Antiretroviral therapy (ART) has significantly prolonged the lifespan of HIV-infected people,1,2 but drug-resistant HIV-1 can arise during the course of ART. The prevalence of antiretroviral drug resistance is increasing among subjects newly infected with HIV-1.3–5 The cause for the emergence of drug-resistant viruses could involve selection due to changes in the immune system. Some studies have shown that ART is associated with a reduction in number of effector cytotoxic T lymphocytes,6 HIV-specific CD4+ T-cell activity,7 the CD8+ noncytotoxic anti-HIV response (CNAR),8 and anti-HIV antibody production.9,10 The specific effects of individual antiretroviral drugs on distinct CD8+ cell anti-HIV responses have not been evaluated. We focused on the CNAR activity that correlates with control of HIV infection.11–13
MATERIALS AND METHODS
The subjects from an established cohort of HIV-infected individuals at the University of California, San Francisco, were analyzed. The study subjects included 12 HIV-1–infected individuals who were receiving combination ART and 14 long-term survivors (LTSs). LTSs are HIV-1–infected subjects who have remained asymptomatic with normal CD4+ lymphocyte counts (>500 cells/μL) without therapy for more than 10 years of infection. The levels of total leukocytes, granulocytes, lymphocytes, monocytes, platelets, and T-cell subsets were determined using a BD FACSort. Measurements of plasma HIV RNA levels were performed using a branched-DNA assay (Siemens Diagnostics, Emeryville, CA) or were self-reported. This study was approved by the University of California, San Francisco Committee on Human Subjects.
Human Primary Cells
Buffy coats from HIV-seronegative subjects were obtained from the Blood Centers of the Pacific, San Francisco, CA. Blood from HIV-infected individuals was collected at University of California, San Francisco. Peripheral blood mononuclear cells were prepared by Histopaque-1077 (Sigma-Aldrich, St. Louis, MO) density gradient centrifugation. Primary CD8+ cells were purified from HIV-infected individuals by positive immunomagnetic isolation using Dynal beads (Invitrogen, Carlsbad, CA). Primary CD4+ cells from the buffy coats were purified by positive selection using Miltenyi immunomagnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of both T-cell populations was >95%. The primary T cells were stimulated for 3 days with phytohaemagglutinin-leukocyte (3 μg/mL) (Sigma-Aldrich).
CD8 + Cell HIV-1 Suppression Assay
The stimulated human CD4+ T cells were acutely infected with different HIV-1 isolates or clones: nevirapine (NVP)-resistant virus [National Institutes of Health (NIH) Cat. No.1392), multidrug-resistant virus (NIH Cat. No. 7391), efavirenz (EFV)-resistant virus clone v7205-2, nelfinavir (NFV)-resistant virus clone v16970-2, or the ritonavir (RTV)-resistant virus clone v40539-1. For measurement of CNAR, CD8+ cells at different CD8+ cell: CD4+ cell input cell ratios (0.5:1, 1:1, and 2:1) were cocultured with HIV acutely infected CD4+ cells in triplicate wells in a 96-well plate with varying concentrations of antiretroviral drugs.11 To measure HIV replication levels in the cultures, 100 μL aliquots of culture supernatants were collected from each well on days 4, 7, and 10 postinfection. Fluids were treated with 1% Triton, and the HIV p24 enzyme-linked immunosorbent assay (NIH AIDS Research and Reference Reagent Program) was performed to evaluate the concentration of p24 antigen in each culture. Alternatively, the collected fluids were centrifuged at 12,000g for 1 hour at 4°C, and the resulting viral pellets were assayed for reverse transcriptase activity as described.14 Results obtained from both assays were comparable.15
Intracellular drugs were measured using liquid chromatography tandem mass spectrometer, consisting of 2 Shimadzu LC-20ADXR pumps, a SIL-20ACXR autosampler, and an AB Sciex API5000 mass spectrometer.16,17 CD8+ cells pretreated with 1 µM or 10 µM NVP, EFV, or NFV were washed twice with 1 × Dulbecco's phosphate-buffered saline and pelleted. The cell pellets were resuspended in 400 µL of 60% methanol with 1% formic acid, vortexed for 1 minute, and sonicated for 10 minutes. The lysed cells were centrifuged at 22,000g for 7 minutes. A 10-µl aliquot of the supernatant was directly injected into the liquid chromatography tandem mass spectrometer system. Ion pair 267/226 for NVP and 568/330 for NFV was used for multiple reaction monitor detection in electrospray ionization-positive mode. For EFV, ion pair 314/68 was used for detection in electrospray ionization-negative mode. The lower limits of quantification for NVP, NFV, and EFV were 0.038 nM (S/N = 15), 0.05 nM (S/N = 91), and 0.5 nM (S/N = 12.7), respectively.
Effect of ART on the CD8+ Cell Anti-HIV Response
CNAR activity was measured before therapy and during the course of ART for up to 5 years in 12 HIV-infected subjects. With treatment, the CD4+ T-cell counts in these subjects declined before ART and increased while on therapy (Fig. 1A). The HIV RNA levels in all these infected patients except subject 11 were undetectable after 1 year of ART (Fig. 1B). For these studies, a conventional CNAR assay was conducted using the CXCR4-tropic, chemokine-resistant virus, HIV-1SF33.11 CNAR significantly declined for 10 subjects who were not in therapy during ART (P < 0.001; Fig. 1C). The data on two subjects were not collected before therapy, but after therapy, a similar decline was observed. The CNAR activity of CD8+ cells from 8 of the 12 subjects was less than 50% after 5 years of treatment (Fig. 1D). And, CNAR activity in 4 of the 12 subjects decreased more than 4-fold during the course of ART.
Effect of Anti-HIV Drugs on the Anti-HIV Response of CD8+ Cells in Culture
Five drug-resistant viruses confirmed to be CXCR4-tropic using the U373-MAGI CXCR4 cell line tropism assay18 (data not shown) were used. The sensitivity of these viruses to the antiviral drugs was first assessed. Replication of the NVP-resistant virus and EFV-resistant virus were not affected by ≤10 μM of the drugs but were significantly reduced in the presence of 25 μM of the drugs (P < 0.01). The replication of a multidrug-resistant virus (NIH Cat. No. 7391) was not affected by ≤5 μM of abacavir, lamivudine, or zidovudine (ZDV) (data not shown). The replication of the NFV-resistant virus and the RTV-resistant virus was not affected by ≤2 μM of NFV or RTV. The plasma drug concentrations in subjects receiving these drugs as ART are equal to or greater than the amount used for these studies.19,20
To determine the effect of antiretroviral medications on CNAR, we isolated CD8+ cells from the 14 HIV-infected drug naive LTSs and cocultured them with CD4+ cells infected by drug-resistant HIV. Cocultures were performed with or without the presence of varying concentrations of the antiretroviral drugs. The concentrations used did not substantially reduce the replication of the drug-resistant viruses. The extent of HIV suppression was calculated by comparing the p24 levels or reverse transcriptase value in the supernatants of the coculture to the average amount of HIV-1 virus replication in CD4+ cells infected with the same virus and exposed to the same concentration of the anti-HIV drug. CD8+ cells from 4 of the 14 LTSs were sensitive to 10 μM of NVP. When exposed to this drug, their antiviral activity measured by CNAR was significantly reduced (P < 0.05; Table 1). No association was observed between the HLA genotype and the sensitivity of the CD8+ T-cell anti-HIV activity to NVP (data not shown). CD8+ cells from 2 of the 14 LTSs were also sensitive to 5 μM of ZDV and showed a reduction in CNAR. However, the difference was not significant (P > 0.05; Table 1). All other antiretroviral medications did not affect CNAR in the 14 subjects (Table 1).
Each drug did not inhibit the proliferation of T lymphocytes in culture (data not shown). And, varying concentrations of the drugs used in this study (eg, 0.5–10 μM) did not show cytotoxicity to CD8+ and CD4+ T cells. The expression of activation markers, such as HLA-DR, CD25, and CD69, on both CD8+ and CD4+ T cells in the coculture was also not affected by 10 μM of NVP (data not shown).
Residual Presence of NVP, EFV, and NFV in Primary CD8+ Cells
The mass spectrometry data showed that 0.33–0.83% of NVP remained in the CD8+ cells after 3 days. The percentages of residual CD8+ cell intracellular concentrations of EFV and NFV were 0.23–2.24% and 1.45–11.6%, respectively (see Figure S1, Supplemental Digital Content, http://links.lww.com/QAI/A395). The reduction of CD8+ cell anti-HIV activity by only NVP suggests that this drug has a specific effect on CD8+ cell function.
Antiretroviral medications can have adverse clinical effects. NVP can cause liver damage, skin reactions, and allergic reactions.21–23 In some cases, hepatic injury progresses despite discontinuation of treatment. ZDV is associated with hematologic toxicity including neutropenia and anemia, particularly in patients with advanced HIV-1 disease. In addition, pure red cell aplasia occurs with ZDV after 6 weeks to 4 years of therapy.24–26
Our data show that the immune system can also be affected by ART. CNAR was significantly reduced during the 5-year clinical course of ART (P < 0.0001; Fig. 1C). The anti-HIV response slowly declined as CD4+ cell counts recovered and viral loads became undetectable (Fig. 1A, B, D).
Although these results could reflect the lack of viral antigen stimulation of CD8+ cell responses, toxicity to the immune system could be involved. To investigate this possibility, CD8+ cells from HIV-infected LTSs were isolated and cocultured with CD4+ cells infected with drug-resistant HIV at different concentrations of the anti-HIV medications. The data showed that the CD8+ cells from 4 of these subjects were very sensitive to 10 μM of NVP (Table 1). The relevant concentration of 10 μM of NVP is 2663 ng/mL. That amount is equal to the lowest plasma concentration of NVP (4500 ± 1900 ng/mL) in individuals on the drug.19 The anti-HIV activity of CD8+ cells from these subjects was markedly inhibited by NVP. This effect did not involve a block in cell proliferation nor activation of CD8+ and CD4+ cells. Two subjects were also sensitive to 5 μM of ZDV (Table 1). The relevant concentration of 5 μM of ZDV is 1336 ng/mL. That amount is in the range of plasma concentrations of ZDV (1220 ± 210 ng/mL) in subjects on the drug.20
Because of the measurement limitation of intracellular concentration of drugs, residual NVP, EFV, and NFV in primary CD8+ cells were evaluated by mass spectrometry. All 3 drugs were found in CD8+ cells. Therefore, reduction of CD8+ cell anti-HIV activity by only NVP suggests that this drug has a specific detrimental effect on CD8+ cell function. Our data indicate that the effect of NVP and perhaps other ART on CD8+ cell anti-HIV responses should be considered when administering these drugs to HIV-infected patients.
In summary, NVP and to some extent ZDV can inhibit the anti-HIV activity of CD8+ cells and could influence the beneficial anti-HIV effects of ART. Drug-resistant viruses still occur especially when patients are treated with NVP alone over time, despite the high anti-HIV efficacy of NVP.27 If the CD8+ T cells from patients on ART still have anti-HIV responses, these immune responses could help prevent HIV replication and the emergence of drug-resistant viruses.
The authors thank Drs S. Bakkour and S. Killian for their helpful discussions. They also thank the AIDS Research and Reference Reagent Program, the NIH, and Dr R. Shafer, Stanford University, for providing drug-resistant HIV-1.
1. Michaels SH, Clark R, Kissinger P. Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. N Engl J Med. 1998;339:405–406.
2. Moh R, Danel C, Messou E, et al.. Incidence and determinants of mortality and morbidity following early antiretroviral therapy initiation in HIV-infected adults in West Africa. AIDS. 2007;21:2483–2491.
3. Shet A, Berry L, Mohri H, et al.. Tracking the prevalence of transmitted antiretroviral drug-resistant HIV-1: a decade of experience. J Acquir Immune Defic Syndr. 2006;41:439–446.
4. Smith D, Moini N, Pesano R, et al.. Clinical utility of HIV standard genotyping among antiretroviral-naive individuals with unknown duration of infection. Clin Infect Dis. 2007;44:456–458.
5. Youmans E, Tripathi A, Albrecht H, et al.. Transmitted antiretroviral drug resistance in individuals with newly diagnosed HIV infection: South Carolina 2005-2009. South Med J. 2011;104:95–101.
6. Ogg GS, Jin X, Bonhoeffer S, et al.. Decay kinetics of human immunodeficiency virus-specific effector cytotoxic T lymphocytes after combination antiretroviral therapy. J Virol. 1999;73:797–800.
7. Pitcher CJ, Quittner C, Peterson DM, et al.. HIV-1-specific CD4+ T cells are detectable in most individuals with active HIV-1 infection, but decline with prolonged viral suppression. Nat Med. 1999;5:518–525.
8. Stranford SA, Ong JC, Martinez-Marino B, et al.. Reduction in CD8+ cell noncytotoxic anti-HIV activity in individuals receiving highly active antiretroviral therapy during primary infection. Proc Natl Acad Sci U S A. 2001;98:597–602.
9. Binley JM, Trkola A, Ketas T, et al.. The effect of highly active antiretroviral therapy on binding and neutralizing antibody responses to human immunodeficiency virus type 1 infection. J Infect Dis. 2000;182:945–949.
10. Killian MS, Norris PJ, Rawal BD, et al.. The effects of early antiretroviral therapy and its discontinuation on the HIV-specific antibody response. AIDS Res Hum Retroviruses. 2006;22:640–647.
11. Mackewicz CE, Ortega HW, Levy JA. CD8+ cell anti-HIV activity correlates with the clinical state of the infected individual. J Clin Invest. 1991;87:1462–1466.
12. Gomez AM, Smaill FM, Rosenthal KL. Inhibition of HIV replication by CD8+ T cells correlates with CD4 counts and clinical stage of disease. Clin Exp Immunol. 1994;97:68–75.
13. Castelli JC, Deeks SG, Shiboski S, et al.. Relationship of CD8(+) T cell noncytotoxic anti-HIV response to CD4(+) T cell number in untreated asymptomatic HIV-infected individuals. Blood. 2002;99:4225–4227.
14. Hoffman AD, Banapour B, Levy JA. Characterization of the AIDS-associated retrovirus reverse transcriptase and optimal conditions for its detection in virions. Virology. 1985;147:326–335.
15. Killian MS, Ng S, Mackewicz CE, et al.. A screening assay for detecting CD8+ cell non-cytotoxic anti-HIV responses. J Immunol Methods. 2005;304:137–150.
16. Chi J, Jayewardene AL, Stone JA, et al.. An LC-MS-MS method for the determination of nevirapine, a non-nucleoside reverse transcriptase inhibitor, in human plasma. J Pharm Biomed Anal. 2003;31:953–959.
17. Colombo S, Beguin A, Telenti A, et al.. Intracellular measurements of anti-HIV drugs indinavir, amprenavir, saquinavir, ritonavir, nelfinavir, lopinavir, atazanavir, efavirenz and nevirapine in peripheral blood mononuclear cells by liquid chromatography coupled to tandem mass spectrometry. J Chromatogr B. 2005;819:259–276.
18. Vodicka MA, Goh WC, Wu LI, et al.. Indicator cell lines for detection of primary strains of human and simian immunodeficiency viruses. Virology. 1997;233:193–198.
19. Pretorius E, Klinker H, Rosenkranz B. The role of therapeutic drug monitoring in the management of patients with human immunodeficiency virus infection. Ther Drug Monit. 2011;33:265–274.
20. Bazzoli C, Jullien V, Le Tiec C, et al.. Intracellular pharmacokinetics of antiretroviral drugs in HIV-infected patients, and their correlation with drug action. Clin Pharmacokinet. 2010;49:17–45.
21. Dong BJ, Zheng Y, Hughes MD, et al.. Nevirapine pharmacokinetics and risk of rash and hepatitis among HIV-infected sub-Saharan African women. AIDS. 2012;26:833–841.
22. Sharma AM, Li Y, Novalen M, et al.. Bioactivation of nevirapine to a reactive quinone methide: implications for liver injury. Chem Res Toxicol. 2012;20:1708–1719.
23. Caixas U, Antunes AM, Marinho AT, et al.. Evidence for nevirapine bioactivation in man: searching for the first step in the mechanism of nevirapine toxicity. Toxicology. 2012;301:33–39.
24. Weinkove R, Rangarajan S, van der Walt J, et al.. Zidovudine-induced pure red cell aplasia presenting after 4 years of therapy. AIDS. 2005;19:2046–2047.
25. Hassan A, Babadoko AA, Mamman AI, et al.. Zidovudine induced pure red cell aplasia: a case report. Niger J Med. 2009;18:332–333.
26. Balakrishnan A, Valsalan R, Sheshadri S, et al.. Zidovudine-induced reversible pure red cell aplasia. Indian J Pharmacol. 2010;42:189–191.
27. Haïm-Boukobza S, Morand-Joubert L, Flandre P, et al.. Higher efficacy of nevirapine than efavirenz to achieve HIV-1 plasma viral load below 1 copy/ml. AIDS. 2011;25:341–344.