Interferon- is a cytokine that has antiviral and immunomodulatory properties [8] and is often proposed for the treatment of chronic HBV infection. In HIV-negative patients, the rate of response to interferon- (loss of serum HBV DNA and HBeAg) is 20% greater than the spontaneous seroconversion rate of 12% after 6 to 12 months of follow-up [9]. However, HIV infection is known to diminish the response to interferon-; these decreased response rates range from 0% [10] to 8% [11].

Therapies for HIV infection are already associated with increased survival [12]. Thus, the frequency of chronic HBV infection, which leads to cirrhosis and its complications, may increase in the population of HIV-infected patients who are coinfected with HBV. Therapies that are more effective against HBV than is interferon- alone are still needed in both HIV-positive and HIV-negative patients.

Lamivudine, the – enantiomer of 2'-deoxy-3'-thiacytidine, an inhibitor of HIV type 1 and HIV type 2 reverse transcriptase [13], has been shown to have antiretroviral activity in HIV-infected patients [14]. Lamivudine is also a potent selective inhibitor of HBV replication because this replication depends on reverse transcription of an intermediate RNA to a minus-stranded DNA, which then serves as a template for the synthesis of plus-stranded DNA [15]. Experimental studies have shown that lamivudine inhibits HBV DNA replication in transfected cell line 2.2.15 and in HBV-infected chimpanzees [15, 16]. Results of a phase II clinical trial [17] showed that lamivudine inhibited HBV replication in patients with chronic HBV infection. However, no information is available about the activity of lamivudine against HBV in HIV-infected patients.

An open-label program of lamivudine therapy (NUCB 3004) has been in progress in France since December 1993. This program is for patients with progressive HIV disease who are refractory to or unable to tolerate therapies other than lamivudine. We evaluated the efficacy of lamivudine against HBV replication in 40 consecutive patients who were infected with both HIV and HBV and who entered this program. We report our results after 1 year of follow-up.

Methods

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Patients

From April to September 1994, 228 HIV-infected patients who were followed at the infectious diseases department at Groupe Hospitalier Pitie-Salpetriere and were eligible for the open-label lamivudine trial were prospectively tested for serologic markers of HBV infection (HBsAg, antibodies to HBsAg, HBeAg, and antibodies to HBeAg), IgG and IgM antibodies to hepatitis delta virus, and antibodies to hepatitis C virus (HCV). Forty of these patients were HBsAg carriers; these patients were consecutively included in our study and were followed for 1 year. Patients were seen at baseline and every 2 months. At each visit, physical examination was done and all clinical events were recorded. Blood samples were obtained for complete blood cell counts, measurement of CD4 lymphocyte counts, serum biochemistry tests, tests for serologic markers of HBV, and measurement of serum HBV DNA concentrations.

Laboratory Assays

Commercially available enzyme immunoassays were used to measure serologic markers of HBV (Abbott Diagnostics, Chicago, Illinois), IgG and IgM antibodies to hepatitis delta virus (Pasteur, Marne la Coquette, France), and antibodies to HCV (Ortho HCV ELISA, Ortho Diagnostic Systems, Inc., Raritan, New Jersey; RIBA HCV, Chiron Corp., Emeryville, California).

Serum HBV DNA was assessed quantitatively and qualitatively. Serum concentrations of HBV DNA were measured by molecular hybridization through hybrid capture (Murex Diagnostics, Dartford, United Kingdom). Hybrid capture has a cut-off point of 5 pg/mL and allows titration to 2000 pg/mL. If the titer is greater than 2000 pg/mL, the test result is positive but exact titration cannot be done. We qualitatively assessed HBV DNA by using PCR at baseline and at months 2, 6, and 12 of lamivudine therapy only if serum concentrations of HBV DNA were less than 5 pg/mL as determined by molecular hybridization. We extracted DNA by heating 100 µL of serum for 45 minutes. The two sets of primer used for the amplification were from the core region (HBV Core; Sorin-Biomedica, Saluggia, Italy). Polymerase chain reaction was done with the extracted DNA, and 35 cycles were run in a programmable thermoblock (Gen Amp PCR system 9600; Perkin Elmer Cetus Corp., Norwalk, Connecticut). To avoid contamination, each step of the PCR assay was done in a separate room and each series was done with at least three negative controls. We identified DNA by using DNA enzyme immunoassay (HBV Core Gene, ETI-K DEIA; Sorin-Biomedica). Single-stranded DNAs were hybridized with an oligonucleotide probe specific for the amplified region and were assayed by a monoclonal antibody that reacted only with double-stranded DNA. This assay can detect seven equivalent genomes per sample [18].

Antiviral Therapies

In the French open-label treatment program, lamivudine was given to patients at a dosage of 300 mg twice daily from December 1993 to May 1995. Then, in May 1995, a protocol amendment recommended reducing the dose by 50% (to 150 mg twice daily). All study patients followed this protocol amendment. Thus, during the 12-month study period, 14 patients received 300 mg of lamivudine twice daily for 1 year and 26 patients received 300 mg of lamivudine twice daily for 9.5 ± 1.2 months followed by 150 mg of lamivudine twice daily for 2.4 ± 1.3 months. Seven patients with high HBV replication at baseline and 3 patients with low HBV replication at baseline received lamivudine in combination with zidovudine, 250 mg/d. The remaining 30 patients received lamivudine alone. Two patients with cytomegalovirus retinitis and no evidence of HBV replication (positivity for antibodies to HBeAg and an HBV DNA concentration < 5 pg/mL) started receiving lamivudine simultaneously with treatment for cytomegalovirus infection (1 patient received foscarnet and 1 received ganciclovir). No patients received interferon- during the course of the study.

Other Concurrent Therapies

At baseline, 38 patients (29 patients with high HBV replication at baseline and 9 patients with low HBV replication at baseline) were receiving sulfamethoxazole (800 mg/d) and trimethoprim (160 mg/d) as prophylaxis for Pneumocystis carinii pneumonia. One patient with high HBV replication and 1 patient with low HBV replication received sulfadiazine (2 g/d) and pyrimethamine (25 mg/d) as prophylaxis for recurrence of cerebral toxoplasmosis. Two patients with high replication and 1 patient with low replication were receiving bleomycin (5 mg/d for 3 days twice monthly) for cutaneous Kaposi sarcoma.

Statistical Analysis

Results are expressed as the mean ±SD. Data were compared using the Mann-Whitney test; a P value less than 0.05 was considered to be significant.

Results

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The clinical and biological characteristics of the 40 study patients are shown in Table 1. No statistically significant differences in age, ratio of men to women, Centers for Disease Control and Prevention clinical stage of HIV infection [19], known duration of HIV infection, or CD4 lymphocyte counts were seen between the high-replication group and the low-replication group (data not shown).

Thirty-four patients had progressive HIV disease (a progressive decrease in CD4 lymphocyte count) despite receiving therapy with zidovudine and didanosine; 6 patients had progressive HIV disease despite receiving therapy with zidovudine and did not tolerate didanosine. All patients carried HBsAg for at least 1 year. In 27 patients, HBsAg and antibodies to HIV were found simultaneously in serum 5.9 ± 2.8 years (range, 1 to 10 years) before lamivudine therapy was started. No patients had a history of initial acute hepatitis or decompensated liver disease or were infected with the hepatitis delta virus. One patient had detectable antibodies to HCV in serum.

At baseline, two groups of patients were retrospectively identified according to serologic markers of HBV and serum concentrations of HBV DNA as measured by molecular hybridization. The first group comprised patients with high HBV replication (patients who were positive for HBeAg and had serum concentrations of HBV DNA > 5 pg/mL as determined by molecular hybridization) (n = 30). Liver biopsy was done in 6 of these patients 27 ± 9 months (range, 12 to 36 months) before lamivudine therapy was started. These 6 patients had histologic findings consistent with chronic HBV infection, had a mean Knodell score [20] of 6 ± 2.7 (range, 4 to 10), and did not lose serum HBV DNA while receiving interferon- therapy (5 million U/m² body surface area three times a week for 6 months). Measurements of serum HBV DNA were available for 17 patients from 12 and 6 months before lamivudine therapy was started and for 4 patients from 6 months before baseline.

The second group was composed of patients with low HBV replication (patients who were positive for antibodies to HBeAg and had serum concentrations of HBV DNA < 5 pg/mL as determined by molecular hybridization) (n = 10). Low serum concentrations of HBV DNA were detectable by PCR in 6 of these 10 patients. In this group, serum concentrations of HBV DNA were available for 3 patients from 12 and 6 months before lamivudine therapy was started and for 2 patients from 6 months before baseline.

Serologic markers of HBV at baseline and 1 year earlier were similar in all of the patients, whether they had high or low HBV replication at baseline.

Patients with High Hepatitis B Virus Replication at Baseline

At baseline, HBV DNA serum concentrations were greater than 2000 pg/mL in 21 patients and were between 18 and 1824 pg/mL in 9 patients. These values were 20 pg/ml to greater than 2000 pg/ml in the 17 patients tested 12 and 6 months before lamivudine therapy was started and the 4 patients tested 6 months before lamivudine therapy was started. The percentages of patients who had serum HBV DNA concentrations greater than 5 pg/mL before baseline, at baseline, and while receiving lamivudine therapy are shown in Figure 1. In 26 of the 27 patients (96.3% [95% CI, 81% to 99.9%]) who completed 12 months of lamivudine therapy, serum concentrations of HBV DNA decreased to less than 5 pg/mL. Among these patients, HBV DNA was detectable in serum only by PCR in 5 patients at month 2 (19% [CI, 6% to 39%]), in 3 patients at month 6 (11% [CI, 2% to 29%]), and in 3 patients at month 12 (11.5% [CI, 2% to 30%]) of lamivudine therapy. In the 6 patients who did not respond to previous treatment with interferon-, HBV DNA was undetectable in serum by both molecular hybridization and PCR during lamivudine therapy. A rebound in the HBV DNA serum concentration (maximum, >2000 pg/mL and 584 pg/mL, respectively) was seen in the 2 patients who stopped receiving lamivudine therapy at months 2 and 8, respectively. One of these patients was an HBeAg carrier and had HBV DNA detectable in serum only by PCR when lamivudine therapy was discontinued. The second patient was an HBeAg carrier but had no HBV DNA detectable in serum by either molecular hybridization or PCR. Serum HBV DNA became undetectable again after this second patient received retreatment with lamivudine.

View larger version (16K): [in this window] [in a new window]   Figure 1. Percentage of patients with high hepatitis B virus (HBV) replication at baseline who had HBV DNA concentrations greater than 5 pg/mL by molecular hybridization before and during lamivudine therapy. * = Number of patients.

Five of the 30 patients who had high HBV replication at baseline lost HBeAg Table 2 in month 2 (n = 3), month 4 (n = 1), or month 6 (n = 1) of lamivudine treatment. Three of these patients seroconverted to become positive for antibodies to HBeAg at months 2, 4, and 6, respectively. No change in HBV serologic findings was seen in the remaining 25 patients (who were positive for both HBsAg and HBeAg).

Mean serum alanine aminotransferase levels were lower at 10 and 12 months of treatment than at baseline (P = 0.02 and P = 0.03, respectively) (Figure 2). An abrupt and transient elevation in alanine aminotransferase levels that was associated with the loss of serum HBV DNA was seen in four patients. Two of these patients seroconverted to become positive for antibodies to HBeAg. Alanine aminotransferase levels returned to baseline values within the next 4 months in these four patients. Mean CD4 lymphocyte counts were low at baseline (113 ± 88 cells/mm³), and no statistically significant difference was seen during treatment (data not shown).

View larger version (12K): [in this window] [in a new window]   Figure 2. Mean alanine aminotransferase levels in patients infected with both the human immunodeficiency virus and hepatitis B virus (HBV) who had high and low HBV replication at baseline. Black squares indicate patients with high HBV replication at baseline; black diamonds indicate patients with low HBV replication at baseline. * = Compared with baseline levels in patients with high HBV replication at baseline. = The mean ±SD of paired differences between alanine aminotransferase values at 12 months and at baseline was 44.4 IU/L (95% CI, 7.2 IU/L to 81.5 IU/L).

Patients with Low HBV Replication at Baseline

After 2 months of lamivudine therapy, 3 of the 10 patients who had had low HBV replication at baseline lost HBsAg. Two of these patients (who also received therapy for cytomegalovirus infection) seroconverted to become positive for antibodies to HBsAg (Table 2). One of these 2 patients was positive for serum HBV DNA by PCR at baseline. In the remaining 7 patients, HBV serologic findings were unchanged (these patients had both HBsAg and antibodies to HBeAg). Serum concentrations of HBV DNA (measured by molecular hybridization) taken 12 months (n = 3) and 6 months (n = 5) before lamivudine therapy was started were similar to concentrations at baseline in all of these patients (<5 pg/mL). No increase in serum HBV DNA concentrations was seen during the study period. As determined by PCR after 2 months of lamivudine therapy, HBV DNA became undetectable in serum in the 6 patients who had been positive for HBV DNA by PCR at baseline. These results were sustained at 6 and 12 months. In the remaining 4 patients, HBV DNA was not detectable in serum at baseline or at months 2, 6, and 12. In 1 of the patients who had HBV DNA detectable in serum by PCR and who seroconverted to become positive for antibodies to HBsAg, the discontinuation of lamivudine therapy at month 8 was not followed by a recurrence of HBV replication at months 10 and 12.

Mean serum alanine aminotransferase levels Figure 2 and CD4 lymphocyte counts (124 ± 103 cells/mm³) (data not shown) did not change during treatment in the patients who had had low HBV replication at baseline (P > 0.2 for both comparisons).

Switching from 300 mg of lamivudine twice daily to 150 mg of lamivudine twice daily did not modify HBV serologic findings, nor did it affect HBV DNA serum concentrations in any patients, whatever their replication status at baseline. Loss of serum HBV DNA and rates of seroconversion to positivity for antibodies to HBeAg did not differ between patients who received lamivudine and zidovudine and patients who received lamivudine alone (Table 3). Except for the lamivudine dose, antiretroviral therapies were not modified during the study. The two patients with cytomegalovirus retinitis received foscarnet (180 mg/kg of body weight per day) or ganciclovir (10 mg/kg per day), respectively, for 1 month. During the ensuing 11 months, doses of both agents were reduced by 50% to prevent relapse.

Adverse Effects and Withdrawal from Study

Lamivudine was generally well tolerated. Transient nausea and mild abdominal pain that did not require the discontinuation of lamivudine therapy occurred in four patients. Three patients dropped out of the study. One patient had frequent vomiting during the first 2 months that was associated with abdominal pain and an increased serum amylase level. These side effects were believed to be related to lamivudine therapy because they promptly disappeared after therapy was withdrawn. One patient stopped receiving lamivudine therapy at month 8 for personal reasons. In another patient, lamivudine therapy was stopped for 1 month at month 8 of treatment, during investigation of a fever of unknown origin. Blood cell counts; serum electrolyte concentrations; and mean serum levels of glutamyltransferase, alkaline phosphatase, and bilirubin did not vary statistically during the study period in any of the patients with high or low HBV replication (data not shown). Three patients died during the study period. One died of respiratory failure secondary to extensive pulmonary Kaposi sarcoma at month 10, one died of respiratory failure secondary to extensive Kaposi sarcoma at month 13, and one died during month 5 after multiorgan failure due to bacterial septicemia.

Discussion

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Although our study was not a randomized, blinded trial, it suggests that lamivudine (300 mg twice daily or 300 mg twice daily followed by 150 mg twice daily) is active against HBV replication in severely immunodeficient HIV-infected patients. As early as month 2 of lamivudine therapy, HBV replication was inhibited in 86% (CI, 69.3% to 96.2%) of patients infected with both HIV and HBV, despite their high degree of immunodeficiency. After a minimum of 6 months of treatment, 96.3% of patients (CI, 81% to 99.9%) had lost serum HBV DNA, as assessed by molecular hybridization. The rate of inhibition of HBV replication with lamivudine, as determined by serum HBV DNA concentrations less than 5 pg/mL, was much higher than that reported with interferon- in both HIV-infected patients (0% to 8%) [10, 11, 21] and patients without HIV infection (40%) [9]. Furthermore, lamivudine was effective in patients in whom HBV DNA had not previously cleared from serum after interferon- therapy.

Lamivudine is a nucleoside analogue that has shown substantial in vitro and in vivo activity against HBV [15-17]. Several 2',3',dideoxycytidine analogues have been examined for their inhibition of HBV replication in experiments using HBV-transfected cell lines 2.2.15 [15]. Among them, lamivudine, 2',3'-dideoxy-3'-thiacytidine, was found to be the most potent [15]. Metabolic studies showed that lamivudine is converted to its monophosphate, diphosphate, and triphosphate. Lamivudine triphosphate shows substantial inhibition of the ß and HBV DNA polymerases [22]. However, in HBV-transfected 2.2.15 cell lines, lamivudine was unable to eliminate intracellular DNA completely; therefore, no evidence indicates that the inhibition of HBV replication is irreversible [15]. Withdrawal of lamivudine in HBV-transfected 2.2.15 cell lines resulted in the reappearance of active HBV replication [23].

As seen in vitro and in most HIV-negative, HBV-infected patients [17], a rebound in the HBV DNA serum concentration occurred in the two HIV-infected patients in our study who were HBeAg carriers and who stopped receiving lamivudine therapy. The rebounds in viral production seemed to be independent of the time of exposure to lamivudine because they occurred after 2 and 8 months of treatment. In contrast, HBV replication did not recur in any patient who continuously received lamivudine therapy for at least 12 months. In most patients, the loss of serum HBV DNA was not associated with seroconversion to positivity for antibodies to HBeAg, and serum HBV DNA remained detectable by PCR in 11.5% of patients (CI, 2% to 30%), showing a low but persistent HBV replication. The persistence of HBeAg and detectable HBV DNA in serum are often predictive of a rebound in HBV replication after a course of interferon- therapy. These findings suggest that continuous lamivudine therapy may be necessary to control HBV replication of HBeAg-positive patients infected with both HIV and HBV. In most cases, prolonged treatment with high doses of lamivudine was well tolerated for 12 months. Major side effects were seen in only one patient, but no hepatotoxicity was recorded.

Although the treatment of HBV infection with other nucleoside analogues has been attempted, results to date have been inconclusive. In immunocompetent HBV-infected persons, treatment with adenine arabinoside 5'-monophosphate has produced conflicting results; it inhibited HBV replication in no more than 50% of cases [24]. Fialuridine showed important activity against HBV replication [25] but induced major side effects [26].

The following evidence suggests that the inhibition of HBV replication seen during lamivudine therapy may have been neither spontaneous nor related to improvement in immunity. First, in HIV-infected patients, the spontaneous rate of disappearance of serum HBV DNA after 3 months of follow-up averages 12% [11], whereas it reached 96.3% in our study after 12 months of lamivudine therapy. Second, none of the tested patients in our study spontaneously lost serum HBV DNA within the 12- or 6-month period before lamivudine therapy was started. Finally, no increase in CD4 lymphocyte counts was seen during the study period.

These results were not influenced by a 50% reduction in the lamivudine dose. In HIV-negative patients, inhibition of HBV replication was obtained in 100% of patients with a minimum daily dose of 100 mg of lamivudine [17]. The optimal dosage of lamivudine for the inhibition of HBV replication in patients infected with both HIV and HBV remains to be established. Zidovudine, an HIV reverse transcriptase inhibitor, had no effect on HBV replication in HIV-infected patients [27]. Zidovudine in combination with lamivudine did not seem to influence the loss of serum HBV DNA and rates of seroconversion to positivity for antibodies to HBeAg. However, any statement about this point is speculative because only a few patients in this study received these two antiretroviral agents simultaneously. The seroconversion to positivity for antibodies to HBsAg seen in two patients who had had low HBV replication at baseline may not be related to lamivudine therapy alone, because these patients also received treatment for cytomegalovirus that may have affected HBV infection in immunocompromised persons [28, 29]. Spontaneous seroconversion to positivity for antibodies to HBsAg remains a possible explanation, although this seroconversion after antiviral therapy is very uncommon in HIV-infected patients [30].

As the survival of HIV-infected patients improves, severe chronic HBV infection, which can lead to cirrhosis, liver failure, and even hepatocellular carcinoma, may occur more frequently. Improvement in hepatic histologic lesions in patients with both HIV and HBV infection who receive lamivudine was not established in this study because we did not do liver biopsies. However, decreases in the histologic evidence of disease activity is commonly associated with the inhibition of HBV replication [31].

The reported risk for death linked to chronic HBV infection in HIV-infected patients indicates that therapy for HBV infection may be appropriate for HIV-infected patients [9]. Our study does not allow firm conclusions to be drawn because it was not designed to evaluate the efficacy of lamivudine against HBV infection, but our data indicate that lamivudine may have a role in HIV-infected patients. At this point, however, lamivudine cannot be recommended for the treatment of HBV infection in these patients. Larger, controlled trials of lamivudine are warranted to confirm these results, assess any improvement in hepatic histologic findings, determine the optimal dose and duration of lamivudine therapy, and establish the incidence of drug toxicity. Furthermore, sustained response rates after discontinuation of lamivudine therapy must be established. Replication of HIV must also be measured, because resistance of HIV to lamivudine might appear during lamivudine therapy and could influence the course of HIV disease. The use of combined agents, such as interferon- and lamivudine, which have different mechanisms of action against HBV, might be a promising approach to future therapy for HBV infection in both HIV-infected and HIV-negative patients.

Drs. Katlama, Dohin, and Tubiana: Service des Maladies Infectieuses, Groupe Hospitalier Pitie-Salpetriere, 47 Boulevard de l'Hopital, 75651 Paris Cedex 13, France.

Dr. Lunel and Ms. Hamm: Laboratoire de Virologie, Professeur Jean Marie Huraux, Groupe Hospitalier Pitie-Salpetriere, 47 Boulevard de l'Hopital, 75651 Paris Cedex 13, France.

Drs. Coutellier and Herson: Service de Medecine Interne, Groupe Hospitalier Pitie-Salpetriere, 47 Boulevard de l'Hopital, 75651 Paris Cedex 13, France.