Epidemiology and Social: CONCISE COMMUNICATION
Lack of effect of compartmentalized drug resistance mutations on HIV-1 pol divergence in antiretroviral-experienced women
Kelley, Colleen Fa; Sullivan, Sharon Tb; Lennox, Jeffrey La; Evans-Strickfaden, Tammyb; Hart, Clyde Eb
aEmory University Department of Medicine, Division of Infectious Diseases, USA
bLaboratory Branch, Division of HIV/AIDS Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
Received 8 December, 2009
Revised 22 February, 2010
Accepted 3 March, 2010
Correspondence to Colleen F. Kelley, Emory University, Division of Infectious Disease, 69 Jesse Hill Jr Dr SE, Atlanta, GA 30303, USA. E-mail: firstname.lastname@example.org
Objective: To examine the persistence of compartmentalized HIV drug resistance mutations (DRM) over time in the female genital tract and its effect on pol gene divergence compared to that in blood.
Design: Longitudinal cohort of 22 antiretroviral-experienced women in the Emory Vaginal Ecology study.
Methods: Blood and vaginal secretions were collected at serial clinic visits. DRM in the HIV reverse transcriptase and protease regions of pol were determined using population based sequencing. Kimura-2 pairwise DNA distances were calculated to measure blood and vaginal secretions divergence in the intervals between clinic visits.
Results: Only eight (36%) women had compartmentalized DRM detected at 14 (31%) of their 45 clinic visits. This compartmentalized resistance was transient; 13 of 14 mutations in blood and all 12 mutations in vaginal secretions were compartmentalized for only one clinic visit. Over time, divergence of both reverse transcriptase and protease were greater in vaginal secretions than in blood. However, divergence in blood, but not in vaginal secretions, increased significantly in the presence of drug resistance or compartmentalized drug resistance.
Conclusion: Compartmentalized DRM between the blood and vaginal secretions are transient in nature, and the presence of DRM does not affect pol gene divergence in the vaginal secretions. Our results provide new evidence that the genital mucosa does not support an independently evolving subpopulation of HIV-1 genomes.
In the United States, transmitted HIV drug resistance is estimated to occur in approximately 8–15% of infected individuals with prevalence varying by geography and risk factor [1–3]. Understanding the sexual transmission of drug-resistant virus is further complicated by reports of viral compartmentalization, or genetic differences in viral genomes detected at distinctive body sites, in the male and female genital tract as compared with the blood [4–12]. Previous studies have demonstrated cross-sectional compartmentalization of drug resistance mutations (DRM) between the blood and female genital tract but have shown variable results as to the actual prevalence [4–6,9]. However, data on the changes in DRM seen over time are restricted to a few reports in the literature with limited follow-up [4,5]. In addition, it is unknown if the presence of DRM affects viral divergence in the female genital tract differently than in blood. Therefore, the objective of this study was to determine changes in DRM over time in the blood and female vaginal secretions and compare the effect of DRM on HIV pol gene divergence.
During 1996–2000, blood and vaginal secretions were collected at 987 clinic visits of 135 HIV-infected women enrolled in the Emory Vaginal Ecology (EVE) Study. These women, aged 18–49 years, had a normal pap smear in the preceding 12 months and were expected to live at least 1 year. Full inclusion and exclusion criteria have been reported previously . A subset of 109 visits for 22 women enrolled in the EVE study, who were antiretroviral experienced with detectable plasma viral loads, were chosen for this analysis.
HIV-1 RNA protease and reverse transcriptase sequences in blood plasma and vaginal secretions
At each clinical examination, venous blood was collected in a CPT vacutainer tube (acid-citrate-dextrose anticoagulant) and vaginal secretions were collected with vaginal lavage by introducing 10 ml of PBS into the vagina and collecting the pooled fluid in the posterior vaginal fornix. Laboratory processing procedures to obtain blood and vaginal secretions supernatant samples used in this analysis and for quantifying cell-free HIV-1 RNA were described previously . The major species sequence of the HIV-1 protease (protease; nucleotides 1–297; entire amino acid sequence, 1–99) and reverse transcriptase (reverse transcriptase; nucleotides 120–741; amino acids 40–247) including DRM were determined in plasma and vaginal secretions supernatant RNA extracts using the TRUGENETM HIV-1 Genotyping Kit and OpenGeneTM DNA Sequencing System (Visible Genetics Inc., Toronto, Canada). Mutations were included in this analysis as DRM if they were determined to be nucleoside reverse transcriptase inhibitor (NRTI) or nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance mutations or major or minor protease inhibitor mutations by the Stanford University HIV Drug Resistance Database . Sequences identified as mixtures of wild type and drug-resistant viruses were reported as DRM.
HIV-1 RNA protease and reverse transcriptase gene divergence
In this analysis, genetic divergence over time in a woman's blood or vaginal secretions was assessed by comparing the pol sequence of the major species to that of the preceding clinic visit and will be referred to as the visit interval. The major species protease and reverse transcriptase sequences from each visit interval were aligned and Kimura-2 parameter pairwise DNA distances were determined using the Pileup and Distance programs included in the GCG Wisconsin package (Accelrys, Madison, Wisconsin, USA). Divergence in a visit interval was quantified and reported as DNA pairwise distance per 100 nucleotides. Statistical analyses were performed using the Instat 3.0 software package (GraphPad Software, Inc, San Diego, California, USA). A P value 0.05 or less was considered significant. Phylogenetic trees with 500 bootstrap replicates were constructed with the MEGA 2.1 program (Tempe, Arizona, USA) using the neighbor-joining method with Kimura-2 parameter matrices.
We analyzed the major species sequence of protease and reverse transcriptase in HIV-1 RNA in blood and vaginal secretions of 22 women who were antiretroviral-experienced. The median visit interval for all women was 3.1 months for blood and 5.4 months for the vaginal secretions (P = 0.2). For the visits with sequences from matched blood and vaginal secretions, the median blood viral load was 4.85 log10 copies/ml and the median vaginal secretions viral load was 4.46 log10 total copies/lavage for these 22 women. A neighbor-joining phylogenetic tree constructed from sequences analyzed showed that vaginal secretions and blood sequences of each woman clustered together without interpatient contamination (data not shown).
Compartmentalization of drug resistance mutations between blood and vaginal secretions is infrequent and transient over time
The assessment of compartmentalized DRM was restricted to 81 study visits of 22 antiretroviral-experienced women where both blood and vaginal secretions sequence data was available; a median of four clinic visits per woman (range 2–13 visits). Overall, only eight (36%) women had compartmentalized DRM detected in 14 (31%) of 45 clinic visits (Table 1). Another six women had DRM detected in blood, which matched those in vaginal secretions at each visit. For the eight women with compartmentalized DRM, all were NRTI experienced, four were NNRTI experienced, and seven were protease inhibitor experienced. The median CD4+ T-cell count was low, 147 cells/μl (range 3–412 cells/μl), which was typical for our patient population at the time (data not shown). Of the 26 compartmentalized DRM, 15 were NRTI, eight were NNRTI, and three were protease inhibitor mutations. There was no indication that compartmentalized DRM were detected more or less often in blood (nine visits) than vaginal secretions (seven visits). The compartmentalization of DRM detected in blood and vaginal secretions of these women was transient; 13 of 14 mutations in blood and all 12 mutations in vaginal secretions were compartmentalized at only one clinic visit. In addition, of the 17 intervals between visits in this study where a change in DRM occurred, only seven resulted in the development of compartmentalized DRM between blood and vaginal secretions.
A higher level of protease and reverse transcriptase divergence in vaginal secretions compared with blood was independent of drug resistance mutations
There was an overall greater nucleotide divergence in protease and reverse transcriptase of women's vaginal secretions compared with blood. The median DNA pairwise distances between clinic visits were significantly higher in vaginal secretions versus blood for all women tested and for those subgroups either with or without DRM or compartmentalized DRM (P < 0.01, Table 2). These higher divergence values in vaginal secretions were not significantly different between women with or without DRM or compartmentalized DRM (P ≥ 0.6, Table 2). In contrast, blood viral divergence was greater for women who had either DRM or compartmentalized DRM detected in blood (P < 0.05). There was no significant difference between the time intervals for collecting blood and vaginal secretions samples used for this pairwise analysis (Table 2).
Our results provide evidence that compartmentalized DRM can be infrequent and transient over time in antiretroviral-experienced women. There was no effect of reverse transcriptase vs. protease mutations on this transience suggesting that the class of drug does not alter this relationship. In addition, sequence analysis revealed an apparent greater divergence of the HIV pol gene over time in vaginal secretions than in blood. Thus, viral subpopulations in the genital tract are reflective of those in the blood over time, which supports the concept of an ongoing migration of infected cell populations from the blood to the genital tract [15–17]. This lack of independent evolution also supports the continuous exchange of viral genomes from the blood to the female genital tract and gives the appearance of greater genetic divergence in the genital tract.
In contrast to our longitudinal analysis, there are previous cross-sectional analyses that have reported more and less extensive occurrences of compartmentalized DRM between the blood and genital tract of infected women [4,6,9,11,18]. The disparate outcomes between these studies may, in part, reflect the differences in techniques used for sampling genital virus or the types of mutations used to define compartmentalization, differences in drug regimens used by participants, and their cross-sectional nature. Across studies, the highest levels of compartmentalization reported in previous studies used swabs to collect genital virus , whereas those with some of the lowest levels of compartmentalization used cervical-vaginal lavages [6,9]. Recent observations show that different HIV-1 quasispecies are more likely found at separate locations within the genital mucosa of an infected woman , suggesting that procedures which sample a large area of the mucosa, such as cervical-vaginal lavage, may produce a more accurate representation of the genital tract quasispecies.
The similarities we observed over time in DRM between virus shed in vaginal secretions and blood occurred while the major species of pol RNA shed in vaginal secretions appeared to be diverging at a faster rate. The apparent higher divergence in vaginal secretions we observed is likely a reflection of the lack of independent proviral evolution within genital tissues of women reported previously . Although there is sufficient evidence that local cellular virus replication in the female genital tract accounts for most HIV-1 shed in vaginal secretions , the majority of those infected genital tract cells replicating virus are believed to have migrated from blood and to have undergone clonal expansion in genital tissues [15–17]. Thus, an ongoing influx and turnover of a subpopulation of virus-infected cells from blood would result in the appearance of a higher level of viral RNA divergence in vaginal secretions between clinic visits. In addition, the increase of pol divergence over time in blood, but not in vaginal secretions, in the presence of DRM or compartmentalized DRM also supports the concept that a temporally linked evolution of HIV-1 is occurring in blood but not in vaginal secretions.
Our data are limited in that more contemporary samples would be expected to show different patterns of DRM. However, the mutations we report are reflective of those most commonly seen in studies of transmitted drug resistance [1–3]. We also did not measure drug concentrations in vaginal secretions or blood. Though previous data do report differences in drug penetration to the female genital tract , the effect on compartmentalized DRM has not been shown . Given the use of bulk sequencing employed in this analysis, which generally detects variants comprising more than 20% of the viral subpopulation, we are unable to make conclusions about the presence of minority DRM, which may be present [6,21]. Nonetheless, our longitudinal data suggest that more sensitive sequencing methodologies might show even less evidence of DRM compartmentalization and a more stochastic relationship between the blood and female genital tract. Finally, in general, the women in this study had high levels of virus shed in the vaginal secretions making inadequate PCR input copies an unlikely cause of the greater divergence we saw.
In summary, we have demonstrated the infrequent and transient nature of compartmentalized DRM over time in the female genital tract. Our results indicate that the majority of HIV-1 transmitted drug resistance, which occurs from exposure to a woman's genital secretions could be predicted from DRMs in her blood. Taken together, the pol divergence and DRM compartmentalization results reported in this study provide new evidence that the genital mucosa does not support an independently evolving subpopulation of HIV-1 genomes.
C.F.K. completed the analysis and writing of the manuscript. S.T.T. began the analysis and writing of the manuscript. J.L.L. developed the study and contributed to the writing of the manuscript. T.E.S. did the blood and vaginal secretion HIV viral loads. C.E.H. developed the study, supervised collection of the virus load and sequence data, and contributed to the analysis and writing of the manuscript.
The findings and conclusions of this manuscript are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.
1. Novak RM, Chen L, MacArthur RD, Baxter JD, Huppler Hullsiek K, Peng G, et al
. Prevalence of antiretroviral drug resistance mutations in chronically HIV-infected, treatment-naive patients: implications for routine resistance screening before initiation of antiretroviral therapy. Clin Infect Dis 2005; 40:468–474.
2. Weinstock HS, Zaidi I, Heneine W, Bennett D, Garcia-Lerma JG, Douglas JM Jr, et al
. The epidemiology of antiretroviral drug resistance among drug-naive HIV-1-infected persons in 10 US cities. J Infect Dis 2004; 189:2174–2180.
3. Hurt CB, McCoy SI, Kuruc J, Nelson JA, Kerkau M, Fiscus S, et al
. Transmitted antiretroviral drug resistance among acute and recent HIV infections in North Carolina from 1998 to 2007. Antivir Ther 2009; 14:673–678.
4. De Pasquale MP, Leigh Brown AJ, Uvin SC, Allega-Ingersoll J, Caliendo AM, Sutton L, et al
. Differences in HIV-1 pol sequences from female genital tract and blood during antiretroviral therapy. J Acquir Immune Defic Syndr 2003; 34:37–44.
5. Ellerbrock TV, Lennox JL, Clancy KA, Schinazi RF, Wright TC, Pratt-Palmore M, et al
. Cellular replication of human immunodeficiency virus type 1 occurs in vaginal secretions. J Infect Dis 2001; 184:28–36.
6. Kemal KS, Burger H, Mayers D, Anastos K, Foley B, Kitchen C, et al
. HIV-1 drug resistance in variants from the female genital tract and plasma. J Infect Dis 2007; 195:535–545.
7. Kemal KS, Foley B, Burger H, Anastos K, Minkoff H, Kitchen C, et al
. HIV-1 in genital tract and plasma of women: compartmentalization of viral sequences, coreceptor usage, and glycosylation. Proc Natl Acad Sci U S A 2003; 100:12972–12977.
8. Philpott S, Burger H, Tsoukas C, Foley B, Anastos K, Kitchen C, Weiser B. Human immunodeficiency virus type 1 genomic RNA sequences in the female genital tract and blood: compartmentalization and intrapatient recombination. J Virol 2005; 79:353–363.
9. Si-Mohamed A, Kazatchkine MD, Heard I, Goujon C, Prazuck T, Aymard G, et al
. Selection of drug-resistant variants in the female genital tract of human immunodeficiency virus type 1-infected women receiving antiretroviral therapy. J Infect Dis 2000; 182:112–122.
10. Sullivan ST, Mandava U, Evans-Strickfaden T, Lennox JL, Ellerbrock TV, Hart CE. Diversity, divergence, and evolution of cell-free human immunodeficiency virus type 1 in vaginal secretions and blood of chronically infected women: associations with immune status. J Virol 2005; 79:9799–9809.
11. Tirado G, Jove G, Kumar R, Noel RJ, Reyes E, Sepulveda G, et al
. Differential virus evolution in blood and genital tract of HIV-infected females: evidence for the involvement of drug and non-drug resistance-associated mutations. Virology 2004; 324:577–586.
12. Diem K, Nickle DC, Motoshige A, Fox A, Ross S, Mullins JI, et al
. Male genital tract compartmentalization of human immunodeficiency virus type 1 (HIV). AIDS Res Hum Retroviruses 2008; 24:561–571.
13. Hart CE, Lennox JL, Pratt-Palmore M, Wright TC, Schinazi RF, Evans-Strickfaden T, et al
. Correlation of human immunodeficiency virus type 1 RNA levels in blood and the female genital tract. J Infect Dis 1999; 179:871–882.
14. Rhee SY, Gonzales MJ, Kantor R, Betts BJ, Ravela J, Shafer RW. Human immunodeficiency virus reverse transcriptase and protease sequence database. Nucleic Acids Res 2003; 31:298–303.
15. Bull ME, Learn GH, McElhone S, Hitti J, Lockhart D, Holte S, et al
. Monotypic human immunodeficiency virus type 1 genotypes across the uterine cervix and in blood suggest proliferation of cells with provirus. J Virol 2009; 83:6020–6028.
16. Poss M, Rodrigo AG, Gosink JJ, Learn GH, de Vange Panteleeff D, Martin HL Jr, et al
. Evolution of envelope sequences from the genital tract and peripheral blood of women infected with clade A human immunodeficiency virus type 1. J Virol 1998; 72:8240–8251.
17. Bull M, Learn G, Genowati I, McKernan J, Hitti J, Lockhart D, et al
. Compartmentalization of HIV-1 within the female genital tract is due to monotypic and low-diversity variants not distinct viral populations. PLoS One 2009; 4:e7122.
18. Frenkel LM, McKernan J, Dinh PV, Goldman D, Hitti J, Watts DH, et al
. HIV type 1 zidovudine (ZDV) resistance in blood and uterine cervical secretions of pregnant women. AIDS Res Hum Retroviruses 2006; 22:870–873.
19. Min SS, Corbett AH, Rezk N, Cu-Uvin S, Fiscus SA, Petch L, et al
. Protease inhibitor and nonnucleoside reverse transcriptase inhibitor concentrations in the genital tract of HIV-1-infected women. J Acquir Immune Defic Syndr 2004; 37:1577–1580.
20. Kwara A, Delong A, Rezk N, Hogan J, Burtwell H, Chapman S, et al
. Antiretroviral drug concentrations and HIV RNA in the genital tract of HIV-infected women receiving long-term highly active antiretroviral therapy. Clin Infect Dis 2008; 46:719–725.
21. Johnson JA, Li JF, Wei X, Lipscomb J, Irlbeck D, Craig C, et al
. Minority HIV-1 drug resistance mutations are present in antiretroviral treatment-naive populations and associate with reduced treatment efficacy. PLoS Med 2008; 5:e158.
drug resistance; evolution; female genital organs; HIV; transmission of infectious diseases
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