Lin, Nina H MD*†; Smeaton, Laura M MS§∥; Giguel, Françoise*‡; Novitsky, Vladimir PhD§‖; Moyo, Sikhulile MS‖; Mitchell, Rebecca M PhD§‖; Makhema, Joseph MDChB‖; Essex, Myron PhD, DVM§‖; Lockman, Shahin MD, MSc†‡§‖; Kuritzkes, Daniel R MD†‡
From the *Division of Infectious Diseases, Massachusetts General Hospital, Boston, MA; †Harvard Medical School, Boston, MA; ‡Section of Retroviral Therapeutics, Brigham and Women's Hospital, Boston, MA; §Department of Biostatistics and Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA; and ‖Botswana Harvard AIDS Institute, Gaborone, Botswana.
Received for publication November 26, 2010; accepted February 9, 2011.
This work was supported in part by Public Health Services grants from the National Institutes of Health (NIH) including K24 RR016482 and U01 AI068636 (an ACTG Virology Support Laboratory contract) (D.R.K.); R01 HD037793 (M.E.); R01 HD044391 (S.L.); a Scholar Award from the Harvard Center for AIDS Research (CFAR) (P30 AI060354), a Harvard Institute for Global Health International Fellowship, a Bristol-Myer's Squibb Virology Fellowship, and a Burroughs Wellcome Fund/American Society of Tropical Medicine and Hygiene post-doctoral fellowship (N.H.L.); the NIH Fogarty grant R24TW007988 (R.M.M); and the CFAR Biostatistical core grant award 2P30AI060354-06 (L.M.S.).
Preliminary data were presented at the 2010 Conference of Retroviruses and Opportunistic Infections, San Francisco, CA, February 8-11, 2010.
The authors have no conflicts of interest to disclose.
Correspondence to: Daniel R. Kuritzkes, MD, Section of Retroviral Therapeutics, Brigham and Women's Hospital, 65 Landsdowne Street, Room 449, Cambridge, MA 02139 (e-mail: email@example.com).
Coreceptor use by HIV-1 is an important determinant of disease progression and response to CCR5 antagonist therapy. Much of the data relating coreceptor use to clinical disease is based on studies of subtype B-infected patients. Most HIV-1 transmission involves CCR5-using (R5) viruses, which predominate in the early phases of infection.1-4 Over time, CXCR4-using viruses emerge in approximately 50% of patients and are associated with an increased risk of disease progression and death.2,5 Less is known about patterns of coreceptor use by HIV-1 subtype C (HIV-1C), which accounts for more than half of new infections worldwide. Studies to characterize coreceptor use of HIV-1C are needed for a better understanding of pathogenesis, treatment, and prevention of infection by this highly prevalent subtype.
Data regarding the prevalence of CXCR4-using HIV-1C are conflicting and come from relatively small, cross-sectional studies. Initial studies suggested that R5 virus predominates in HIV-1C infection throughout the course of disease.6-9 Subsequent studies identified CXCR4-using virus in a small number of treatment-naive HIV-1C-infected patients with advanced HIV disease10-13 with some studies showing that 30% to 50% of patients with AIDS harbor CXCR4-using virus.14-16 One study suggested a possible association of antiretroviral therapy (ART) with higher rates of CXCR4-using virus,14 but the relationship between ART and emergence of CXCR4-using virus was inferred by the comparison of prevalence rates between small cohorts of treatment-naïve and -experienced subjects. To obtain a better estimate of the prevalence of CXCR4-using virus in HIV-1C infection and its relationship to ART, we determined coreceptor use of plasma virus from a large cohort of HIV-1C-infected women from Botswana with advanced HIV-1 disease.
Subjects selected for testing were women participating in the Mashi study, a randomized clinical trial of prevention of mother-to-child transmission conducted in southeastern Botswana from 2001 to 2003 (clinicaltrials.gov identifier: NCT00197587).17,18 All participating women received zidovudine from 34 weeks gestation and were randomized to single-dose nevirapine or placebo intrapartum. Combination ART became available through the Botswana government to participating women with CD4 less than 200 cells/mm3 or AIDS-defining illness beginning in 2002, 19 months into the study. Participants who completed the Mashi study were offered enrollment into a follow-up study, in which clinical status, HIV-1 RNA, and CD4 cell count were monitored at 3-month intervals.19 For those who initiated ART, virologic failure was defined as confirmed HIV-1 RNA greater than 400 copies/mL 6 or more months after starting ART or failure to achieve at least a 1-log10 drop in HIV-1 RNA by 12 weeks.19 Plasma HIV-1 RNA was assessed using the standard COBAS Amplicor HIV-1 Monitor assay (Roche Molecular Systems, Bradenburg, NJ); CD4 cell counts were measured by standard techniques. The use of Mashi samples for this study was approved by the ethics committees and Institutional Review Boards at the Botswana Health Ministry, the Harvard School of Public Health, and Partners HealthCare System. All participants had provided written informed consent for use of stored samples for approved HIV-related research.
HIV-1 Coreceptor Use Assay
Coreceptor use of plasma virus was determined using a sensitive single-cycle assay validated for subtype C viruses20 at screening or enrollment into Mashi from women with screening CD4 counts below 200 cells/mm3, and for women who initiated ART, at the time of virologic failure. Viruses that used only CCR5 were considered R5; those that used only CXCR4 were considered X4; and those that used both coreceptors were considered to have dual or mixed (DM) coreceptor use. The study was powered to estimate the prevalence of CXCR4-using virus among treatment-naïve patients with a precision of ± 6.1% (95% confidence interval), which required a sample size of at least 100 patients.
Associations of viral tropism with clinical parameters were explored by univariable analyses using Fisher exact test for categorical parameters and the Wilcoxon rank-sum test for continuous parameters. The Cochran-Armitage exact test of trend was used to identify significant differences in prevalence across increasing categories of baseline (pre-ART) CD4 cell count and HIV-1 RNA. All reported P values are two-sided. Changes in HIV-1 RNA and CD4 cell count over time were explored using Type 3 Tests of fixed effects using SAS Version 9.1 (SAS Institute Inc, Cary, NC).
In the Mashi study, 206 women had screening CD4 cell counts below 200 cells/mm3. Of the 165 women with samples available for tropism testing, 12 were excluded because they had initiated ART before enrollment, leaving 153 samples for testing. Baseline characteristics of the 153 women with samples tested for coreceptor use were similar to those of the 53 women who were not tested, except that baseline median viral load was higher (P = 0.01) and the time to ART initiation was longer among women with a tested sample (P < 0.001; Table 1). The exclusion of the 12 women who initiated ART before enrollment likely contributed to the difference in time to ART initiation between the two groups. Among those tested, the median baseline CD4 count was 130 cells/mm3 (interquartile range, 95-171 cells/mm3) and the plasma HIV-1 RNA level was 5.02 log10copies/ml (interquartile range, 4.51-5.44 log10 copies/mL).
Prevalence of CXCR4-Using Viruses and the Characteristics of the Women by Coreceptor Use
The coreceptor use assay generated results for 148 of 153 (96.7%) women. Among those with an assay result, 22 (14.9%) had DM virus and 126 (85.1%) had R5 virus; no samples were X4 (Table 1). A maximum likelihood tree built from sequences of the C2-V4 region of a subset of env amplicons (N = 61) confirmed that each sequence was unique and clustered with reference subtype C sequences (bootstrap value 98%).
Median baseline viral load, time to initiation of ART, death, and mother-to-child transmission did not differ significantly between women with DM versus R5 virus, but women with DM virus were significantly older than women with R5 virus (median age, 33.1 years versus 28.1 years, respectively; P = 0.025). Comparison of the median baseline CD4 cell count in women harboring DM virus with those with exclusively R5 virus showed differences that approached statistical significance (P = 0.08) (Table 1). The distribution of women infected with DM virus varied significantly across the range of CD4 cell counts between 0 and 200 cells/mm3 with the highest proportion having baseline CD4 cell counts below 50 cells/mm3 (Cochran-Armitage exact test of trend, P = 0.026) (Fig.1).
CXCR4-Using Virus as a Predictor of Clinical Progression
The association of baseline coreceptor use with change in CD4 cell count and virus load from study entry to ART initiation was examined, but the number of women not on ART decreased substantially over time; the majority started ART shortly after enrollment when ART became available in 2002 through the Botswana government treatment program. By 12 months after enrollment, 82% of women infected with R5 or DM virus had initiated ART; among those who remained treatment-naïve, the mean CD4 cell count was 130 cells/mm3 for DM virus (n = 4) compared with 170 cells/mm3 for R5 virus (n = 23). The mean CD4 cell counts at sequential 3-month intervals were always lower among women with DM virus compared with those with R5 virus, but these differences did not achieve statistical significance (P = 0.149).
Change in Coreceptor Use Over Time
Paired samples from 24 women collected before ART and at virologic failure were tested for HIV coreceptor use to determine the association of ART exposure with emergence of CXCR4-using virus. The mean interval between entry and virologic failure was 28 months (range, 8-64 months). The majority of women (n = 17 [71%]) had R5 virus at both time points. In four women (17%) with R5 virus at entry, DM virus emerged after ART exposure. Seven women without a sample at study entry had R5 virus at virologic failure.
This study determined the prevalence and clinical correlates of CXCR4-using virus in a cohort of ART-naïve HIV-1 C-infected women in Botswana. Because previous studies suggested a much lower frequency of CXCR4-using virus in HIV-1C infection, we selected women with low CD4 cell counts for testing to increase the likelihood of identifying DM or X4 virus. Because some studies suggest that ART may select for DM or X4 virus, the prevalence of CXCR4-using virus was examined in ART-naïve women.
Nearly 15% of women with CD4 cell counts below 200 cells/mm3 harbored DM virus. By contrast, studies in subtype B-infected patients with similarly advanced HIV-1 disease show a prevalence of CXCR4-using virus of 50% or more.2,3,5,21,22 Previous studies among treatment-naïve patients infected with HIV-1C show a prevalence of DM virus of 0% to 30%. These studies were generally based on small numbers of patients in Africa with the largest having 40 patients.6-9,11,14,15,23-25 One study of 174 HIV-1C-infected patients in India showed a prevalence of 3.5% for CXCR4-using virus in treatment-naïve patients who had a wide range of CD4 cell counts.26 Because our study focused on patients with lower CD4 cell counts, the prevalence of DM and X4 virus among HIV-infected patients in Botswana overall most likely is considerably lower than 15%. The relatively low frequency of CXCR4-using virus even among persons with advanced disease suggests that CCR5 antagonists such as maraviroc may be particularly useful in the treatment of HIV-1C infection. These data also support evaluating a role for CCR5 antagonists as agents for the prevention of HIV-1 transmission in areas of the world where HIV-1 C predominates.
The association of HIV-1 coreceptor use and disease progression is well established in HIV-1 subtype B infection but less so for other subtypes.4,27-29 Because our study only examined HIV-1 coreceptor use among patients within a narrow range of CD4 cell counts, it was not possible to assess fully the association between CD4 cell count and presence of CXCR4-using virus. Nevertheless, even among patients with CD4 counts below 200 cells/mm3, there was a significant trend toward higher prevalence of CXCR4-using HIV-1 among patients with the lowest CD4 counts (P = 0.02).
In cross-sectional studies of subtype B-infected patients, CXCR4-using virus is detected in approximately 20% of treatment-naive patients but in up to 50% of treatment-experienced patients.27,29-32 This difference suggests to some that ART selects for CXCR4-using viruses independent of CD4 cell count. However, after adjusting for nadir CD4 cell count, the association of ART and CXCR4 is no longer statistically significant.33,34 In a cross-sectional study of 28 HIV-1C-infected patients, CXCR4-using virus was not detected in any ART-naïve patients but was found in 50% of ART-experienced patients. This finding led the authors to propose that ART exerts a greater effect on coreceptor use in HIV-1C infection as compared with subtype B.14 In the current study, the majority of women with R5 virus at the start of ART remained R5 at the time of virologic failure; DM virus emerged in only four (19%) women. This rate is consistent with observations from natural history studies, in which DM or X4 virus emerged in 23% of patients over time.22
This study has several limitations. As noted, the inclusion only of women with low CD4 cell counts may have limited the ability to detect significant associations between coreceptor use and other clinical factors. In addition, data were obtained from pregnant women only; gender and pregnancy, however, are not known to affect the emergence of CXCR4-using virus. The phenotypic assay used in the current study can detect minority CXCR4-using variants present at 1% to 5% of the population, depending on the plasma virus load. It is possible that a somewhat higher frequency of CXCR4-using virus would have been detected using the enhanced-sensitivity Trofile assay (Monogram BioSciences, South San Francisco, CA), which has a threshold of detection of 0.3% for CXCR4-using variants.35
In summary, DM virus was readily detected in HIV-1C-infected women with advanced disease, but its prevalence was considerably lower than that reported in subtype B- or D-infected patients with similarly advanced disease.36,37 Exposure to ART did not appear to be a major factor in emergence of CXCR4-using virus in the cohort studied here. Such knowledge is essential for an improved understanding of the biologic and clinical differences between HIV-1 subtypes and for the design of therapeutic agents and preventive vaccines targeted against the most prevalent HIV-1 subtype.
We thank all the participants and the study staff of the Mashi study for making this study possible. We thank Erik van Widenfelt and Jean Leidner for assistance in sample identification and data management; Keikantse Matlhagela for sharing the treatment failure samples; Roger Shapiro and the Harvard Initiative for Global Health (HIGH) for providing support for overseas work; Daniel Negusse for assistance in developing the tropism assay; and Zixin Hu, Noriaki Hosoya, Athe Tsibris, and Manish Sagar for valuable scientific insights.
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