HIV-1 uses cysteine–cysteine receptor 5 (R5) and/or cysteine–X-cysteine receptor 4 (X4) for cell entry. Individuals with X4-utilizing strains have a more rapid clinical and immunological deterioration compared with those with R5-tropic virus [1–4], although the underlying mechanism remains uncertain.
The level of T-cell immune activation is established early in the course of HIV-1 infection and is independently predictive of HIV-1 progression [5–8]. Cell surface molecules CD38 and HLA-DR are upregulated on activated T cells. Estimation of T-cell activation can be undertaken by measuring the percentage of CD4 and CD8 T cells expressing either one or both markers, or median density of each activation marker per cell (median fluorescent intensity, MFI). We compared T-cell activation in individuals infected with R5 compared with X4 viruses sampled during primary HIV-1 infection (PHI), to assess whether this may contribute to the faster immunological decline observed in those with X4 variants .
One hundred twenty of 149 participants enrolled from UK clinical sites to the SPARTAC (Short Pulse Anti Retroviral Therapy at HIV Seroconversion) trial, a randomized controlled trial comparing short-course antiretroviral therapy (ART) versus no therapy in PHI , had available samples for T-cell activation and tropism testing. Participants were ART-naive at the time of analysis and estimated to be within 6 months of HIV-1 seroconversion .
Viral tropism was inferred using genotypic analysis of the HIV-1 gp120 V3 loop region from stored plasma samples. Edited sequences were submitted to the Geno2pheno (coreceptor) predictive algorithm . The false-positive rate was set at 5.75% in accordance with published clinical outcome data [11,12].
The following T-cell activation markers were assessed on CD8 and CD4 T cells: CD38 expression (% and MFI), HLA-DR expression (% and MFI) and dual CD38 and HLA-DR expression (%). Cryopreserved peripheral blood mononuclear cells were thawed and incubated with CD3-pacific orange, CD4-Alexa 700, CD8-PE-Cy5 (all BD Biosciences, San Diego, California, USA), HLA-DR–PE-Cy7 (eBiosciences, San Diego California, USA) and CD38-Qdot 605 (Invitrogen, Grand Isle, New York, USA) and live/dead stain (Pacific Blue; Invitrogen). Fluorescent antibody staining was evaluated using an LSR-II flow cytometer (BD Sciences). Data were analysed using FloJo software, version 8.8.6 (TreeStar Inc., Ashland, Oregon, USA).
Median levels of T-cell activation markers were compared between individuals with R5 and X4-using viruses using Mann–Whitney tests. If a marker was significantly associated with tropism (P < 0.05), its relationship with baseline variables (viral load, CD4 T-cell count, age, sex and estimated time since seroconversion) was examined using linear regression. Variables were included in multivariate models if the P value was less than 0.1 in univariate models, and backwards selection was used to assess an independent association. Marker values were log10 transformed to normalize data distribution.
Ten of 120 (8.3%) participants had X4-using virus with no significant difference in baseline characteristics between those infected with X4-tropic and R5-tropic variants. The majority (97%) were men and infected with subtype B virus (88%). Overall median age was 34.5 years [interquartile range (IQR) 28.9–41.5]. Estimated time from seroconversion was 73 days (IQR 53–95). Six (5%) participants, all infected with R5-tropic virus, had transmitted drug resistance (Stanford 4–5).
Median baseline viral load and CD4 T-cell count was similar at 4.8 and 4.5 log copies/ml, and 550 and 520 cells/μl, in participants with R5 and X4 virus, respectively.
Median levels of CD4 T-cell activation, measured by dual CD38 HLA-DR expression and HLA-DR expression, were significantly higher in participants with X4 versus R5 virus (CD38 HLA-DR dual expression: 9.3% (IQR 6.5–15.5) vs. 4.9% (IQR 3.3–8.8), P = 0.01; HLA-DR% expression: 19.6% (IQR 11.6–27.8) vs. 10.6% (IQR 6.7–14.1), P = 0.01; HLA-DR MFI: 76.9 (IQR 55.9–100) vs. 49.5 (IQR 35.9–59.7), P = 0.005). There was weak evidence that CD4 CD38 expression was also higher in those with X4 virus [CD4 CD38%: 57.2% (IQR 51.2–73.1) vs. 50.1% (IQR 37.0–65.1), P = 0.1; CD38 MFI 591.5 (474–919) vs. 451.5 (IQR 310–579), P = 0.06]. No significant differences in CD8 T-cell activation were observed, although there was a trend towards higher CD8 HLA-DR expression in those with X4 variants [HLA-DR%: 41.2% (IQR 26.5–73.4) vs. 30.9% (IQR 18.9–43.0), P = 0.06; HLA-DR MFI: 262.5 (IQR 153–885) vs. 178.5 (IQR 108–303), P = 0.07].
Table 1 shows factors associated with CD4 HLA-DR% expression and CD4 CD38 HLA-DR dual expression on linear regression analysis. Results for CD4 HLA-DR MFI were similar to those of CD4 HLA-DR% (not shown). There was strong evidence to suggest that X4 tropism was associated with higher levels of both activation markers after adjusting for other variables. T-cell activation was also associated with HIV-1 RNA levels.
We demonstrate increased levels of CD4 T-cell activation in individuals with X4-tropic compared with R5-tropic HIV variants in PHI. This may reflect underlying pathogenic processes and account for the increased rate of immunological decline associated with X4 tropism.
We found no association between tropism and viral load and CD4 T-cell count, in keeping with another study reporting no difference in these parameters in PHI despite faster subsequent disease progression in individuals with X4 virus . We did not assess disease progression because a significant proportion of participants were randomized to start ART at baseline as part of the SPARTAC trial protocol.
Guidelines endorsing genotypic tropism testing have been developed by national and European consensus panels [13,14] and data in PHI show high concordance with phenotypic tropism results . We found an 8.3% prevalence of X4 variants measured using the geno2pheno algorithm (FRP 5.75%), comparable to previous studies .
CCR5 is expressed on 15–30% of CD4 T-cells, whereas CXCR4 is expressed on nearly all CD4 T-cells including naïve cells . Our study supports data showing higher levels of CD4 T cells positive for Ki67, a marker of T-cell division, in individuals with emergence of X4 variants . Faster depletion of the naive CD4 T-cell pool through increased activation, proliferation and apoptosis may explain the faster disease progression associated with X4 viruses . It possible that X4 viruses, directly or indirectly, predispose to higher levels of CD4 T-cell activation or that, conversely, individuals with high levels of immune activation are more susceptible to infection with X4-tropic variants, or more likely to switch from R5-tropic to X4-tropic virus following HIV-1 acquisition. This is the first time an association has been shown between viral tropism and specific markers of CD4 T-cell activation in PHI.
Author contributions: E.H., K.P. and S.F. designed the study. E.H. and S.H. performed flow cytometry; S.K., N.M. and M.R. carried out tropism testing and interpretation; K.P. and E.H. performed the statistical analysis; E.H. wrote the first draft of the paper. All authors contributed to subsequent drafts and approved the final version.
We are grateful to the NIHR BRC for their support. We would like to thank the participants of the SPARTAC trial.
SPARTAC investigators: Trial Steering Committee (Independent Members): A. Breckenridge (Chair), P. Clayden, C. Conlon, F. Conradie, J. Kaldor*, F. Maggiolo, F. Ssali. (Country Principal Investigators): D.A. Cooper, P. Kaleebu, G. Ramjee, M. Schechter, G. Tamussi, J. Weber. Trial Physician: S. Fidler. Trial Statistician: A. Babiker. Data and Safety Monitoring Committee (DSMC): T. Peto (Chair), A. McLaren (in memoriam), V. Beral, G. Chene, J. Hakim. Co-ordinating Trial Centre: MRC Clinical Trials Unit, London (A. Babiker, K. Porter, M. Thomason, F. Ewings, M. Gabriel, D. Johnson, K. Thompson, A. Cursley*, K. Donegan*, E Fossey*, P. Kelleher*, K. Lee*, B. Murphy*, D. Nock*). Central Immunology Laboratories and Repositories: The Peter Medawar Building for Pathogen Research, University of Oxford, UK (R. Phillips, J. Frater, L. Ohm Laursen*, N. Robinson, P. Goulder, H. Brown). Central Virology Laboratories and Repositories: Jefferiss Trust Laboratories, Imperial College, London, UK (M. McClure, D. Bonsall*, O. Erlwein*, A. Helander*, S. Kaye, M. Robinson, L. Cook*, G. Adcock*, P. Ahmed*). Clinical Endpoint Review Committee: N. Paton, S. Fidler. Investigators and Staff at Participating Sites: Australia: St Vincent's Hospital, Sydney (A. Kelleher), Northside Clinic, Melbourne (R. Moore), East Sydney Doctors, Sydney (R. McFarlane), Prahran Market Clinic, Melbourne (N. Roth), Taylor Square Private Clinic, Sydney (R. Finlayson), The Centre Clinic, Melbourne (B. Kiem Tee), Sexual Health Centre, Melbourne (T. Read), AIDS Medical Unit, Brisbane (M. Kelly), Burwood Rd Practice, Sydney (N. Doong) Holdsworth House Medical Practice, Sydney (M. Bloch) Aids Research Initiative, Sydney (C. Workman). Coordinating centre in Australia: Kirby Institute University of New South Wales, Sydney (P. Grey, D.A. Cooper, A. Kelleher, M. Law). Brazil: Projeto Praça Onze, Hospital Escola São Francisco de Assis, Universidade federal do Rio de Janeiro, Rio de Janeiro (M. Schechter, P. Gama, M. Mercon*, M. Barbosa de Souza, C. Beppu Yoshida, J.R. Grangeiro da Silva, A. Sampaio Amaral, D. Fernandes de Aguiar, M de Fátima Melo, R. Quaresma Garrido). Italy: Ospedale San Raffaele, Milan (G. Tambussi, S. Nozza, M. Pogliaghi, S. Chiappetta, L. Della Torre, E. Gasparotto), Ospedale Lazzaro Spallanzani, Roma (G. D’Offizi, C. Vlassi, A. Corpolongo). South Africa: Cape Town: Desmond Tutu HIV Centre, Institute of Infectious Diseases, Cape Town (R. Wood, J. Pitt, C. Orrell, F. Cilliers, R. Croxford, K. Middelkoop, L.G. Bekker, C. Heiberg, J. Aploon, N. Killa, E. Fielder, T. Buhler). Johannesburg: The Wits Reproductive Health and HIV Institute, University of Witswatersrand, Hillbrow Health Precinct, Johannesburg (H. Rees, F. Venter, T. Palanee), Contract Laboratory Services, Johannesburg Hospital, Johannesburg (W. Stevens, C. Ingram, M. Majam, M. Papathanasopoulos). Kwazulu-Natal: HIV Prevention Unit, Medical Research Council, Durban (G. Ramjee, S. Gappoo, J. Moodley, A. Premrajh, L. Zako). Uganda: MRC/Uganda Virus Research Institute, Entebbe (H. Grosskurth, A. Kamali, P. Kaleebu, U. Bahemuka, J. Mugisha*, H.F. Njaj*). Spain: Hospital Clinic-IDIBAPS, University of Barcelona (J.M. Miro, M. López-Dieguez*, C. Manzardo, J.A. Arnaiz, T. Pumarola, M. Plana, M. Tuset, M.C. Ligero, M.T. García, T. Gallart, J.M. Gatell). UK and Ireland: Royal Sussex County Hospital, Brighton (M. Fisher, K. Hobbs, N. Perry, D. Pao, D. Maitland, L. Heald), St James's Hospital, Dublin (F. Mulcahy, G. Courtney, S. O’Dea, D. Reidy), Regional Infectious Diseases Unit, Western General Hospital and Genitourinary Dept, Royal Infirmary of Edinburgh, Edinburgh (C. Leen, G. Scott, L. Ellis, S. Morris, P. Simmonds), Chelsea and Westminster Hospital, London (B. Gazzard, D. Hawkins, C. Higgs), Homerton Hospital, London (J. Anderson, S. Mguni), Mortimer Market Centre, London (I. Williams, N. De Esteban, P. Pellegrino, A. Arenas-Pinto, D. Cornforth*, J. Turner*), North Middlesex Hospital (J. Ainsworth, A. Waters), Royal Free Hospital, London (M. Johnson, S. Kinloch, A. Carroll, P. Byrne, Z. Cuthbertson), Barts & the London NHS Trust, London (C. Orkin, J. Hand, C. De Souza), St Mary's Hospital, London (J. Weber, S. Fidler, E. Hamlyn, E. Thomson*, J. Fox*, K. Legg, S. Mullaney*, A. Winston, S. Wilson, P. Ambrose), Birmingham Heartlands Hospital, Birmingham (S. Taylor, G. Gilleran). Imperial College Trial Secretariat: S. Keeling, A. Becker. Imperial College DSMC Secretariat: C. Boocock. Asterisk (*) shows people who left the study team before the trial ended.
Conflicts of interest
The SPARTAC trial was funded by Wellcome Trust grants WT069598MA and 069598/Z/02/B.
There are no conflicts of interest.
1. Schuitemaker H, Koot M, Kootstra NA, Dercksen MW, de Goede RE, van Steenwijk RP, et al. Biological phenotype of human immunodeficiency virus type 1 clones at different stages of infection: progression of disease is associated with a shift from monocytotropic to T-cell-tropic virus population. J Virol 1992; 66:1354–1360.
2. Tersmette M, Lange JM, de Goede RE, de Wolf F, Eeftink-Schattenkerk JK, Schellekens PT, et al. Association between biological properties of human immunodeficiency virus variants and risk for AIDS and AIDS mortality. Lancet 1989; 1:983–985.
3. Koot M, Keet IP, Vos AH, de Goede RE, Roos MT, Coutinho RA, et al. Prognostic value of HIV-1 syncytium-inducing phenotype for rate of CD4+ cell depletion and progression to AIDS. Ann Intern Med 1993; 118:681–688.
4. Raymond S, Delobel P, Mavigner M, Cazabat M, Encinas S, Souyris C, et al. CXCR4-using viruses in plasma and peripheral blood mononuclear cells during primary HIV-1 infection and impact on disease progression. AIDS 2010; 24:2305–2312.
5. Liu Z, Cumberland WG, Hultin LE, Prince HE, Detels R, Giorgi JV. Elevated CD38 antigen expression on CD8+ T cells is a stronger marker for the risk of chronic HIV disease progression to AIDS and death in the Multicenter AIDS Cohort Study than CD4+ cell count, soluble immune activation markers, or combinations of HLA-DR and CD38 expression. J Acquir Immune Defic Syndr Hum Retrovirol 1997; 16:83–92.
6. Hazenberg MD, Otto SA, van Benthem BH, Roos MT, Coutinho RA, Lange JM, et al. Persistent immune activation in HIV-1 infection is associated with progression to AIDS. AIDS 2003; 17:1881–1888.
7. Deeks SG, Kitchen CM, Liu L, Guo H, Gascon R, Narvaez AB, et al. Immune activation set point during early HIV infection predicts subsequent CD4+ T-cell changes independent of viral load. Blood 2004; 104:942–947.
8. Bofill M, Mocroft A, Lipman M, Medina E, Borthwick NJ, Sabin CA, et al. Increased numbers of primed activated CD8+CD38+CD45RO+ T cells predict the decline of CD4+ T cells in HIV-1-infected patients. AIDS 1996; 10:827–834.
9. Fidler S. The effect of short-course ART in Primary HIV Infection: final results from an international randomised controlled trial; SPARTAC. In: Proceedings of the 6th IAS Conference on HIV Pathogenesis, Treatment and Prevention; July 2011; Rome.
11. McGovern RA, Thielen A, Mo T, Dong W, Woods CK, Chapman D, et al. Population-based V3 genotypic tropism assay: a retrospective analysis using screening samples from the A4001029 and MOTIVATE studies. AIDS 2010; 24:2517–2525.
12. McGovern R, Dong W, Zhong X, Knapp D, Thielen A, Chapman D, et al.Population-based sequencing of the V3-loop is comparable to the enhanced sensitivity trofile assay in predicting virologic response to maraviroc of treatment-naïve patients in the MERIT trial. In: Proceedings of the 17th Conference on Retroviruses and Opportunistic Infections; 2010; San Francisco; abstract 92.
13. Vandekerckhove LPR, Wensing AMJ, Kaiser R, Brun-Vezinet F, Clotet B, De Luca A, Dressler S, et al. Consensus statement of the European guidelines on clinical management of HIV-1 tropism testing. J Int AIDS Soc 2010; 13 (Suppl 4):O7.
14. Geretti AM, Mackie NE. BHIVA guidelines on determining HIV-1 tropism in routine clinical practice. 2009. http://www.bhiva.org/Tropism.aspx
. [Accessed 17 March 2011]
15. Jekle A, Keppler O, De Clercq E, Schols D, Weinstein M, Goldsmith M. In vivo evolution of human immunodeficiency virus type 1 toward increased pathogenicity through CXCR4-mediated killing of uninfected CD4 T cells. J Virol 2003; 77:5846–5855.
16. Hazenberg MD, Otto SA, Hamann D, Roos MT, Schuitemaker H, de Boer RJ, et al. Depletion of naive CD4 T cells by CXCR4-using HIV-1 variants occurs mainly through increased T-cell death and activation. AIDS 2003; 17:1419–1424.
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