Persistence of HIV-1 Structural Proteins and Glycoproteins in Lymph Nodes of Patients Under Highly Active Antiretroviral Therapy
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Persistence of HIV-1 structural proteins and glycoproteins in lymph nodes of patients under highly active antiretroviral therapy Mikulas Popovic*†, Klara Tenner-Racz‡, Colleen Pelser*, Hans-Jurgen Stellbrink§, Jan van Lunzen§, George Lewis*, Vaniambadi S. Kalyanaraman¶, Robert C. Gallo*†, and Paul Racz‡ *Institute of Human Virology, University of Maryland Biotechnology Institute, University of Maryland, Baltimore, MD 21201; ‡Bernhard-Nocht Institute for Tropical Diseases, 2000 Hamburg 4, Germany; §University Hospital Eppendorf, D-20246 Hamburg, Germany; and ¶Advanced BioScience Laboratories, Kensington, MD 20895 Contributed by Robert C. Gallo, August 12, 2005 Here we report a long-term persistence of HIV-1 structural proteins a slow and incomplete process in a number of long-term treated and glycoproteins in germinal centers (GCs) of lymph nodes (LNs) patients (19–22). in the absence of detectable virus replication in patients under Earlier in vitro studies explored interactions of mononuclear cells highly active antiretroviral therapy (HAART). The persistence of from peripheral blood with native or recombinant HIV-1 structural viral structural proteins and glycoproteins in GCs was accompanied proteins and glycoproteins, with the matrix protein HIV-1p17 (23, by specific antibody responses to HIV-1. Seven patients during the 24), and particularly with the HIV-1Env (gp120͞160) (25). These chronic phase of HIV-1 infection were analyzed for the presence of extensive studies of B and T cell interactions with HIV-1Env and the capsid protein (HIV-1p24), matrix protein (HIV-1p17), and HIV-1p17 showed a broad spectrum of changes in cell surface envelope glycoproteins (HIV-1gp120͞gp41), as well as for viral RNA markers, cytokine production, B cell maturation, and increased T (vRNA) in biopsy specimens from LNs obtained before initiation of cell proliferation and HIV-1 replication in the virus-infected T cell therapy and during HAART that lasted from 5 to 13 months. In cultures (23–25). However, the significance of these studies has parallel, these patients were also monitored for viremia and been in question because of the absence of clear-cut in vivo evidence specific anti-HIV-1 antibody responses to structural proteins and demonstrating persistence of HIV-1 structural proteins and glyco- glycoproteins both before and during treatment. Before-therapy proteins accessible to mononuclear cells. -viral levels, as determined by RT-PCR, ranged from 3 ؋ 103 to 6.3 ؋ Observations from earlier studies demonstrated that, in un 105 copies of vRNA per ml, whereas during treatment, vRNA was treated individuals infected with HIV-1, the gag proteins (the under detectable levels (<25 copies per ml). The pattern of vRNA capsid HIV-1p24 and -p17) can be consistently detected in germinal detection in peripheral blood was concordant with in situ hybrid- centers (GCs) of the lymphoid tissue (26–30). Double immunola- ization results of LN specimens. Before treatment, vRNA associated beling for the HIV-1gag proteins and for either markers of follicular with follicular dendritic cells (FDCs) was readily detected in GCs of dendritic cells (FDCs) or IgM revealed colocalized staining on the LNs of the patients, whereas during therapy, vRNA was consis- surface of FDCs (28). Because IgM is only bound to and not tently absent in the GCs of LN biopsies of treated patients. In produced by FDCs, the finding indicates that the HIV-1gag protein contrast to vRNA hybridization results, viral structural proteins and in the GC is located on FDCs extracellularly and very likely, these glycoproteins, evaluated by immunohistochemical staining, were antigen–antibody complexes are accessible to mononuclear cells. present and persisted in the GC light zone of LNs in abundant Importantly, long-term retention of antigen–antibody complexes on amounts not only before initiation of therapy but also during FDCs was documented in experimental animal studies during HAART, when no vRNA was detected in GCs. Consistent with immunization (31). immunohistochemical findings, specific antibody responses to HIV- Since the introduction of HAART, HIV-1 infection has been 1p17, -p24, and -gp120͞gp41, as evaluated by ELISA and virus effectively controlled in a large number of patients for years. In neutralization, persisted in patients under therapy for up to 13 treated patients, HIV-1 RNA [viral RNA (vRNA)] in blood months of follow-up. The implications of these findings are dis- frequently declines below detectable levels (11, 12). Although it is cussed in relation to HIV-1 persistence in infected individuals and well established that HIV-1 infection and replication take place the potential role of chronic antigenic stimulation by the deposited mainly in the lymphatic tissue, a limited number of systematic structural proteins in GCs for AIDS-associated B cell malignancies. studies have been performed analyzing HIV-1 status in lymph node (LN) biopsies from patients before and during long-term therapy. IV infection is characterized by a severe impairment of both In these studies, the persistence of HIV-1p24 was observed in GCs MEDICAL SCIENCES Hcellular and humoral immunity. Both T and B cell compart- of LNs in patients chronically infected with HIV-1 who were under ments are profoundly altered (1, 2). Parallel with immune- HAART and exhibited vRNA below detectable levels in blood and persistent activation of these compartments (1–4), HIV-infected LN specimens (32–36). These studies did not address the persis- individuals show decreased humoral responses to antigens (5–7). tence of other HIV-1 structural proteins and glycoproteins in The alteration of B cells is manifested by hypergammaglobulinemia HIV-1 infected patients under long-term antiretroviral therapy. (1, 2), increased spontaneous antibody secretion in vitro (8), en- Taking into consideration earlier in vitro studies on the capacity of hanced levels of autoantibodies (9), and increased incidence of B HIV-1p17 and -1Env to induce a broad spectrum of changes in cell lymphomas (10). The widespread use of highly active antiret- mononuclear cells (23–25), it was important to establish the long- roviral therapy (HAART) has substantially modified the natural history of HIV-1 infection. The effects of this therapy are mani- These data were presented at the International Meeting of the Institute of Human fested by a strong suppression of viral replication in the peripheral Virology, Sept. 29–Oct. 3, 2003, Baltimore, MD. blood and in lymphoid tissue in individuals infected with HIV-1 (11, Abbreviations: LN, lymph node; GC, germinal center; FDC, follicular dendritic cell; FH, 12). As a result, CD4ϩT cell counts increase, T cell activation follicular hyperplasia; HAART, highly active antiretroviral therapy; vRNA, viral RNA. decreases, and antigen-specific and nonspecific T cell function †To whom correspondence may be addressed. E-mail: [email protected] or improves (13–18). Similarly, B cell responses are normalized, [email protected]. although this reversal of profound alteration of immune system is © 2005 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0506857102 PNAS ͉ October 11, 2005 ͉ vol. 102 ͉ no. 41 ͉ 14807–14812 Downloaded by guest on October 2, 2021 term persistence of these viral structural protein and glycoproteins line phosphatase antialkaline phosphatase technique, with New in patients under HAART. In this paper, we provide evidence Fuchsin as red chromogen (30, 32). demonstrating the long-term persistence of HIV-1p17, -p24, and -gp120͞gp41 in GCs of LNs in seven HIV-1-infected patients under In Situ Hybridization. HIV-1 RNA detection was performed on HAART. These viral structural proteins and glycoproteins persist paraffin and frozen sections using a 35S-labeled single-stranded in LNs in the absence of detectable HIV-1 replication and were (antisense) RNA probe (Lofstrand Laboratories, Gaithersburg, accompanied by the presence of specific anti-HIV-1 antibodies in MD). The probe was from 1.4- to 2.7-kb fragments representing patients’ sera. Ϸ90% of the HIV-1 genome (37). The in situ hybridization pro- cedure was described in detail elsewhere (32, 37). From each biopsy, Materials and Methods 18–50 sections were hybridized, because it is known that in patients Patients. A total of seven patients chronically infected with HIV-1 who respond well to HAART, cells expressing HIV-1 RNA are were included in this study. The patients’ code numbers, dates of rare (38). As a positive control, cytospin preparations of H9 cells procurement of blood, plasma, sera, and biopsy specimens from infected with HIV-1 were hybridized with the same probe. As a LNs obtained before treatment and during HAART are listed in negative control, sections from each LN specimen were hybridized Table 1, which is published as supporting information on the PNAS with a radiolabeled RNA sense-strand probe. Slides were dipped web site. Five patients were from the clinical study ‘‘COSMIC’’ (35) into photo emulsion (NTB2; Kodak) and exposed in the dark at 4°C and were treated by the following antiviral drugs: stavudine, for 7 days. The slides were developed in a developer (D19, Kodak), lamivudine, nelfinavir, saquinavir, and recombinant IL2. Results of fixed, counterstained with hematoxylin, and mounted. Examination this clinical trial have been reported (35). Two patients were from of the slides was performed with a Zeiss Axiophot microscope the clinical study ‘‘PEGI’’ and were treated with zidovudine, equipped with epiluminescent illumination,