aLaboratoire d'Immunologie Cellulaire et Tissulaire INSERM U543, bService d'Hépatologie, cLaboratoire de Virologie, and dService de Maladies Infectieuses, Centre Hospitalier Universitaire Pitié-Salpêtrière, Paris, France.
See also pp. 1, 59, 67, 75, 131
Sponsorship: This work was supported by ANRS, the French agency for AIDS research.
Received: 27 June 2003; revised: 17 July 2003; accepted: 11 August 2003.
Anti-hepatitis C virus (HCV) treatment combining IFN-α and ribavirin (IFN+R) has still poorly defined effects on immune responses. Frequencies of T helper (Th) type 1 cells specific for HCV, cytomegalovirus (CMV) and HIV were measured in chronically HCV-infected patients with or without HIV co-infection during IFN+R treatment. Although scarce, peripheral blood HCV-specific Th1 cells did not change significantly, while frequencies of HIV and CMV-specific Th1 cells decreased in co-infected patients independently of CD4 cell count changes. This suggests that IFN+R therapy might compromise virus-specific immune defences in immunosuppressed patients.
The treatment of chronic hepatitis C virus (HCV) is based on a combination of ribavirin (nucleoside analogue of guanosine) with IFN-α (IFN+R) or its pegylated forms. Although their mechanisms of action are not yet completely understood, both compounds exert immunoregulatory effects. IFN-α is known to mediate anti-inflammatory effects , to inhibit leukocyte proliferation while modulating both T helper (Th) type 1 and Th2 immune responses. Ribavirin reduces hepatic inflammation and decreases proliferation and to an extent the production of IFN-γ by Th1 lymphocytes . Although this combination became the standard of care for chronic HCV infection, the immune consequences of this treatment are still poorly defined. As the immune control of virus infections depends upon strong IFN-γ-producing Th1 responses, the question is raised as to whether the immune control of both HCV and other viruses can be altered during this treatment. HCV infection is frequently associated with an HIV co-infection, and this question becomes particularly relevant in HCV patients co-infected with HIV, in whom the immune status to those pathogens remains fragile even during antiretroviral therapy.
In patients chronically infected with HCV, with or without HIV co-infection, the peripheral blood Th1 responses to HCV are known to be weak and narrowly focused on core-proteins [3–5], except in HIV long-term non-progressors co-infected with HCV who respond better to HCV than patients infected with HCV alone . The numbers of circulating HIV-specific CD4 T cells are also low in chronic HIV infection with or without antiretroviral therapy, whereas higher frequencies of CMV-specific Th1 cells persist durably . The weak T-cell proliferative responses to HCV core, HIV and tuberculin were shown to decrease transiently during the first weeks of IFN+R treatment in HCV–HIV co-infected patients, followed by a return to baseline values , whereas few HCV-specific Th1 responses were amplified in patients displaying genotype 3 HCV clearance . Although suggesting a transient impact of IFN+R treatment on immune responses to these pathogens; however, a quantitative effect on the numbers of Th1 cells specific for those pathogens remains to be defined.
To address this question, an ELISpot IFN-γ assay quantifying frequencies of CD4 Th1 cells specific for viruses was used, as previously described , with several viral recombinant proteins from HCV (core, NS3 and NS4), and HIV (p24) together with CMV antigens. CD4 cells were obtained from the peripheral blood of two groups of HCV-infected patients: 12 HIV positive (median CD4 cell counts 428 cells/mm3, range 186–846) and nine HIV negative (median CD4 cell counts 885 cells/mm3, range 296–1899), before and at 6 months of IFN-α (3 MIU three times a week) plus ribavirin (800 mg/day) therapy. All HIV-positive patients were treated with highly active antiretroviral therapy for 6 months or more before study entry and had undetectable HIV plasma loads (< 2.3 log copies/ml).
Briefly, peripheral blood mononuclear cells (PBMC; 2 × 105 cells/well for HCV and 1 × 105 for CMV and HIV) were stimulated for 40 h with three recombinant HCV proteins: core, NS3 and NS4 (10 μg/ml; Chiron Corporation, Emeryville, CA, USA), one recombinant HIV p24 protein (2 μg/ml; Protein Sciences, Meriden, CT, USA) and one CMV antigen (10 μg/ml; BioWitteker). Phytohaemagglutinin (1 μg/ml; Murex, Paris, France) and medium alone served as positive and negative controls, respectively. The following reagents were used: anti-human IFN-γ antibodies for capture and detection (IgG1/B-B1 and B-G1, respectively; Diaclone, Strasbourg, France), strepatavidin-alkaline-phosphatase and 5-bromo-4-chloro-3-indolyl-phosphate/4-nitrobleutetrazolium (Sigma, Saint-Quentin Fallavier, France). Antigen-specific spot forming cell (SFC) frequencies were measured on an automated microscope (Zeiss, Munick, Germany) and counted as positive if 50 or more SFC/1 × 106 PBMC were detected above the background.
Before IFN+R treatment, HCV-specific Th1 responses were detectable in two out of 12 HIV-positive patients (against core and NS3) and in three out of nine HIV-negative patients (all against core) with low frequencies of Th1-specific cells (below 250 SFC/million PBMC in all cases). Anti-HIV Th1 cells were detectable in five out of the 12 co-infected patients with a median frequency of 177 SFC/1 × 106 PBMC (range 83–1113). In contrast, higher frequencies of Th1 cells specific for CMV were detectable in 14 cases (5/9 HIV negative and 9/12 HIV positive) at similar levels in both groups, with a median of 580 and 446 SFC/1 × 106 PBMC, respectively (range 63–2000).
At 6 months of IFN+R treatment, virological responses (negativization of HCV polymerase chain reaction) were observed in three HIV-positive patients and in three HIV-negative patients, independently of the HCV genotypes and of baseline HCV-specific Th1 cell frequencies. After 6 months of treatment the CD4 cell counts showed a median decrease, although non significant, of −54 and −130 CD4 cells/mm3 in the HIV-co-infected and HCV-alone groups, respectively. The HIV viral load remained undetectable in all co-infected patients. When detected at baseline, HCV-specific Th1 cell frequencies decreased in all cases (n = 5) in both the HCV-alone and co-infected groups. Of note was the fact that core-specific anti-HCV Th1 cells became detectable in two HIV- negative patients who cleared HCV (C2 and C8), and an NS3-specific Th1 cell response was also detected in one HIV-positive patient who did not respond to treatment (P12). In the remaining patients (9/12 HIV positive and 5/9 HIV negative), HCV-specific Th1 cells were below the ELISpot threshold. In contrast, the frequencies of HIV-specific Th1 cells decreased by at least threefold in four co-infected patients and increased in only one patient, while remaining undetectable in six out of 12 HIV-positive patients (Fig. 1b). Considering the unknown CMV status at baseline, we have only described the evolution of CMV-specific Th1 responses in patients with detectable Th1 responses at baseline. The frequencies of CMV-specific Th1 cells increased or remained stable in three out of the five HIV-negative and in only one out of the nine HIV-positive patients. A significant decrease from baseline of CMV-specific Th1-cell frequencies was more pronounced in HIV-positive than in HIV-negative patients, and was observed in six out of nine HIV-positive versus two out of five HIV-negative patients (P < 0.001; Fig. 1b,d), despite a lack of changes in the CD4 cell counts.
Our results, although limited in numbers, show that the frequencies of CD4 Th1 cells specific for viruses such as HIV and CMV decrease after 6 months of IFN+R therapy in patients co-infectected with HCV and HIV, despite the preservation of CD4 cell counts. This decrease in virus-specific immune responses might be less frequent in patients infected with HCV alone, suggesting that IFN+R therapy in HIV-positive patients might compromise the fragile immune equilibrium and restoration of immune function allowed by antiretroviral therapy. In addition, in co-infected patients no induction of Th1 responses to HCV were observed, except in one patient whereas the concomitant development of an immune reactivity to HCV core and of a virological response to anti-HCV treatment was observed in only two HIV-negative patients. Our results also show that HCV-specific immune responses evolved independently of the HCV outcome during IFN+R therapy, suggesting that the virological response to IFN+R therapy depends on many host and viral parameters. Therefore, altogether our findings suggest that particular attention should be paid to the risk of infectious complications during treatment with IFN+R in immune-suppressed HIV-infected patients with low CD4 cell counts. Extended follow-up studies co-evaluating immune responses to viruses in correlation with clinical outcomes have still to better define this risk during the treatment of HCV co-infection.
1. Tilg H. New insights into the mechanisms of interferon alpha: an immunoregulatory and anti-inflammatory cytokine. Gastroenterology 1997, 112:1017–1021.
2. Meier V, Bürger E, Mihm S, Sale B, Ramadori G. Ribavirin inhibits DNA, RNA, and protein synthesis in PHA-stimulated human peripheral blood mononuclear cells: possible explanation for therapeutic efficacy in patients with chronic HCV infection. J Med Virol 2003, 69:50–58.
3. Alatrakchi N, Di Martino V, Thibault V, Autran B, and the ALT and IMMUNE-VIRC ANRS Study Groups. Strong CD4 Th1 responses to HIV and hepatitis C virus in HIV-infected long-term non-progressors co-infected with hepatitis C virus. AIDS 2002, 16:713–717.
4. Cramp ME, Rossol S, Chokshi S, Carucci P, Williams R, Naoumov NV. Hepatitis C virus-specific T-cell reactivity during interferon and ribavirin treatment in chronic hepatitis C. Gastroenterology 2000, 118:346–355.
5. Legrand E, Neau D, Galperine T, Trimoulet P, Moreau JF, Pitard V, et al. CD4 T lymphocyte proliferative responses to hepatitis C virus (HCV) antigens in patients coinfected with HCV and human immunodeficiency virus who responded to anti-HCV treatment. J Infect Dis 2002, 186:302–311.
6. Pitcher CJ, Quittner C, Peterson DM, Connors M, Koup RA, Maino VC, Picker LJ. 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, 5:483–484.#m AcknowledgementsThe authors would like to thank E. Lefranc and C. Baude from the Laboratory of Cellular Immunology for their technical support.
© 2004 Lippincott Williams & Wilkins, Inc.