Hepatitis B Virus DNA in Peripheral-blood Mononuclear Cells in Chronic Hepatitis B After HBsAg Clearance

ANDREW MASON,1, BORIS YOFFE,33 43 CHRISTINE NOON AN,^, MARY MEARNS,~ CAROLYN CAMPBELL,l, AMANDA KELLEY1* AND ROBERT P. PERRILLOl.

'Gastroenterology Section, Veterans Affairs Medical Center and 2Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63106; and 3Veterans Affairs Medical Center and 4Division of Molecular Virology

and 5Department of Internal Medicine, Baylor College of Medicine, Houston, Texas 77030

In this study, peripheral-blood mononuclear cells from patients with chronic hepatitis B and sponta- neous or therapy-induced disappearance of HBsAg were examined for HBV DNA. Samples were evaluated by in situ hybridization and polymerase chain reaction both before and after clearance of HBsAg. By in situ hybridization, positive signals were observed in 2 of 13 samples collected after HBsAg loss, in 8 of 15 samples before HBsAg loss and in 0 of 4 control patients without serological markers of active or prior HBV infection. When polymerase chain reaction analyses were per- formed, HBV DNA was detected in 5 of 12 HBsAg- negative samples and 10 of 15 HBsAg-positive samples from the study group. Testing of mononuclear cells after disappearance of HBsAg revealed that two of eight patients were HBV DNA positive by in situ hybridization and by polymerase chain reaction, whereas two additional patients were positive by polymerase chain reaction alone. Mononuclear cell- associated HBV DNA was detected between 2 and 9 mo after the disappearance of circulating HBsAg by in situ hybridization and as long as 4 yr later by polymerase chain reaction. These data indicate that patients who have undergone HBsAg seroconversion may none- theless harbor HBV DNA in their peripheral-blood mononuclear cells for prolonged periods. (HEPATOLOGY 1992;16:36-41.)

Like many human blood-borne viruses, HBV may replicate in cells of hemopoietic origin. Single-stranded replicative intermediates of HBV DNA have been dem- onstrated by Southern-blot hybridization in peripheral- blood mononuclear cells (PBMCs) (1-4), as have other molecular forms of HBV DNA (5-131, HBV RNA (14) and HBV proteins (11, 12). The infection of PBMCs is part

Received December 3, 1991; accepted March 4, 1992. Dr. Yoffe is supported by grants from the American College of Gastroenter-

ology, a Baylor College of Medicine-administered Biomedical Research Support Grant from the National Institutes of Health and a grant from Schering Corp (Kenilworth, NJ).

Dr. Noonan is supported by a grant from the Gulf Coast Regional Blood Center.

Address reprint requests to: Robert P. Penillo, M.D., Veterans Affairs Medical Center (111 JC), 915 N. Grand Blvd., St. Louis, MO 63106.

31/1/37864

of a diffuse extrahepatic life cycle of hepadnaviruses that occurs during states of acute and chronic infection. We recently demonstrated that HBV DNA can be detected in spleen, lymph node and other extrahepatic tissues (15-17). Similar observations have been made in chim- panzees infected with human HBV and in woodchucks and Pekin ducks infected with hepadnaviruses (18-20). However, the biological relevance of the extrahepatic life cycle of these viruses is still poorly understood, espe- cially with regard to viral latency in PBMCs.

Persistent detection of HBV DNA in peripheral lymphoid cells commonly occurs in HBsAg-negative immunocompromised patients, even in the absence of circulating markers for previous HBV infection (4,6,8). In contrast, persistent HBV DNA has infrequently been demonstrated in immunocompetent patients who have cleared the virus (5,7,21). However, the latter group has not been studied extensively, and the data are noncom- parable because assays of different sensitivities have been used. Recently HBV DNA was detected by poly- merase chain reaction (PCR) in the livers of patients with chronic hepatitis B as long as 5 yr after clearance of HBsAg from serum (22, 23). In that study, the great majority of serum samples obtained at the time of liver biopsy were HBV DNA negative by PCR (22). We have now expanded these preliminary studies to determine whether HBV DNA can also be detected in PBMCs by both PCR and in situ hybridization.

MATERIALS AND METHODS Study Population. The study group consisted of eight

patients (three women and five men; mean age 44 & 12 yr) who had participated in one of three different clinical research protocols on antiviral therapy for chronic hepatitis B. All participants in this study had histological evidence of chronic hepatitis, persistent elevations in serum ALT and AST, detectable HBV DNA or DNA polymerase in serum and both HBsAg and HBeAg reactivity for at least 6 mo. Disappearance of HBsAg generally occurred in each patient months to years after HBeAg seroconversion. Two of these patients were untreated, five patients had been treated with interferon and one patient had been treated with a combination of short-term prednisone and adenosine arabinoside monophosphate. Loss of

36

Vol. 16, No. 1, 1992 HBV DNA IN PBMCs AFTER LOSS OF HBsAg 37

HBsAg was associated with improvement of liver histological appearance and sustained normalization of serum aminotrans- ferase levels in each case. One of the study group patients was also positive for antibody to human immunodeficiency virus- 1 but had no features consistent with AIDS.

Thirteen PBMC samples were harvested from the study group at intervals of 2 to 67 mo after the disappearance of HBsAg and 3 to 87 mo after the loss of HBeAg. Seven of the patients displayed protective levels of antibody to HBsAg (anti-HBs) ( > l o mIU/ml) at the time of disappearance of HBsAg. These antibody responses were sustained throughout the study interval, with the exception of one patient.

Fifteen additional PBMC samples were harvested from these patients while circulating HBsAg was still detectable. In this instance, PBMCs were obtained while the patients were HBeAg and HBV DNA positive (n = 10) or HBeAg and HBV DNA negative (n = 5). PBMCs were obtained from two patients with chronic non-A, non-B hepatitis and from two uninfected, healthy volunteers and used as controls. All control patients were negative for serological markers of active or past HBV infection. In Situ Hybridization. The methods for riboprobe prepa-

ration and in situ hybridization have been described in detail elsewhere (24). A 35S-labeled riboprobe, specific for HBV DNA, was synthesized from a 310-bp complementary DNA of the HBV (uyw) surface antigen gene (a kind donation from D. A. Shafritz, M.D., Albert Einstein College of Medicine, New York, NY). Likewise, 36S-labeled riboprobes were synthesized as “antisense” and “sense” probes from a 350-bp complementary DNA segment of the human heat-shock protein 90 gene (Hu hsp 90) (a kind donation from N. Rebbe, Ph.D., Washington University School ofMedicine, St. Louis, MO) to act as positive and negative controls, respectively.

PBMCs were isolated by Ficoll-Hypaque (Sigma Chemical Co., St. Louis, MO) density-gradient centrifugation and stored in liquid nitrogen until use. After thawing, the PBMCs were washed with PBS (pH 7.4), and approximately 5 x lo5 cells were pipetted onto poly-L-lysine (Sigma Chemical Co.)- treated slides. Each sample was analyzed four times with the HBsAg gene probe and once each with the “sense” and “antisense” Hu hsp 90 gene probes. After air drying, the PBMCs were fixed with 4% paraformaldehyde for 20 min and washed in PBS. Before hybridization, the cells were digested with proteinase-K, pretreated with acetic anhydride and heated at 95” C for 10 min to denature the DNA. Hybridization with the radiolabeled riboprobes was performed for 14 hr at 50” C. After high-stringency washes, unhybridized riboprobe was dlgested with RNAse. The slides were then coated with autoradiographid emulsion and incubated for 6 wk at 4” C. After development, the samples were counterstained with hematoxylin and eosin (24). PCR. PBMCs were washed three times in PBS (pH 7.4);

then total cellular DNA was isolated as described previously (25). The cell supernatant was saved as a control for HBV DNA contamination of the PBMCs. The oligonucleotide primers used in the amplification reaction were derived from the genomic sequence of a highly conserved region of the HBcAg gene (adw subtype of HBsAg [251). Both the cell supernatant and 250 ng of cellular DNA from each PBMC sample were amplified for 30 cycles in a total reaction volume of 50 pl with a GeneAmp kit (Perkin-Elmer Cetus, Norwalk, CT) and automated instrumentation (DNA Thermal Cycler, Perkin- Elmer Cetus) according to the manufacturer’s instructions. In the first cycle, DNA was denatured for 3 min at 94” C, whereas in subsequent cycles a 1-min denaturation was performed at the same temperature. The annealing and extension steps

were performed for 30 sec at 49“ C and 1 min ( + 5 sedcycle) a t 72” C, respectively. Well-defined internal negative and positive control samples were included in each experiment. The amplified products were separated by agarose-gel electro- phoresis, stained with ethidium bromide, transferred to nylon filters by capillary transfer and then hybridized under condi- tions of high stringency to a full-length, 32P-labeled HBV DNA probe. The levels of sensitivity of the assay were monitored by the titration of cloned HBV DNA and were estimated to be at least 0.00 1 genome equivalents/cell for agarose-gel electro- phoresis and ethidium bromide staining and up to 0.00001 genome equivalents/cell for DNA/DNA hybridization analyses of amplified cell-associated DNA.

RESULTS The results of the in situ hybridization and PCR

procedures are summarized in Table 1. Of the 28 PBMC samples from the study group that were analyzed by in situ hybridization, 10 were positive for HBV DNA. Two of these samples were harvested while the patients were HBsAg negative and eight were taken while patients were HBsAg positive. Notably, a strong hybridization signal was observed in the 10 HBV DNA-positive samples (Fig. 1A and B). In contrast, a weak hybrid- ization signal was observed in all the PBMCs studied with the Hu hsp 90 “antisense” probe (positive control), and the Hu hsp 90 “sense” probe (negative control) gave uniformly negative results (data not shown). None of the four HBV negative control samples showed HBV DNA reactivity.

PCR amplification and subsequent Southern-blot hybridization revealed the presence of HBV DNA in 5 of 12 available PBMC samples that were harvested when the study patients were HBsAg negative and in 10 of the 15 samples obtained when patients were HBsAg pos- itive. None of the four negative controls nor the supernatant of the PBMCs contained detectable HBV DNA. Overall, four of the eight patients in the study group (A, C, D, F) had detectable HBV DNA in their PBMCs when serum was negative for HBsAg. In each case, detection of the HBV-specific sequences required Southern-blot hybridization analysis of PCR-amplified products, suggesting that low levels of HBV DNA were present in the cells. In two of these patients (A and D), the HBV DNA was also detected by in situ hybridization at 2 and 9 mo, respectively, after the disappearance of HBsAg. In the two remaining individuals (C and F) the HBV DNA could only be detected by PCR at 3 and 46 mo, respectively, after HBsAg loss.

In the HBsAg-positive study group samples, an ap- parent association was seen between HBeAg positivity and the number of PBMCs displaying a hybridization signal. When HBeAg was positive in serum, approxi- mately 5% of PBMCs were HBV DNA positive, whereas after HBeAg seroconversion only an occasional positive cell could be detected. Similarly, only HBeAg-positive patients had HBV DNA detectable by ethidium bromide-agarose gel electrophoresis analysis of PCR- amplified DNA. After HBeAg clearance, the HBV DNA in the PCR-amplified product could only be detected by the more sensitive technique of Southern-blot hybrid-

38 MASON ET AL. HEPATOLOGY

TABLE 1. Detection of HBV DNA in PBMCs by in eifu hybridization and PCR amplification Time after HBeAg Time after HBsAg

study W U P clearance (mo) clearance (mo) I n eihr hybridization PCR amplitication

Individual patients A 4 2 + +

- - 7 5

B 47 2 C 38 3 D 35 9 + + E 13 12

14 13 F 3 1

48 46 - G“ 47 47 H 34 14 - NA

87 67 15 HBsAg-positive samples in patients A-H - - 8/15 10115

+ 26 24 - - -

+ -

- - - - - -

+ - -

- -

2 NANBH samples - - 012 012 2 healthy, uninfeded controls - - 012 012

NA = not available; NANBH = non-A, non-B hepatitis. “Patient waa positive for human immunodeficiency virus-1 .

ization (Fig. 2). No apparent association was found between the expression of anti-HBs and the detection of HBV DNA in the PBMCs.

DISCUSSION In this study we show that HBV can persist in PBMCs

for several years after the disappearance of HBeAg and HBsAg. PCR analyses revealed that 50% of the patients who cleared serum HBsAg had HBV DNA in their PBMCs even though the serum aminotransferases had returned to normal and liver histological appearance had improved (26). In these patients, HBV DNA could be detected, by in situ hybridization and PCR, as long as 9 mo and 4 yr, respectively, after disappearance of HBsAg. Our findings are similar to those obtained with other lymphotrophic viruses where viral specific sequences have been detected in the PBMCs months or years after resolution of the original illness (27-29).

In other studies, HBV DNA has been detected in approximately 30% of PBMC samples analyzed by both Southern-blot (7) and in situ hybridization (14) up to 1 yr after HBsAg loss. In the single study we could find that examined PBMCs by PCR, HBV DNA was not detected in cells derived from two patients within a year of HBsAg conversion (21). In agreement with the findings of Hadchouel et al. (14), we found that approx- imately 5% of PBMCs from HBeAg-positive patients had levels of HBV DNA that could be detected by in situ hybridization, but only a few positive cells were observed after HBeAg seroconversion. Although PCR is a more sensitive detection technique than in situ hybridization, the latter is complementary because it permits detection of multiple copies of the viral genome in relatively few cells and also establishes the percentage of cells har- boring HBV in a given population.

We recently demonstrated that six of seven patients in this study group had HBV DNA detectable in the liver by PCR at a time when most patients were negative for

HBV DNA in serum by the same technique (22). It is noteworthy that in the only case in which HBV DNA could not be demonstrated in liver tissue (patient A), PBMCs were positive by PCR when the liver specimen was obtained (24 mo after loss of HBsAg; Table 1). Conversely, in three other cases (patients B, G and H; Table l ) , PBMCs were found to be negative for HBV DNA although the liver samples contained HBV DNA by PCR. A discrepancy between the detection of HBV DNA in PBMCs and liver tissue was also observed in a study by Pasquinelli and associates (7) in which samples from patients with acute and chronic hepatitis B were evaluated by Southern-blot hybridization. Thus the data taken together indicate that HBV DNA can be indepen- dently sequestered in hepatocytes, lymphocytes or both, years after HBsAg clearance from serum.

The broad extent of the extrahepatic life cycle of hep- nadnaviruses in the lymphoid system has been appre- ciated in woodchucks, Pekin ducks and chimpanzees (18-20); HBV has been isolated in lymphoid tissues and other extrahepatic sites and in PBMCs in humans. Romet-Lemonne et al. (30) reported that HBsAg could be demonstrated by immunocytochemical study in bone marrow cells from an HBsAg-positive patient and that these cells could support viral replication and produce Dane particles after 25 passages in culture. Replicative intermediates of HBV DNA have been detected in the spleen and lymph nodes of humans by Southern blotting (15, 161, and recently HBV DNA and HBV RNA were observed in bone marrow, spleen and lymph nodes by in situ hybridization in patients with HBV infection (17). These data provide evidence for persistent HBV in- fection of the lymphoid tissue, which may result in periodic “seeding” of the peripheral blood, accounting for the intermittent detection of PBMC-associated HBV DNA that we observed in some of our patients.

In other blood-borne viral diseases, infected lympho- cytes may act as “Trojan horses” and circulate to

Vol. 16, No. 1, 1992 HBV DNA IN PBMCs AFTER LOSS OF HBsAg

W

39

FIG. 1. Autoradiographs of PBMCs after in situ hybridization with an HBsAg genc-specific %-labeled riboprobe from (A) a patient in the study group with HBsAg-positive, HBeAg-positiveiHBV DNA-positive chronic hepatitis B and (B) an HBsAg-negative patient in the study group who recovered from chronic hepatitis B (H & E counterstain; original magnification x 400).

susceptible organs while protected from the immune of hepatitis B immune globulin and performed serial system (29). This mechanism was also suggested to Southern-blot analyses for HBV DNA in liver, serum apply to HBV by Zignego et al. (121, who treated and PBMCs (12). Liver-graft reinfection eventually HBV-infected liver transplant recipients with high doses developed in all patients; in some instances, replicative

40 MASON ET AL.

PLC Fi Fii Hii Pos D C Ncg Ai Aii Aiii Ei G Eii Pos

HEPATOLOGY

FIG. 2. Southern-blot hybridization analyses of PCR-amplified DNA isolated from PBMCs. Autoradiographic exposure time was 3 days. Letters (A to G) = patients who cleared HBAg from serum; Pos = the HBsAg-positive patients in the study p u p ; Neg = controls without serological markers of HBV infection; PLC = 100 ng of total HBV DNA obtained from PLCIpFW5, an HBV-positive hepatoma cell line.

intermediates of HBV DNA were found only in PBMCs and not in serum (12).

Although the biological significance of the demon- stration of HBV in PBMCs, the lymphoid system and other extrahepatic tissue is poorly understood, the extrahepatic life cycle of HBV is clearly advantageous for the virus. Once the HBV is established in the lymphoid system, it may avoid immune destruction by a variety of mechanisms, such as nonspecifidy decreasing cytokine and antibody production (31) and specifically inhibiting anti-HBs synthesis (321, both of which may have implications for viral persistence. The lymphoid system may thus act as a reservoir for the latent virus that can lead to future reactivated infection in a fashion similar to that which occurs for other blood-borne viruses such as cytomegdovirus (27,281.

Our data shows that HBV DNA can still be found in PBMCs as long as 4 yr after recovery of chronic hepatitis B. However, the transcriptional activity and the mo- lecular state of the HBV DNA were not determined. Additional studies such as detection of HBV RNA by reverse PCR are necessary to establish whether the HBV DNA sequences detected in our patients are transcrip- tionally active. Furthermore, the small amounts of HBV DNA derived from the PBMCs of patients who lost HBsAg precluded restriction enzyme analysis of the HBV DNA to establish its molecular form. Evaluating the transcriptional activity and physical state of the virus in the PBMCs would help predict whether these irdhted PBMCs can play a role in transmission of the infection to susceptible hosts. Some authors have sug- gested that the HBV can integrate into the lymphocyte host genome, because high-molecular-weight bands have been seen on Southern blots performed on DNA derived from PBMCs (5-9). Southern-blot studies have also revded replicative intermediates of HBV DNA in PBMCs, which suggests the presence of infectious particles (1-4).

The potential for infectivity by PBMCs may be particularly important because transfusion of blood

products from HBsAg-negative blood donors has been implicated in HBV-related posttransfusion hepatitis (33, 34). In a recent study involving 200 HBsAg-negative patients, most with serological evidence of past HBV infection, Wang and associates found that 9% had detectable HBV DNA by PCR (35). Although our study is considerably smaller, 50% of our patients had HBV DNA detected in their PBMCs by PCR at a time when this method could not detect serum HBV DNA in most (22). Thus there may be a role for PBMCs in addition to serum in the potential transmission of HBV when blood or organ donors have remote histories of infection. Clearly, additional studies will be required to establish the biological significance of these phenomena.

Acknowledgments: The authors thank Dr. G. Malizia and Dr. E. Aragona for their generous assistance and advice in the development of the in situ hybridization technique.

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