Combination therapy with lamivudine and adenovirus causes transient suppression of chronic woodchuck hepatitis virus infections

Abstract

Treatment of hepatitis B virus carriers with the nucleoside analog lamivudine suppresses virus replication. However, rather than completely eliminating the virus, long-term treatment often ends in the outgrowth of drug-resistant variants. Using woodchucks chronically infected with woodchuck hepatitis virus (WHV), we investigated the consequences of combining lamivudine treatment with immunotherapy mediated by an adenovirus superinfection. Eight infected woodchucks were treated with lamivudine and four were infected with approximately 10(13) particles of an adenovirus type 5 vector expressing beta-galactosidase. Serum samples and liver biopsies collected following the combination therapy revealed a 10- to 20-fold reduction in DNA replication intermediates in three of four woodchucks at 2 weeks after adenovirus infection. At the same time, covalently closed circular DNA (cccDNA) and viral mRNA levels both declined about two- to threefold in those woodchucks, while mRNA levels for gamma interferon and tumor necrosis factor alpha as well as for the T-cell markers CD4 and CD8 were elevated about twofold. Recovery from adenovirus infection was marked by elevation of sorbitol dehydrogenase, a marker for hepatocyte necrosis, as well as an 8- to 10-fold increase in expression of proliferating cell nuclear antigen, a marker for DNA synthesis, indicating significant hepatocyte turnover. The fact that replicative DNA levels declined more than cccDNA and mRNA levels following adenovirus infection suggests that the former decline either was cytokine induced or reflects instability of replicative DNA in regenerating hepatocytes. Virus titers in all four woodchucks were only transiently suppressed, suggesting that the effect of combination therapy is transient and, at least under the conditions used, does not cure chronic WHV infections.

FIG. 1

Adenovirus infection causes a transient suppression of WHV viremia. Chronically infected woodchucks 320 and 321 were inoculated i.v. with ∼5 × 10¹¹ PFU of the adenovirus vector. WHV titers in serum were determined by Southern blot assays for viral DNA. All titers are normalized to the titer at the time of adenovirus infection (ca. 10⁹ per ml of serum). Arrows along the abscissa show the timing of liver biopsies, described further in the footnote to Table 1.

FIG. 2

Adenovirus inoculation was followed by transient elevation in the serum of the liver enzyme SDH. Adenovirus inoculation was carried out at the indicated time after initiation of lamivudine therapy. Mock-infected animals received PBS rather than adenovirus.

FIG. 3

Adenovirus infection leads to a prolonged suppression of WHV viremia. (A) Infected woodchucks. Adenovirus was inoculated at the indicated time after initiation of lamivudine therapy (St. LAM). WHV titers in the serum were determined by Southern blot assays for viral DNA. (B) Woodchucks inoculated with PBS rather than adenovirus. Lamivudine administration was continued until the end of the study. Arrows along the abscissa show the timing of liver biopsies, described further in the legend to Fig. 4 and Table 2, footnote a. To test that the response to the adenovirus was statistically significant, the woodchuck viremia data for the adenovirus- and mock-infected woodchucks were evaluated separately, before and after infection. We first used the data collected from 1 week before initiation of lamivudine therapy through to the time of infection. Second-degree polynomials (20) were fitted to the data from each set of animals, and the sum of squared errors (ss) committed for each group was determined: ss(treated) and ss(mock), and the sum ss(treated) +ss (mock). We next fitted the data for all eight animals by a single second-order curve and determined the sum of squared errors [ss(both)] using just one rather than two curves. The ratio ss(both)/[ss(treated) + ss(mock)] is necessarily greater than 1.0. To assess the significance of this ratio, we permuted the data for the eight woodchucks so that the treated group was not the original but one of 69 other permutions of eight animals. The above ratio was determined for all 69 permutions. Under the null hypothesis that the two groups are identical, the true ratio (the one determined by the actual data) is uniformly distributed, in rank order, among the 70 values obtained. It was the 15th largest. Hence, the null hypothesis is accepted at the 30% level, using a two-sided test. The same procedure was repeated on the posttreatment data, except that a one-sided test was used to determine the probability that the responses to mock infection and adenovirus infection were identical. This time the ratio obtained from the original data was the largest of the set of 70. Thus, the null hypothesis that the treatment response was the same for both groups (P = 1/70 = 0.0143) was rejected.

FIG. 4

Analysis of WHV DNA in livers of adenovirus (AD)-infected woodchucks. Total DNA and cccDNA were extracted and subjected to Southern blotting following electrophoresis in 1.5% agarose gels. The filters were hybridized with a ³²P-labeled probe representing the complete viral genome. In the upper panel, cccDNA collected from 5 × 10⁵ liver cells, as determined by nuclear counts, was loaded into each lane of the gel; 2.5 μg of total liver DNA was loaded in each lane in the lower panel. cccDNA (ccc) and total DNA copy numbers (equivalents of 3.3 kbp of double-strand WHV DNA) were quantified using a Fuji phosphorimager to detect radioactivity bound to the filters. Copy numbers were calculated assuming that the liver is comprised of 70% hepatocytes (hep). The infiltration of lymphocytes did not appear to cause a major alteration in this estimation (≤2-fold [Fig. 9A]). RC-DNA, relaxed circular DNA; SS-DNA, single-stranded DNA.

FIG. 4

Analysis of WHV DNA in livers of adenovirus (AD)-infected woodchucks. Total DNA and cccDNA were extracted and subjected to Southern blotting following electrophoresis in 1.5% agarose gels. The filters were hybridized with a ³²P-labeled probe representing the complete viral genome. In the upper panel, cccDNA collected from 5 × 10⁵ liver cells, as determined by nuclear counts, was loaded into each lane of the gel; 2.5 μg of total liver DNA was loaded in each lane in the lower panel. cccDNA (ccc) and total DNA copy numbers (equivalents of 3.3 kbp of double-strand WHV DNA) were quantified using a Fuji phosphorimager to detect radioactivity bound to the filters. Copy numbers were calculated assuming that the liver is comprised of 70% hepatocytes (hep). The infiltration of lymphocytes did not appear to cause a major alteration in this estimation (≤2-fold [Fig. 9A]). RC-DNA, relaxed circular DNA; SS-DNA, single-stranded DNA.

FIG. 5

Adenovirus infection produced a decline in viral core antigen in the liver. Immunoperoxidase staining of fixed liver tissue for detection of viral core (nucleocapsid) antigen was carried out as described in Materials and Methods. Two biopsies are illustrated, one collected 1 month before adenovirus or mock infection the other collected 15 days after. The major effect noticed for woodchuck 326 was a decline in the number of hepatocytes with a strong staining reaction. A lesser decline was observed with woodchuck 328 (not shown). No appreciable decline was observed in woodchucks 331 and 338 or in any of the mock-infected controls (for example, 336 shown here). Magnification, ×200.

FIG. 6

Viral replication may still be suppressed 6 months after adenovirus infection. Immunoperoxidase assays for viral core antigen in liver sections were carried as described in Materials and Methods. As illustrated, the majority of hepatocytes in two woodchucks (326 and 328) still expressed little or no core antigen 6 months after adenovirus infection. At the end of the study, the majority were positive in all of the woodchucks.

FIG. 7

Adenovirus (AD) infection induced a slight decline in viral mRNAs. RNA was extracted from liver biopsy specimens, and 10 μg was subjected to Northern blot analysis. Filters were hybridized with a ³²P-labeled DNA probe representing the entire viral genome. The results were quantified with a Fuji image analyzer and are summarized in Table 2. mRNA signals for woodchucks 336 and 337 were low but detectable at 1 month before mock infection.

FIG. 8

Adenovirus (AD) infection is associated with infiltration of CD3⁺ leukocytes and an elevation in the fraction of hepatocytes with PCNA-positive nuclei. CD3⁺ leukocytes (A) and PCNA-positive hepatocytes (B) were determined as described previously (14) and in Materials and Methods. The percent CD3⁺ cells in the lobule is the intralobular count of CD3⁺ cells divided by the number of hepatocytes times 100.

FIG. 9

Adenovirus infection induced hepatic inflammation and expression of inflammatory cytokines. ³²P-labeled probes for woodchuck cytokine mRNAs were produced, and RNase protection assays were carried out as described previously (14) and in Materials and Methods. Following gel electrophoresis, radioactive signals were quantified using a Fuji image analyzer. Relative signal intensities are shown at the bottom. (A) Adenovirus infected; (B) mock infected. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.