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Macrophage Activation in HIV-2–Infected Patients Is Less Affected by Antiretroviral Treatment—sCD163 in HIV-1, HIV-2, and HIV-1/2 Dually Infected Patients Bo L. Hønge, MD,*†‡ Morten N. Andersen, MD,§k Sanne Jespersen, MD, PhD,†‡ Candida Medina, MD,¶ Faustino G. Correira, MD,¶ Martin R. Jakobsen, PhD,k Alex Laursen, MD, PhD, DMSc,‡ Christian Erikstrup, MD, PhD,* Holger J. Møller, MD, PhD, DMSc,§ and Christian Wejse, MD, PhD,†‡# for the Bissau HIV cohort study group

Abstract: The course of disease among HIV-2, HIV-1, and HIV-1/2 dually infected patients is different. We investigated the macrophage activation marker soluble CD163 (sCD163) dynamics in 212 HIV-1, HIV-2, and HIV-1/2 dually infected patients. There were no differences in sCD163 levels at baseline or during follow-up without antiretroviral therapy (ART). At follow-up on ART, median sCD163 levels were decreased for HIV-1–infected patients (P , 0.001), but not among HIV-2 (P = 0.093) or HIV-1/2 dually infected patients (P = 0.145). The larger decrease in sCD163 levels among HIV-1–infected patients during ART may indicate an HIV type–dependent differential effect of ART on macrophage activation during HIV infection. Key Words: HIV, HIV-1, HIV-2, AIDS, CRP, sCD163, GuineaBissau, Africa (J Acquir Immune Defic Syndr 2016;72:254–258)

INTRODUCTION Human immunodeficiency virus type 2 (HIV-2) is endemic in West Africa. Compared with HIV-1, HIV-2 is associated with a lower viral load and with a slower rate of Received for publication August 31, 2015; accepted January 19, 2016. From the *Department of Clinical Immunology, Aarhus University Hospital, Denmark; †Bandim Health Project, Indepth Network, Bissau, Guinea-Bissau; Departments of ‡Infectious Diseases; §Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark; kDepartment of Biomedicine, Aarhus University, Denmark; ¶National HIV Programme, Ministry of Health, Bissau, Guinea-Bissau; and #GloHAU, Center for Global Health, School of Public Health, Aarhus University, Aarhus, Denmark. The Global Fund to Fight AIDS, TB, and Malaria supported data collection through the Secretariado Nacional de Luta contra Sida in Guinea-Bissau during 2009–2010. The Bissau HIV Cohort was supported by the International Epidemiologic Databases to Evaluate AIDS (IeDEA) network and the West African Platform for HIV Intervention Research (WAPHIR). The National Cancer Institute (NCI), the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), and the National Institute of Allergy and Infectious Diseases (NIAID) of the US National Institutes of Health (NIH), as part of the International Epidemiologic Databases to Evaluate AIDS (IeDEA) under Award Number U01AI069919. The authors have no conflicts of interest to disclose. B.L.H. and M.N.A. contributed equally to the writing of this article. Correspondence to: Bo L. Hønge, MD, Department of Clinical Immunology, Aarhus University Hospital, Palle Juul-Jensens Boulevard 99, 8200 Aarhus N, Denmark (e-mail: [email protected]). Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.

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both CD4 cell decline and clinical progression.1,2 The West African country Guinea-Bissau has experienced a rise in HIV1 prevalence and, at the same time, holds the world’s highest prevalence of HIV-2–infected individuals.3 The hemoglobin scavenger receptor CD163 is expressed almost exclusively on monocytes and macrophages.4 Extracellular toll-like receptor activation leads to shedding of membrane-associated CD163, which becomes soluble CD163 (sCD163).5,6 Macrophages are involved in HIV-1 disease progression, and macrophage activation is affected by HIV-1.7 In a recent study of HIV-1–infected patients, sCD163 levels were higher among patients with low CD4 cell count.8 Another study found that sCD163 levels decreased in parallel with HIV-1 RNA levels during antiretroviral therapy (ART), and discontinuation of treatment caused both sCD163 and HIV-1 RNA levels to increase rapidly.9 Among tuberculosis and HIV-coinfected patients from Guinea-Bissau, higher levels of sCD163 levels were associated with elevated risk of mortality, and the association was stronger among HIV-1– infected patients than among HIV-2–infected patients.10 However, blood levels of sCD163 in HIV-2–infected patients, or the potential clinical utility of sCD163 in HIV disease management, are not established.9 Because macrophages may play a role in the progression of HIV disease and response to ART, we aimed to investigate the dynamics of macrophage activation marker sCD163 in HIV-1, HIV-2, and HIV-1/2 dually infected patients with and without ART.

METHODS Setting and Patients Patients from the Bissau HIV Cohort at Hospital Nacional Simão Mendes, Bissau, Guinea-Bissau were included in this study. Patient care was based on the HIV guidelines in Guinea-Bissau and the cohort has previously been described.11,12 In this follow-up study, we included patients from whom 2 blood samples had been collected, as either (1) 1 sample at HIV diagnosis and 1 sample after 6–24 months of follow-up without ART or (2) 1 sample at HIV diagnosis and 1 sample after 6–24 months of ART. Patients

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with samples taken between July 27, 2007, and June 18, 2012, were included in the study.

HIV Testing HIV diagnosis was made at the HIV clinic in Bissau by screening for HIV antibodies using the rapid test Determine HIV-1/2 (Abbott Laboratories, Abbott Park, IL). HIV antibody confirmation and initial HIV type discrimination were performed using SD Bioline HIV 1/2 3.0 (Standard Diagnostics Inc., Kyonggi-Do, South Korea).13 We have shown that the initial HIV type determination may be inaccurate14; hence, all patients had another HIV type determination performed at Department of Clinical Immunology, Aarhus, Denmark, using ImmunoComb HIV 12 Bispot (Orgenics, Yavne, Israel).15 In 7 cases, the ImmunoComb II HIV 1&2 Bispot test result was inconclusive, and HIV type was determined by INNO-LIA HIV I/II Score (Fujirebio, Gent, Belgium).

Soluble CD163 in HIV-1/HIV-2–Infected Patients

association between biomarkers at baseline and follow-up was assessed using paired t tests. Linear regression analysis was used when adjusting for confounders. For comparing nonnormally distributed continuous variables between 2 groups, Wilcoxon rank-sum test was used. All statistical analyses were performed using Stata IC 13.0 (StataCorp, Texas). Figure 1 was made using Prism 5.0 (GraphPad Software, CA).

Ethics The Bissau HIV Cohort has been approved by the National Ethics Committee in Guinea-Bissau (Parecer NCP/

Laboratory Methods Peripheral blood CD4 cell counts were measured at the National Public Health Laboratory in Bissau by Partec Cyflow SL3 (Partec, Munster, Germany). C-reactive protein (CRP) concentrations were measured in plasma at the Department of Clinical Biochemistry, Aarhus University Hospital, Denmark, according to standard clinical method (Roche Cobas 6000, Roche Diagnostics GmbH, Mannheim, Germany). Plasma sCD163 concentrations were measured in all samples, by an in-house enzyme-linked immunosorbent assay as described in detail previously,16 with the exception that in this study, samples were analyzed using an automated system (BEP 2000; Siemens Healthcare Diagnostics, Munich, Germany). Briefly, microtiter wells (Maxisorp, Nunc; Thermo Scientific, Roskilde, Denmark) were coated with 2.0-mg/L polyclonal rabbit anti-human CD163 (by courtesy of Professor Søren Moestrup, Aarhus University). After washing, 100 mL of patient plasma sample diluted 1:101 in phosphate-buffered saline/albumin buffer was incubated in the wells for 1.5 hours. Wells were washed and then incubated with detection antibody for 1 hour (1.0 mg/L, clone GHI/61, BD Biosciences Pharmingen, Erembodegem, Belgium). Wells were washed again, and 0.125 mg/L of peroxidase-conjugated polyclonal goat anti-mouse immunoglobulin antibodies was added (P0447; Dako, Glostrup, Denmark). After 1-hour incubation, and wash, 100 mL of TMB ONE substrate containing H2O2 (Kem-En-Tec, Taastrup, Denmark) was added, and the reaction was stopped after 3 minutes using 1 M of H3PO4, and the plates were read at 450/620 nm. All incubation steps were performed at 37°C. The interassay variation in this study was ,6% coefficient of variation (15 runs).

Statistical Methods

Categorical variables were compared using x2 test. Continuous variables were compared among groups based on HIV type using analysis of variance. Biomarker levels of sCD163 and CRP were compared among groups using analysis of variance on logarithmically transformed values, and

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FIGURE 1. A, Changes in sCD163 levels for patients on followup without ART. B, Changes in sCD163 levels for patients on follow-up with ART. www.jaids.com |

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Hønge et al

No.15/2007). On inclusion, patients provided a voluntary, signed, and dated informed consent or fingerprint if illiterate.

RESULTS Baseline Characteristics In total, 212 HIV-infected patients (51.4% HIV-1, 26.4% HIV-2, and 22.2% HIV-1/2) were included in the study (Table 1). There were no differences in baseline levels of CRP or sCD163 between groups (median sCD163 among HIV-1: 2.53 mg/L, HIV-2: 2.56 mg/L, and HIV-1/2: 2.85 mg/ L, respectively, P = 0.861). This was also the case after adjusting for age, sex, and baseline CD4 cell count (P = 0.781). CD4 cell count at baseline correlated with levels of both sCD163 and CRP (r2 = 0.036, P = 0.006 and r2 = 0.054, P = 0.001, respectively). Concentrations of sCD163 and CRP were also correlated (r2 = 0.041, P = 0.003).

Patients at Follow-Up Without ART Among 123 (58.0%) patients on follow-up without ART, the median follow-up time was 365 days [interquartile range (IQR) 228–463 days], and there was no difference in follow-up time between HIV groups (P = 0.569). In all HIV groups, the median CD4 cell count declined [median values: HIV-1, 70 (IQR 162–13); HIV-2, 64 (IQR 150–18); and HIV-1/2, 66 (IQR 189–41) cells/mL], but there was neither an overall nor an HIV type–related change in sCD163 levels (Fig. 1A) or in CRP levels (data not shown). The change in CD4 cell count was not associated with change in levels of CRP [regression coefficient (P = 0.561) or sCD163 (P = 0.559)].

Patients at Follow-Up on ART A total of 89 (42.0%) patients (50 HIV-1, 22 HIV-2, and 17 HIV-1/2) initiated ART after HIV diagnosis. The median time between baseline CD4 cell count and initiation of ART was 14 days (IQR 9–33 days). Patients on ART had a median

follow-up time of 481 days (IQR 401–583 days). During this period, patients with HIV-1 infection had a median increase in CD4 cell count of 174 (IQR 91–263) cells/mL, patients with HIV-2: 133 (IQR 70–219) cells/mL and HIV-1/2: 134 (IQR 53–159) cells/mL (P = 0.190). There was no difference in follow-up time between patient groups (P = 0.878). During the follow-up period, the decrease in CRP level was statistically significant for HIV-1–infected patients (P = 0.025), but not for HIV-2 (P = 0.093) or HIV-1/2 dually infected patients (P = 0.283). Only HIV-1–infected patients on ART had a significant decline in sCD163 levels (P , 0.001, Fig. 1B). When comparing HIV types, HIV-1–infected patients had a larger decline in median sCD163 levels than HIV-2–infected patients (21.03 vs. 20.59 mg/L, P = 0.008) and HIV-1/2 dually infected patients (21.03 vs. 20.71 mg/L, P = 0.032). When adjusting for the influence of age, sex, and baseline CD4 cell count on sCD163 levels, the association between decrease in sCD163 and HIV type remained statistically significant, HIV-1 vs. HIV-2: regression coefficient = 0.237 (95% CI: 0.014 to 0.460, P = 0.038) and HIV-1 vs. HIV-1/2: regression coefficient = 0.145 (95% CI: 0.027 to 0.263, P = 0.017).

Influence of ART Regimen on sCD163 Most (68.0%) HIV-1–infected patients received zidovudine (AZT), lamivudine (3TC), and nevirapine (NVP). Among HIV-2 and HIV-1/2 dually infected patients, the most frequently used combination was AZT, 3TC, and ritonavirboosted indinavir (IDV/r). Despite being initially diagnosed with HIV-2 or HIV-1/2 dual infection, 4 patients initiated ART with AZT, 3TC, and efavirenz (EFV) or NVP. By the confirmatory HIV type discrimination, 5 patients with HIV-2 or HIV-1/2 infection received either EFV or NVP. Different ART regiment may influence changes in sCD163 levels differently. For all HIV types, NVP-based vs. EFV-based ART was not associated with change in sCD163 levels (P = 0.160). But after adjusting for age, sex, and baseline CD4 cell count, patients receiving EFV had an as-significant larger decrease (P = 0.049). For all patients,

TABLE 1. Characteristics of HIV-Infected Patients at Baseline Total, N = 212 Sex (%) Male Female Age (%) ,30 yrs 31–40 yrs .40 yrs Body mass index (%) #18.5 kg/m2 .18.5 kg/m2 Unknown CD4 cell count (%) #200 cells/mL 201–350 cells/mL .350 cells/mL

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HIV-1, n (%) = 109 (51.4)

HIV-2, n (%) = 56 (26.4)

HIV-1/2, n (%) = 47 (22.2)

P 0.780

53 (25.0) 159 (75.0)

29 (26.6) 80 (73.4)

14 (25.0) 42 (75.0)

10 (21.3) 37 (78.7)

67 (31.6) 69 (32.6) 76 (35.8)

48 (44.0) 37 (33.9) 24 (22.0)

8 (14.3) 13 (23.2) 35 (62.5)

11 (23.4) 19 (40.4) 17 (36.2)

52 (24.5) 154 (72.6) 6 (2.8)

30 (27.5) 77 (70.6) 2 (1.8)

13 (23.2) 40 (71.4) 3 (5.4)

9 (19.2) 37 (78.7) 1 (2.1)

75 (35.4) 48 (22.6) 89 (42.0)

44 (40.0) 25 (22.9) 40 (36.7)

18 (32.1) 12 (21.4) 26 (46.4)

13 (27.7) 11 (23.4) 23 (48.9)

,0.001

0.553

0.508

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treatment with a protease inhibitor (PI) vs. a non-PI–based treatment was not associated with change in sCD163 in the unadjusted analyses (P = 0.133) or in the adjusted analyses (P = 0.975).

DISCUSSION In this follow-up study, we compared HIV-1, HIV-2, and HIV-1/2 dually infected patients on follow-up with regard to CD4 cell count and concentrations of CRP and sCD163. We confirmed the association of sCD163 with CD4 cell count.8 Although CD4 cell count declined during follow-up, CRP and sCD163 levels did not change significantly in any of the HIV groups without ART. For patients on ART, we found that sCD163 levels decreased significantly for HIV-1–infected patients but not for HIV-2–infected patients.

Soluble CD163 in HIV-1/HIV-2–Infected Patients

time of infection among HIV-2–infected patients may have exhausted their macrophage response or that the regimen used in HIV-2 treatment is less efficient. ART regimen may influence changes in sCD163 levels. In a study of HIV-1–infected patients, individuals receiving protease inhibitors (PIs) had a higher decline in sCD163 levels than patients not receiving PIs.22 In our study, a larger proportion of HIV-2–infected patients received PIs than HIV1–infected patients, but we found no difference in sCD163 levels between patients with and without PI-based ART. Also, we do not have reason to believe that patients receiving a PI-based regimen would have been more vulnerable to virological failure than those receiving a non-nucleoside reverse transcriptase inhibitor-based regimen.23

CONCLUSION Strengths and Weaknesses

This is, to our knowledge, the first study to report and compare sCD163 levels among HIV-1, HIV-2, and HIV-1/2 dually infected patients both with and without ART. The lower pathogenicity of HIV-2 is not entirely understood, and studies comparing immune response to different HIV types may potentially provide information as to why this is.17 We included patients in this study based on availability from the biorepository among patients with at least one follow-up sample. sCD163 levels are stable for long periods when stored at freezer temperature.18 Soluble CD163 levels have been shown to correlate with HIV-1 viral load both at diagnosis and during ART,9 but no studies so far have investigated the association between HIV-2 RNA and sCD163 levels. Unfortunately, viral load measurements were not available in Guinea-Bissau during the study period,19 and hence we were not able to detect development of treatment failure among the patients on ART. However, many HIV-2– infected individuals have undetectable RNA levels even without ART.20

Relations to Other Studies and Potential Biological Mechanisms CD163 is shed from cells of monocyte/macrophage linage,16 but data regarding sCD163 in HIV-2–infected patients are scarce.21 An increased monocyte activation in HIV-2 infection has previously been described. This was accompanied by upregulation of the immunoinhibitory molecules PD-L1/PD-1, leading the authors to suggest that this may be an explanation for the more benign outcome in those with HIV-2 infection.21 In this study, we found no differences in sCD163 levels between HIV types at baseline or at follow-up among patients without ART. However, for patients on ART, HIV-2–infected patients had a significantly lower decrease in sCD163 levels than HIV-1–infected patients. This may reflect a differential macrophage activation within HIV-1 and HIV-2. Owing to the lower pathogenic potential of HIV-2, many HIV-2– infected patients have lived with their infection longer than HIV-1–infected patients. We speculate that a longer carriage Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.

We confirmed the previously reported association of sCD163 levels to CD4 cell count and found that sCD163 levels decreased significantly more in HIV-1, than in HIV-2 and HIV-1/2 dually infected patients on ART. This may reflect differences in macrophage activation in HIV-1 and HIV-2 infection during ART.

ACKNOWLEDGMENTS The authors thank health care personnel at the HIV clinic at Hospital Nacional Simão Mendes. The Bissau HIV Cohort comprises Peter Aaby, David da Silva Té, Zacarias José da Silva, Christian ErikstrupBo Langhoff Hønge, Anders Fomsgaard, Alex Laursen, Candida Medina, Inés Oliviera, Lars Østergaard, Amabelia Rodrigues, Sanne Jespersen, and Christian Wejse (chair). REFERENCES 1. Rowland-Jones SL, Whittle HC. Out of Africa: what can we learn from HIV-2 about protective immunity to HIV-1? Nat Immunol. 2007;8: 329–331. 2. Campbell-Yesufu OT, Gandhi RT. Update on human immunodeficiency virus (HIV)-2 infection. Clin Infect Dis. 2011;52:780–787. 3. Da Silva ZJ, Oliveira I, Andersen A, et al. Changes in prevalence and incidence of HIV-1, HIV-2 and dual infections in urban areas of Bissau, Guinea-Bissau: is HIV-2 disappearing? AIDS. 2008;22:1195–1202. 4. Kristiansen M, Graversen JH, Jacobsen C, et al. Identification of the haemoglobin scavenger receptor. Nature. 2001;409:198–201. 5. Etzerodt A, Maniecki MB, Møller K, et al. Tumor necrosis factor a-converting enzyme (TACE/ADAM17) mediates ectodomain shedding of the scavenger receptor CD163. J Leukoc Biol. 2010;88:1201–1205. 6. Tuluc F, Meshki J, Spitsin S, et al. HIV infection of macrophages is enhanced in the presence of increased expression of CD163 induced by substance P. J Leukoc Biol. 2014;96:143–150. 7. Herbein G, Gras G, Khan KA, et al. Macrophage signaling in HIV-1 infection. Retrovirology. 2010;7:34. 8. Noel N, Boufassa F, Lécuroux C, et al. Elevated IP10 levels are associated with immune activation and low CD4+ T-cell counts in HIV controller patients. AIDS. 2014;28:467–476. 9. Burdo TH, Lentz MR, Autissier P, et al. Soluble CD163 made by Monocyte/Macrophages is a novel marker of HIV activity in early and chronic infection prior to and after anti-retroviral therapy. J Infect Dis. 2011;204:154–163. 10. Knudsen TB, Gustafson P, Kronborg G, et al. Predictive value of soluble haemoglobin scavenger receptor CD163 serum levels for survival in verified tuberculosis patients. Clin Microbiol Infect. 2005;11:730–735.

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11. Jespersen S, Hønge BL, Oliveira I, et al. Cohort profile: the bissau HIV Cohort–a cohort of HIV-1, HIV-2 and co-infected patients. Int J Epidemiol. 2015;44:756–763. 12. Hønge BL, Jespersen S, Nordentoft PB, et al. Loss to follow-up occurs at all stages in the diagnostic and follow-up period among HIV-infected patients in Guinea-Bissau: a 7-year retrospective cohort study. BMJ Open. 2013;3:e003499. 13. Hønge BL, Bjarnason Obinah MP, Jespersen S, et al. Performance of 3 rapid tests for discrimination between HIV-1 and HIV-2 in GuineaBissau, West Africa. J Acquir Immune Defic Syndr. 2014;65:87–90. 14. Hønge BL, Jespersen S, Pétur Bjarnason MO, et al. Inter-observer variation of the rapid test SD bioline HIV 1/2 3.0 for HIV type discrimination: experiences from Guinea-Bissau. J Acquir Immune Defic Syndr. 2015;68:e23–e25. 15. Andersson S, da Silva Z, Norrgren H, et al. Field evaluation of alternative testing strategies for diagnosis and differentiation of HIV-1 and HIV-2 infections in an HIV-1 and HIV-2-prevalent area. AIDS. 1997;11:1815–1822. 16. Møller HJ. Soluble CD163. Scand J Clin Lab Invest. 2012;72:1–13. 17. Esbjörnsson J, Månsson F, Kvist A, et al. Inhibition of HIV-1 disease progression by contemporaneous HIV-2 infection. N Engl J Med. 2012; 367:224–232.

18. Møller HJ, Hald K, Moestrup SK. Characterization of an enzyme-linked immunosorbent assay for soluble CD163. Scand J Clin Lab Invest. 2002; 62:293–299. Available at: http://informahealthcare.com/doi/abs/10.1080/ 003655102760145852. Accessed July 15, 2015. 19. Jespersen S, Hønge BL, Oliveira I, et al. Challenges facing HIV treatment in Guinea-Bissau: the benefits of international research collaborations. Bull World Health Organ. 2014;92:909–914. 20. Popper SJ, Sarr AD, Travers KU, et al. Lower human immunodeficiency virus (HIV) type 2 viral load reflects the difference in pathogenicity of HIV-1 and HIV-2. J Infect Dis. 1999;180:1116–1121. 21. Cavaleiro R, Tendeiro R, Foxall RB, et al. Monocyte and myeloid dendritic cell activation occurs throughout HIV type 2 infection, an attenuated form of HIV disease. J Infect Dis. 2013;207: 1730–1742. 22. Beltrán LM, Muñoz Hernández R, de Pablo Bernal RS, et al. Reduced sTWEAK and increased sCD163 levels in HIV-infected patients: modulation by antiretroviral treatment, HIV replication and HCV coinfection. PLoS One. 2014;9:e90541. 23. Jespersen S, Tolstrup M, Hønge B, et al. High level of HIV-1 drug resistance among patients with HIV-1 and HIV-1/2 dual infections in Guinea-Bissau. Virol J. 2015;12:41.

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