Search

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.

Little is known about the immune responses induced by recombinant adenoviral (Ad) vectors in humans. The humoral and cellular immune responses were therefore analyzed in 21 patients with localized malignancy (mesothelioma), who received intrapleurally high doses of a replication-defective Ad5 vector carrying a suicide gene. Eight of 21 patients had pretreatment titers of neutralizing antibodies (NAb) to Ad at > or =1:100. Peripheral blood mononuclear cells (PBMCs) proliferated in response to adenoviral 5 structural proteins before treatment in 17 of 21 patients. Preexisting humoral and cellular immunity did not preclude gene transfer. Vector instillation induced high titers of nonneutralizing and neutralizing anti-Ad antibody (4- to 341-fold increase in 18 of 20 patients) in a dose-dependent manner. Three patients generated antibodies to the transgene, herpes simplex virus thymidine kinase. Ad5-specific proliferation of PBMCs increased significantly (>3-fold) after vector administration in 12 of 21 patients in a dose-dependent manner. Thus, replication-defective Ad5 administered intrapleurally induced significant humoral and cellular immune responses that induced no obvious adverse clinical sequelae.

The effect of rev (art/trs) gene on the level of HIV-1 envelope (env) expression using recombinant adenovirus was investigated. Recombinant adenoviruses expressing either the envelope or the rev gene of the human immunodeficiency virus type 1 (HIV-1) were constructed by inserting the gene into an expression cassette. The expression cassette contained the adenovirus type 7 major late promoter, followed by leader 1 of the adenovirus tripartite leader and a portion of intron between leaders 1 and 2, leaders 2 and 3, and a hexon polyadenylation signal. The cassette was then inserted at the terminal region between the E4 and ITR regions of the adenovirus 7 genome with a concomitant E3 region deletion (80-87 m.u.). A549 cells infected with the recombinant virus containing the env gene produced the envelope glycoproteins gp160, gp120, and gp41. HIV-1 envelope gene expression was greatly enhanced (20- to 50-fold) in the cells that were simultaneously infected with the recombinant adenovirus containing the rev gene as measured by ELISA and Western blotting. Interestingly, this effect was observed despite the lack of the 5' down splice site for rev and seems to be post-transcriptional. Another recombinant adenovirus which contains both the rev and the env genes was constructed by inserting the rev gene in the deleted E3 region and the env gene in the terminal cassette. This double recombinant virus expressed high levels of env antigen in A549 cells similar to those attained upon co-infection with two separate recombinant viruses containing the rev or env gene. Furthermore, the rev gene nucleotide sequence could be altered without altering the amino acid sequence and its sequences truncated by 17 amino acids from the C-terminus had no effect of rev function.

Types 4 and 7 adenovirus are currently used as live, oral vaccines for the prevention of adenovirus respiratory disease in military recruits. These vaccine strains have been genetically engineered in order to express HIV-1 or HBV antigens in infected cells. A dog model was developed to evaluate the immunogenicity of these recombinant vaccines. Dogs inoculated with live adenovirus-HBV recombinant vaccine produced antibody against hepatitis B surface antigen.