determination. A genotypic tropism prediction tool in HIV-2, based on plasma RNA gp105 V3 loop sequence, has been recently established [13]. However, most of HIV-2-infected patients naturally displayed undetectable plasma viral load in the absence of antiretroviral treatment [14], and so, tropism cannot be determined in plasma. In these cases, analysis of proviral DNA V3 loop sequence issued from peripheral blood mononuclear cells (PBMC) samples might be useful to predict HIV-2 tropism determination and to assess maraviroc eligibility.
In this study, we assessed HIV-2 tropism in paired plasma and PBMC samples to evaluate concordance between HIV-2 proviral DNA and plasma RNA genotypic tropism predictions.
Among the 216 samples issued from the ANRS (Agence Nationale de Recherches sur le SIDA et les hépatites virales) CO5 HIV-2 Cohort collected during the years 2007 and 2008 for which paired plasma and PBMC specimens were available in the Virology department of the Bichat-Claude Bernard hospital, we selected the first sample of all patients with detectable HIV-2 viral load (>100 copies/ml) (n = 77); and the 31 first patients with undetectable HIV-2 viral load (<100 copies/ml). Written informed consents were obtained for all patients at the time of inclusion in the ANRS CO5 HIV-2 Cohort.
Plasma RNA and PBMC DNA were extracted with QIAsymphony Virus/Bacteria Midi Kit and QIAsymphony DNA Mini Kit, respectively (Qiagen, Hilden, Germany). V3 loop was amplified by a first-step DNA PCR or RNA reverse transcription-PCR (RT-PCR) followed by nested-PCR using primer pairs 1S/1AS and EB2/EB5, respectively, as previously described [13,15]. GenSearch software (PhenoSystems, Lillois, Belgium) was used to align the nucleotide sequences. HIV-2 tropism was interpreted using the four V3 loop major determinants of C-X-C chemokine receptor type 4 coreceptor use (L18Z, V19K/R, V3 global net charge >+6, insertions at position 24) that we previously described [13]. A McNemar chi-square test was used to compare HIV-2 tropism in plasma and PBMC. Wilcoxon rank tests were performed to compare plasma RNA level and CD4 cell counts.
The rate of V3 loop successful RNA amplification was 6% in patients with undetectable viral load (n = 31), 23% in those with viral load between 100 and 700 copies/ml (n = 40), and 95% when viral load was above 700 copies/ml (n = 37).
The rate of V3 loop successful DNA amplification was 55, 63 and 97% in patients with undetectable viral load, with a viral load between 100 and 700 copies/ml, and with a viral load above 700 copies/ml, respectively.
Paired RNA and DNA sequences were obtained for 43 patients, including 27 infected with HIV-2 Group A, 15 with HIV-2 Group B and one with HIV-2 Group H. Among the 43 patients, 56% were women, 77% from West Africa, and 86% were infected through heterosexual contacts.
Genotypic prediction of plasma HIV-2 RNA tropism was as follows: 31 were classified as R5 (72%) and 12 as X4/dual-mixed (28%). Genotypic prediction of HIV-2 DNA tropism in paired PBMC specimens was as follows: 20 were classified as R5 (47%) and 23 as X4/dual-mixed (53%). The proportion of X4/dual-mixed tropic virus was significantly higher in proviral DNA than in plasma RNA (P = 0.001).
Among the 43 study samples, tropism concordance between plasma RNA and proviral DNA was 74% [95% confidence interval (CI) 61–87). All discordant samples (n = 11) were attributable to the prediction of R5 in plasma RNA and of X4/dual-mixed in proviral DNA.
No significant difference was observed in tropism distribution depending on HIV-2 group (P = 0.39).
Median CD4 cell count of patients with R5 and X4/dual-mixed viruses in plasma were 307 cells/μl [interquartile range (IQR) = 166–433) and 262 cells/μl (IQR = 201–390), respectively (P = 0.86). Median HIV-2 RNA level of patients with R5 and X4/dual-mixed viruses in plasma was not statistically different (1616 vs. 4956 copies/ml, respectively; P = 0.24) (Fig. 1). No difference was observed regarding time since HIV-2 diagnosis in patients with R5 or X4/dual-mixed tropism in plasma and PBMC (71 vs. 86 months, P = 0.86; and 82 vs. 81 months, P = 0.64, respectively).
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Fig. 1: HIV-2 RNA viral load and CD4 cell count median values in patients with concordant and discordant HIV-2 tropism in paired plasma and peripheral blood mononuclear cell samples from the ANRS CO5 HIV-2 Cohort, 2007–2008.ANRS, Agence Nationale de Recherches sur le SIDA et les hépatites virales; X4/DM, X4/dual-mixed.
Regarding patients with concordant or discordant tropism in plasma and PBMC, no significant differences were observed in HIV-2 RNA level, CD4 cell count and time since HIV-2 diagnosis.
In this population, we were able to obtain HIV-2 V3 loop sequences from proviral DNA in 63 and 55% of patients with plasma HIV-2 RNA level between 100 and 700 copies/ml and below 100 copies/ml, respectively. This is the first study comparing HIV-2 tropism in paired plasma/PBMC specimens, and we showed that tropism obtained from both specimens were concordant in 74% of cases.
HIV-2 infection is characterized by lower titers of plasma RNA level than in HIV-1 infection [16]. Indeed, at the time of inclusion in the ANRS CO5 HIV-2 Cohort, 63% of the patients displayed undetectable viral load [14]. For this reason, we were interested in assessing HIV-2 viral tropism in PBMC specimens. In the present series, V3 loop DNA sequences were successfully amplified in 55% of samples with undetectable plasma viral load. Furthermore, R5 tropism observed in proviral DNA has a concordance with R5 plasma tropism of 100% in our population, and X4/dual-mixed tropism in proviral DNA has a concordance with X4/dual-mixed plasma tropism of 52%. All observed discordances were due to the presence of X4/dual-mixed tropic viruses in cellular compartment and of R5-tropic viruses in plasma. So, there is a reduced risk of false R5 tropism prediction in PBMC that might compromise patient's prognosis by maraviroc misuse.
These findings underlined the higher proportion of X4/dual-mixed tropic viruses detected in PBMC than in plasma, as previously observed in HIV-1 [17–19]. This can result from different mechanisms, including the existence of distinct cellular niches for R5 and X4 viruses. Currently, the clinical significance of these data is unclear and warrants further investigation, likely by using next-generation sequencing technologies.
In conclusion, we showed that tropism prediction on the basis of proviral DNA issued from PBMC specimens is a suitable tool for tropism genotypic prediction in HIV-2-infected patients with low or undetectable viral load, the most frequent case observed in HIV-2 infection.
Acknowledgements
The study was supported by ANRS (French National Agency for research on AIDS and viral hepatitis) and by the European Community's Seventh Framework Programme (FP7/2007–2013) under the project ‘Collaborative HIV and Anti-HIV Drug Resistance Network (CHAIN)’ (grant no. 223131).
We thank clinical and virological investigators of the HIV-2 Cohort Study (ANRS CO5 VIH-2).
C.C., F.D., B.V., D.D. and F.B.V. contributed to this study concept. B.V. and L.L. performed the virological analysis. A.T. and A.B. performed the statistical analysis. B.V., F.D., C.C., A.B., F.B.V., S.M. and D.D. contributed to the analysis and interpretation of the data. B.V., C.C., F.D., A.B., G.C. and D.D. contributed to the writing of the manuscript. All authors contributed to the critical reviewing of the manuscript.
Financial support for this study was provided by the French National Agency for Research on AIDS and viral hepatitis (ANRS) and by the European Community's Seventh Framework Programme [FP7/2007–2013) under the project ‘Collaborative HIV and Anti-HIV Drug Resistance Network (CHAIN)’ (grant no. 223131)].
Conflicts of interest
The authors have not declared any conflicts of interest.
This study was presented in part at the International Workshop on HIV & Hepatitis Virus Drug Resistance and Curative Strategies, Sitges, Spain, June 2012 (Abstract 70).
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