Analysis of T cell receptor Vbeta gene usage during the course of disease in patients with chronic hepatitis B.

The T cell receptor (TCR) is a heterodimeric molecule expressed on the surface of T cells and recognizes foreign peptides presented by the major histocompatibility complex on the surface of antigen-presenting cells or virus-infected cells. Analysis of TCR usage by T cells which recognize hepatitis B virus (HBV) provides further insight into the participation of T cell populations during the course of disease. In this study, we examined the T-cell-proliferative response and the TCR Vbeta gene usage of peripheral blood mononuclear cells in 3 patients with clinical evidence typical of chronic hepatitis B. All 3 patients had significant T-cell proliferative responses against HBV core antigen (HBcAg) during the remission stage, while no responses were detected during the acute exacerbation stage. In addition, the TCR Vbeta7 gene was utilized more frequently in T cells recognizing HBcAg during remission, while TCR Vbeta1 and Vbeta2 were utilized at a higher percentage during acute exacerbation. On the contrary, the T cell proliferative response against HBV surface antigen was undetectable and no specific Vbeta gene was utilized more frequently by all 3 patients, regardless of disease state. Our longitudinal studies, although based on a small sample of patients, demonstrate that the population of HBcAg-activated T cells alters during the course of disease in chronic hepatitis B patients.

Characterization of Trichomonas vaginalis virus proteins in the pathogenic protozoan T. vaginalis.

The 4.6-kb double-stranded (ds) RNA of Trichomonas vaginalis virus (TVV)-T1 has been shown to encode two overlapping genes, cap and pol. In this study, a serum for specifically detecting viral cap gene product was raised against a recombinant protein, and sera for specifically detecting pol gene product were raised against synthetic oligopeptides. A 75-kDa major protein and a 160-kDa minor protein were detected by anti-CAP serum in a TVV-T1 sample, indicating that the 75-kDa protein is the viral capsid protein. The 160-kDa protein alone was also detected by two distinct anti-POL sera, indicating that the pol gene is expressed as a CAP-POL fusion protein. These results suggest that the TVV-T1 genome is arranged into a cap-pol organization in a manner similar to that of viruses in family Totiviridae.

An ELISA for RNA helicase activity: application as an assay of the NS3 helicase of hepatitis C virus.

A convenient enzyme-linked immunosorbent assay (ELISA) for RNA helicase activity was developed with principles similar to the standard assay. The helicase ELISA utilizes a non-radioactive double-stranded substrate with a biotin-labeled template (long) strand hybridized to a digoxigenin (DIG)-labeled release (short) strand. The template strand binds to the wells of streptavidin-coated microtiter plates (SA-MTP) where the helicase catalyzes the unwinding reaction. Substrate not unwound retains the DIG-labeled release strand and is detected using anti-DIG coupled to horseradish peroxidase. Chromogenic detection follows. Absorbance measurement allows determination of unwinding efficiency of reactions. To demonstrate effectiveness, the ELISA-based assay was used to study the unwinding activity of the hepatitis C virus (HCV) NS3 helicase. Using a known inhibitor of NS3 helicase activity and two mutant HCV helicases, the ability of the assay to screen potential anti-helicase drugs and putative helicases is illustrated. The helicase ELISA is more convenient than the standard helicase assay and is especially suited for the testing of large numbers of samples.

Improvement of hepatitis B virus DNA vaccines by plasmids coexpressing hepatitis B surface antigen and interleukin-2.

DNA vaccines encoding a viral protein have been shown to induce antiviral immune responses and provide protection against subsequent viral challenge. In this study, we show that the efficacy of a DNA vaccine can be greatly improved by simultaneous expression of interleukin-2 (IL-2). Plasmid vectors encoding the major (S) or middle (pre-S2 plus S) envelope proteins of hepatitis B virus (HBV) were constructed and compared for their potential to induce hepatitis B surface antigen (HBsAg)-specific immune responses with a vector encoding the middle envelope and IL-2 fusion protein or with a bicistronic vector separately encoding the middle envelope protein and IL-2. Following transfection of cells in culture with these HBV plasmid vectors, we found that the encoded major protein was secreted while the middle protein and the fusion protein were retained on the cell membrane. Despite differences in localization of the encoded antigens, plasmids encoding the major or middle proteins gave similar antibody and T-cell proliferative responses in the vaccinated animals. The use of plasmids coexpressing IL-2 and the envelope protein in the fusion or nonfusion context resulted in enhanced humoral and cellular immune responses. In addition, the vaccine efficacy in terms of dosage used in immunization was increased at least 100-fold by coexpression of IL-2. We also found that DNA vaccines coexpressing IL-2 help overcome major histocompatibility complex-linked nonresponsiveness to HBsAg vaccination. The immune responses elicited by HBV DNA vaccines were also modulated by coexpression of IL-2. When restimulated with antigen in vitro, splenocytes from mice that received plasmids coexpressing IL-2 and the envelope protein produced much stronger T helper 1 (Th1)-like responses than did those from mice that had been given injections of plasmids encoding the envelope protein alone. Coexpression of IL-2 also increased the Th2-like responses, although the increment was much less significant.

Identification of humoral antigenic determinants in the hepatitis C virus NS3 protein.

Immunoblot analysis on serum samples from 90 patients with chronic hepatitis C virus infection revealed four putative immunogenic regions within the NS3 protein of the virus: E (around aa 1250/ 1251), A (within aa 1250-1334), A/B (around aa 1323 and 1334), and B/C (around aa 1407 and 1412). Among them, region E was most immunodominant, and region A was recognized much less frequently by patients with cirrhosis than by those with chronic hepatitis (10% vs. 46%, chi 2 = 12.05, P < .01). The results suggest that region A might be a potential prognostic marker to differentiate chronic hepatitis from cirrhosis.

Prominent proliferative response of peripheral blood mononuclear cells to a recombinant non-structural (NS3) protein of hepatitis C virus in patients with chronic hepatitis C.

The proliferative response of peripheral blood mononuclear cells (PBMC) to a recombinant non-structural (NS3) protein of hepatitis C virus (HCV) was studied in 41 patients with chronic hepatitis C. Of them, 28 had chronic persistent hepatitis (CPH) and 13 chronic active hepatitis (CAH). The positive proliferation rate of PBMC to the recombinant NS3 protein, T9Ag, was 66% in the 41 patients (77% in CAH versus 61% in CPH; P > 0.05) when stimulation index (SI) = 4 was set as the cut-off value. However, mean SI of CAH patients was significantly higher than that of CPH patients (8.3 +/- 5.2 versus 5.1 +/- 3.6; P < 0.05). Six other chronic hepatitis patients who were repeatedly negative for anti-HCV antibody but positive for serum HCV RNA also had an SI of > or = 4.0. The frequency of cellular immune response to the T9Ag is among the highest results obtained by using HCV antigens tested so far. Our studies thus indicate that NS3 is an immunologically important region of HCV for T cells. Moreover, the proliferative response to T9Ag may help to establish hepatitis C etiology in chronic hepatitis patients who are seronegative with currently available anti-HCV assays.