Persistence of HIV-1 Structural Proteins and Glycoproteins in Lymph Nodes of Patients Under Highly Active Antiretroviral Therapy

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, a 3CD camera, and a laminvudine, neviraprine, and nelfinavir. The cases for the study personal computer-based image analysis system (KS 4000; Kontron, were selected from these clinical trials according to the following Esching, Germany). Cells expressing HIV-1 RNA were counted, criteria: (i) the availability of paraffin-embedded as well as frozen the area of the section measured, and the frequency of RNA- tissue specimens in liquid nitrogen; (ii) the presence of a marked producing cells per mm2 of tissue section calculated. follicular hyperplasia (FH) in LNs at the baseline; (iii) the follow-up biopsies should show the persistence of FH in some patients and HIV-1 Antibodies to HIV-1p17, HIV-1p24, and HIV-1env Detected by normalization of the follicles in others. From the PEGI clinical trial, ELISA. Antibody titers to HIV-1IIIBp17, -p24, and -gp120͞160 were one case was with FH and one with nonenlarged GCs. Biopsies of determined by ELISA according to the manufacturer’s instructions axillary LNs were performed in patients before initiation of treat- (Advanced BioScience Laboratories). Briefly, Nunc Maxi-sorp ment and during therapy lasting 5–13 months, when plasma viremia (Nalge Nunc) plates were coated with protein (1 ␮g͞well) in 100 ␮l exhibited Ͻ25 copies per milliliter. As controls, stored LN tissues of coating buffer [Protein Detector horseradish peroxidase (HRP) with FH not related to HIV-1 infection were selected from our files. ELISA kit, Kirkgaard & Perry Laboratories] overnight at 4°C. The plate was then emptied and blocked with 300 ␮l of blocking buffer Lymphocyte Subsets. Lymphocyte subsets were evaluated on fresh (BSA) for1hatroom temperature. After blocking, sera were tested whole blood samples from these patients by flow cytometry (FAC- atdilutionstartingfrom1:100inblockingsolutionfor1hat37°C. SCalibur, Becton Dickinson) by using monoclonal antibodies di- ϩ The plate was then washed five times with wash buffer and rected against CD3ϩ, CD4 , and CD8ϩ (Becton Dickinson), as incubated with the secondary antibody (goat anti-mouse, rabbit, or described (35). Values of the lymphocyte subsets determined ␮ ͞ ␮ before and during HAART are listed in Table 1. human HRP antibody, 1 g ml in 100 l of BSA diluents) for 1 h at 37°C. After incubation, the plates were washed again five times ␮ ͞ HIV-1 RNA Assay. Viremia was measured in plasma samples by with wash buffer, and 100 l of a 1:1 solution of HRP substrate detection of HIV-1 RNA using Roche Amplicor PCR (Roche H2O2 was added. The substrate was incubated for 10 min at room Diagnostics, Mannheim, Germany). Before initiation of the anti- temperature before adding the stop solution, and the reading was retroviral therapy as well as subsequently in the course of therapy, done at the absorbance 450 nm. Values were considered positive HIV-1 RNA copy numbers were monitored in a single laboratory when the readings of the absorbance in the tested samples were from 6 to 13 months. When values fell below 400 copies of vRNA 2-fold higher that those in control samples. per milliliter, an ultrasensitive modification of the assay was applied by using 1 ml of plasma (Cobas Amplicor HIV-1 Monitor, Version Neutrialization of R5 and X4 HIV-1. Neutralization of R5 and X4 was ␤ 1.5, ultrasensitive protocol, Roche Diagnostics). The detection limit tested in a -galactosidase chemiluminescent assay that has been ͞ ͞ of vRNA was Ͻ25 viral copies per milliliter of plasma. Detected described (39). Briefly, U373 CD4 MAGI cells expressing either HIV-1 copy numbers before and during therapy are listed in CCR5 or CXCR4 were allowed to attach overnight to 96-well Table 1. flat-bottom tissue culture plates. The next day, the medium was replaced with serial dilutions of antibody in culture medium and 50 Light Microscopy. Biopsies of LN tissues were fixed in 4% neutral- TCID50 (tissue culture 50% of infective dose) per well of either the buffered formalin overnight, embedded in paraffin, and stained HIV-193BR020 (R5) or the HIV-1MN (X4) laboratory isolates. After with hematoxylin͞eosin, Giemsa, or Gomori silver impregnation 24 h, the cells were washed and maintained in fresh culture medium for routine histology. Portions of LNs were embedded in tissue- for 6–7 days. Infectivity was then determined by a chemilumines- freezing medium (Leica, Nussloch, Germany), snap-frozen in liquid cence assay (Applied Biosystems) according to the manufacturer’s nitrogen, and stored at Ϫ70°C until use. protocol.

Immunohistochemistry. Before staining, frozen sections of LNs were Results cut, fixed in 4% paraformaldehyde, and incubated with primary Detection of HIV-1 RNA in Patients Before and During Treatment with antibodies against HIV-1 proteins: p17 (BT-2, a mouse monoclonal HAART. As shown in Table 1, HIV-1 viremia was monitored by antibody, Advanced BioScience Laboratories) and p24 (DAKO) at RT-PCR detection of vRNA in plasma samples obtained from room temperature for 30 min. To detect HIV-1Env tissue, sections seven patients before initiation of antiretroviral therapy and during of LNs were incubated with ant-gp120 mouse monoclonal antibod- HAART. The detected base-line values in these patients for HIV-1 ies M85–2 or M777B3–4 (Advanced BioScience Laboratories) at in plasma before therapy were in the range from 3 ϫ 103 to 6.3 ϫ 4°C overnight. Antibody binding was visualized by using the alka- 105 vRNA copies per ml. During antiretroviral therapy, vRNA in all

14808 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0506857102 Popovic et al. Downloaded by guest on October 2, 2021 Fig. 1. Changes in HIV RNA burden in the LNs during HAART. In situ hybridization for vRNA (green) before HAART (A) shows diffuse hybridization signals in the GC, representing FDC-bound vRNA and many virus-producing cells (arrow). The former is under limit of detection in the follow-up biopsy (B). Cells with HIV-1 gene expression are rare (arrow).

seven patients was consistently below detectable levels (Ͻ25 copies per ml).

Detection of HIV-1 RNA in LNs by in Situ Hybridization. In contrast to HIV-1 antigens, the amounts and distribution of HIV-1 RNA detected by in situ hybridization showed marked differences before initiation of treatment and during HAART. As shown in Fig. 1A, heavy diffuse autoradiographic signals representing FDC-bound HIV-1 RNA were seen in the GCs of LNs from patients before treatment. In patients under HAART, the diffuse labeling was absent even in GCs with well developed light zones (see Fig. 1B). Before treatment, single cells showing HIV-1 gene expression were always present, mainly but not exclusively in the GCs. The frequency of these cells per mm2 of tissue varied between 0.7 and 3.7. This contrasted sharply with follow-up biopsies in which cells with vRNA were very rare (0–0.005 HIV-1 RNA-positive cells per mm2 of tissue section) and showed no preferential localization within the microenvironment of the LN. Furthermore, all productively infected cells showed fewer silver grains than cells in the first LN biopsies from patients before therapy. Because the amount of hybridization signals parallels the amount of HIV-1 RNA produced by cells (11), the finding suggests that under HAART, only low levels of HIV-1 are produced per cell. Thus, the in situ hybridization data, namely the detection of HIV-1 vRNA in GCs of LNs prior to the therapy of these patients, coincided with viremia. Fig. 2. Persistence of HIV-1 structural proteins in the GC of patients under Morphology and Immunohistochemical Analyses of Patients’ LNs. As HAART. Frozen sections were immunolabeled for the capsid p24 (A and B), the described above, the vRNA detections, LN biopsies from the seven matrix p17 (C and D), and the envelope glycoprotein gp120 (E and F). The patients with chronic HIV-1 infection, were also investigated for the amounts of HIV-1p24 and -p17 deposited in the GCs show no differences at

presence of viral structural proteins and glycoproteins before baseline (A and C) and in the follow-up biopsy (B and D). The HIV-1gp120 was MEDICAL SCIENCES treatment and during HAART (see Table 1). In five patients from also detected in the follow-up biopsy (E) but not in GCs of tonsils obtained one study (COSMIC), LN biopsies were obtained while patients from an HIV-1 antibody-negative patient (F). were under antiretroviral therapy that lasted 6–13 months. In two patients from the second study (PEGI), biopsies were obtained performed on frozen sections by using highly specific mouse after 5 months of therapy. The conventional histology of LN biopsies showed a marked FH at the baseline. The large irregularly monoclonal antibodies. In all seven patients, the major capsid shaped follicles occupied up to two-thirds of the cross-sectional area HIV-1p24, the matrix HIV-1p17 proteins, and the viral envelope of the LNs. The dark zone of the GC was broad and contained protein HIV-1gp120 were readily detected in the LN tissues ob- numerous centroblasts, many of them in mitosis, and tangible body tained before as well as during treatment (see also Table 1). The macrophages. In the follow-up biopsies, the histology was un- staining patterns of these HIV-1 proteins in tissue specimens of LNs changed or improved toward normal LN structure that manifested from patients before and under HAART are depicted in Fig. 2 itself by a slight enlargement of one or two regularly shaped GCs. A–D. The detection of HIV-1gp120 in the tissue specimen is shown In two subjects, the FH resolved. In these cases, the LNs showed from a patient under treatment in parallel with a control tissue active but nonenlarged and regularly shaped GCs. exhibiting FH not related to HIV-1 infection (see Fig. 2 E and F). Immunohistochemical staining of tissue specimens from LNs for Large deposits of the HIV-1 antigens were present in the light zone detection of HIV-1 structural proteins and glycoproteins was of the GCs containing centrocytes, some CD4ϩ T cells, and a dense

Popovic et al. PNAS ͉ October 11, 2005 ͉ vol. 102 ͉ no. 41 ͉ 14809 Downloaded by guest on October 2, 2021 mesh of FDC. As expected, the staining for structural proteins and glycoproteins showed a reticular pattern resembling the FDC network in LNs. In follow-up biopsies, HIV-1 antigens were always detected, even in cases that showed remission of the FH. The dark zone of GC showed no staining for HIV-1 antigens, and preparations were negative for these antigens so long as tissue sections did not include the light zone. As shown in Fig. 2, the intensity of staining and its pattern for the HIV-1 proteins (-p24 and -p17) and also for the envelope glycoprotein HIV-1gp120 (untreated not shown) did not noticeably differ in tissue specimens taken from patients either before initiation of therapy or during treatment. Thus, the results from the immunohistochemical analyses strongly suggest that HIV-1 structural proteins and glycoproteins persist in GCs of LNs in patients under HAART for a long time in the absence of detectable virus replication, because both the GCs of LNs and peripheral blood samples were consistently negative for vRNA.

Antibody Responses to HIV-1 Structural Proteins and Glycoproteins. To substantiate the findings of long-term persistence of HIV-1 structural proteins and glycoproteins in GCs of LNs in these patients under HAART, the presence of specific antibodies directed to HIV-1p17, -p24, and Env (gp160) was assessed in sera by ELISA. Antibody titers were determined by end-point dilution to each of these HIV-1 antigens in sera samples obtained before treatment and during therapy that lasted from 5 to 13 months (see also Table 1). As shown in Fig. 3, significant differences in antibody titers were found in sera samples among the patients as well as in antibody responses of a patient to each HIV-1 antigen. Antibodies directed against p17 antigen exhibited the lowest titers in these patients and were in the range from 1:100 to 1:3,200 (see Fig. 3A). Antibody titers against p24 exhibited a very broad range from 1:400 to 1: 4,000,000 (see Fig. 3B). The strongest antibody responses in a more narrow range, from 1:512,000 to 1:2,048,000, were found against HIV-1gp160 (see Fig. 3C). In most cases, however, sera samples from patients obtained before treatment with high antibody titers against gp160 had also higher titers against p24 and p17. A serum sample (code no. 6-B70) from one patient under HAART was negative for anti-p17 antibodies and exhibited lower titer to p24 (1:200) and higher titer to gp160 (1:256,000). In this particular case, serum was available only from the time of therapy. In general, there was a tendency toward a decline in antibody titers in sera obtained from patients under HAART. As shown in Fig. 3, in most cases, an Ϸ4-fold decrease was observed in antibody titers against p24 and Ϸ p17 and less to gp160 ( 2-fold). As pointed out by others in earlier Fig. 3. Detection-speciﬁc anti-HIV-1 antibodies directed against the matrix studies, the observed differences between p24 and gp160 antibody protein p17 (A), the capsid protein p24 (B), and the envelope glycoproteins titers and kinetics may reflect different mechanism in the regulation (gp160) (C). Note the persistence of the HIV-1 antibody titers evaluated by of anti-p24 and -gp120 responses. It is possible that the generation ELISA. There was an Ϸ2- to 4-fold decrease in antibody titers against these of anti-HIV-1p24 and also -HIV-1p17 antibodies is helper T HIV-1 structural proteins and glycoproteins under HAART. cell-dependent, whereas anti-HIV-1gp120 antibody production is helper T cell-independent and more directly responsive to the amount of viral antigen (19, 40). titers exhibited 2- to 4-fold decreases in those sera obtained from patients during treatment. Presence of HIV-1 Neutralizing Antibodies. We have further charac- In conclusion, patients under long-term antiretroviral therapy terized gp160 antibodies for their capacity to neutralize HIV-1 R5 not only retained HIV-1 structural proteins and glycoproteins and X4 isolates. The results from virus-neutralization experiments deposited in GCs of lymph-nodes but also maintained specific are shown in Fig. 4. The HIV-193BR020 isolate of R5 phenotype antibody responses to these HIV-1 antigens in the absence of and the HIV-1MN isolate of X4 phenotype were used in these detectable virus replication. experiments, respectively. The neutralization capacity of sera was lower for the HIV-1 R5 isolate and reached its plateau for most sera Discussion samples at dilution 1:80, whereas neutralizing titers for the The results described in this paper provide evidence that, in HIV-1 X4 isolate were at 1:320 dilution with these sera. These data addition to the capsid protein HIV-1p24, the matrix protein HIV- are in agreement with earlier observations that an HIV-1 isolate of 1p17 and the envelope glycoproteins HIV-1Env (gp120͞gp41) are X4 phenotype adapted for growth in T cell lines is more sensitive also accumulated in the GC light zone of LNs in patients who were to neutralizing antibodies (41, 42). Comparing neutralizing capacity treated with antiretroviral drugs for the entire period of this study, of paired sera samples obtained from patients before and during up to 13 months. Previous studies focused on immunohistochemical HAART, the same pattern of decline was found as in the case of detection of only one viral structural protein, namely p24, in anti-HIV-1gp160 antibody detection by ELISA. The neutralizing patients under HAART (32–36). Although limited to the detection

14810 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0506857102 Popovic et al. Downloaded by guest on October 2, 2021 Long-lived GCs with persisting antigens were also observed in mice after immunization with a recombinant vesicular stomatitis virus (VSV)-glycoprotein. VSV-specific B cells were associated with the persisting antigen and also produced immunoglobulins, hence strongly suggesting that FDC-bound viral antigens in the long-lived GCs most probably are involved in the maintenance of serological IgG memory (43, 44). The persisting humoral response to HIV-1 structural proteins and glycoproteins in patients under HAART very likely represent ‘‘conserved’’ antibodies, because they were generated and maintained mainly by the FDC antigenic pool from the virus before its replication was suppressed by therapy. Consequently, this ‘‘conserved’’ humoral response could provide an advantage for emergence of new HIV-1 variants resistant to the ‘‘historical’’ neutralizing antibodies. It will be important to compare patients’ sera samples obtained before and during HAART and characterize in detail the reactivity of these ‘‘conserved’’ antibodies against HIV-1gp120͞gp41, particularly in those cases when HIV- 1-replicating variants emerge after long-term therapy. More de- tailed analyses of these antibodies can contribute to a better understanding of HIV-1 persistence in vivo. A characteristic feature of HIV infection is B cell hyperactivity, manifested by marked hypergammaglobulinemia (1, 2). In the peripheral circulation of HIV-1-infected patients, B cells secrete abundant amounts of immunoglobulins, a fact that was also demonstrated by HIV-1 antibody production in vitro (8) and expression of B cell activation markers (3, 4). Several mechanisms have been suggested for excessive B cell activation in HIV-1 disease, including the direct stimulatory effects of viral proteins on B cells and induction of B cell stimulatory cytokines associated with HIV-1 infection (25). In addition, enhanced T cell help has been implicated in the induction of polyclonal B cell activation (45, 46). It has been demonstrated that the envelope glycoproteins induce differentia- Fig. 4. Detection of virus neutralization antibodies against the R5 (A) and the X4 (B) HIV-1 isolates. Also the virus neutralizing antibodies persisted tion of B lymphocytes from normal volunteers into Ig-secreting cells during the long-term HAART. (20, 25). The induction of B cell differentiation is a complex process and is T cell-dependent, requiring contact between T cells exposed to HIV-1Env and B cells (20). Although several cell-surface molecules have been implicated into cell-to-cell contact-dependent of one viral structural protein for a short period, these studies have interaction in HIV-1-induced B cell activation, the exact nature of already suggested the persistence of HIV-1 proteins and glycopro- the cell-surface molecules involved is not clear. In this context, the teins in GCs of LNs, because vRNA diminished to undetectable recent work of Hunziker et al. (47) using an animal model contrib- levels in patients’ plasma and LN biopsies within 2–3 months from utes to a better understanding of the mechanism of hypergamma- initiation of therapy (33–36). We extended this observation and globulinemia in virus infection caused by pathogens that do not firmly established that all major components of viral structural infect B cells. In mice infected with lymphocytic choriomeningitis proteins and glycoproteins persist in GCs of LNs in patients virus (LCMV), a polyclonal hypergammaglobulinemia developed. chronically infected with HIV-1 in the absence of detectable virus More than 90% of the IgG-producing cells were nonspecific for replication under HAART for long periods of time. The persis- LCMV. Hypergammaglobulinemia did not depend on IL-6 or B cell tence of these HIV-1 antigens in GCs of LNs was accompanied by receptor specificity. The increase in IgG production resulted from the presence of specific antibodies recognizing HIV-1p17, -p24, and switching natural IgM specificities to IgG in preimmune B cells with -gp120͞gp41, respectively. receptors that were not specific for the LCMV antigen. The process In this study, all seven patients responded well to HAART. The depended on CD4ϩT cells and the CD40L molecule. Hypergam- results of the in situ hybridization coincided with the data of the viral maglobulinemia did not develop in mice deficient either in TCR or load detection in plasma. Because in these patients the peripheral LD40L molecules. It is well established that CD40L is critical for MEDICAL SCIENCES virus burden was consistently below the limit of detection for at least GC formation and the Ig isotype switch that takes place mainly in 6 months, HIV-1 antigens could not be captured from the blood the GC of LNs (48, 49). It is noteworthy that both viruses, HIV-1 circulation. Although residual HIV-1 replication occurs in patients and LCMV, show tropism for lymphoid organs and macrophages, with undetectable vRNA both in plasma and in GC LNs (35, 38), and hypergammaglobulinemia develops during infection with these viruses when high amounts of viral antigens are generated. it is unlikely that few productively infected cells found in LN Viremia in HIV-1-infected people is associated with generalized biopsies of these patients (in the present study, a maximum of six B cell dysfunction, resulting in the appearance of a phenotypically RNA positive cells per 50 sections) provided the amounts of viral distinct subpopulation of B cells (20). This B cell subpopulation with proteins and glycoproteins that could replenish the FDC antigen– plasmacytoid morphology and reduced expression of CD21 is a antibody pool (see also Results). All LNs examined were consis- poor responder to B cell stimuli and secretes high levels of immu- tently negative for FDC-bound vRNA. It is most likely that HIV-1 noglobulins (20). During effective antiretroviral therapy, levels of structural proteins and glycoproteins detected in the follow-up serum immunoglobulins and frequency of Ig-secreting cells are biopsies represent immune complexes captured by FDCs mainly normalized in HIV-1-infected patients, thereby strongly suggesting before the initiation of the therapy. Thus, the GCs of LNs positive that viremia drives B cell hyperreactivity in vivo (20, 50). Despite for HIV-1 antigens are long-lived where the viral structural proteins effective elimination of viremia, the immune system in a number of and glycoproteins persist for a long time. patients is not fully recovered to normal, and there is a discrepancy

Popovic et al. PNAS ͉ October 11, 2005 ͉ vol. 102 ͉ no. 41 ͉ 14811 Downloaded by guest on October 2, 2021 between the rapid decline of circulating Ig-secreting cells and a slow of cytokines and chemokines that are critical in activation, recruit- decline of IgG in the sera of patients during therapy. Patients ment, and cell proliferation (23–25). successfully treated with anti-HIV-1 drugs generate specific anti- Introduction of HAART has significantly prolonged and im- bodies to HIV-1 in the absence of detectable vRNA, suggesting proved the quality of life in individuals infected with HIV-1. This persistent immune system activation by retention of HIV-1 antigens potent therapy has effectively controlled not only HIV-1 load and over long periods (19, 21). Based on these observations, Morris et replication but also the occurrence of AIDS-associated malignan- al. (50) postulated that production of polyclonal IgG molecules in cies such as Kaposi sarcoma and B cell lymphoma of the CNS (10, some compartment that is not in equilibrium with other compo- 53, 54). It is well established that B cell stimulation and prolonged nents of the immune system is sustained for some time. Residual immune deficiency are main risk factors for non-Hodgkin’s lym- HIV-1 replication and the presence of persistent antigen trapped on phoma (NHL) in people with AIDS (10). Given that at least the FDCs were suggested as complementary factors sustaining immune viral structural glycoproteins HIV-1gp120͞gp41 and the matrix response under HAART. Our study has established that HIV-1 protein HIV-1p17 possess a capacity to actively interact with cells proteins and glycoproteins are trapped and persist in GCs of LNs of the immune system (23–25), the long-term presence in GCs of in patients before and after containment of virus by therapy. LNs points to the fact that at least one risk factor for NHL was not In recent years, there has been discussion of the role of FDC- eliminated by antiretroviral therapy. Earlier observations showed bound antigens in specific B cell responses, mainly in the regulation that the serum-soluble CD23 level correlates with the subsequent of serum IgG. Some researchers question the role of FDC-bound development of AIDS-related NHL (55, 56), and colocalization of antigen in the GC reaction and maintenance of serological memory CD23 expression in FDCs bounding the HIV-1 antigen–antibody (51). A majority of researchers in this field, however, emphasize the importance of FDC and the GC reaction in maintaining serological complexes in GCs of LNs was also demonstrated (32). memory (44, 48, 49). In this context, it should be pointed out that HIV-1 transgenic rats harboring an HIV genome deleted in p24 This paper is dedicated to Dr. Jan Svoboda, D.Sc, on the occasion of his 70th birthday. We thank Irene Kalisz, Sonya Geborgi, Petra Meyer, generate enlarged GCs in lymphatic tissues, morphologically almost Gudrun Grossschupff, and Birgit Raschdoff for excellent technical indistinguishable from those in HIV-1 infected individuals (52). assistance. This investigation was supported by the National Institutes of Consequently, we have focused our efforts on studies of p17 and Health, National Institute of Allergy and Infectious Diseases Grant Env persistence in GCs of LNs. These findings may be relevant to 1R03AIo55345-01; Concerned Parents for AIDS Research, Cigarette lymphopathogenesis involving GC formation and B cell activation, Restitution Funds; Fondazione Cassa di Risparmio di Perugia (Italy); because both these viral structural protein and glycoproteins inter- and the German Ministry of Education and Research (Bundesministe- act with mononuclear cells and induce and͞or enhance a number rium fu¨r Bildung und Forschung), Contract KompNet 01KI0211.

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