SummarySuperantigens, in association with class II major histocompatibility complex (MHC) molecules, activate T cells bearing particular/~ chain variable domains of the T cell receptor (TCR). Unlike conventional peptide antigens, superantigens bind as intact proteins to TCR and MHC molecules outside their peptide binding sites. To characterize these interactions at the molecular level, random point mutations were generated in the gene encoding toxic shock syndrome toxin 1, a bacterial superantigen associated with toxic shock syndrome. Functionally impaired mutants were identified based on their lack of murine and human T cell stimulatory activities, and experiments analyzing binding to human histocompatibility leukocyte antigen-DR molecules differentiated residues involved in MHC from TCR binding. The results showed that the great majority of mutations are clustered in two distinct regions of the toxic shock syndrome toxin 1 molecule. The class II MHC binding site is located in the hydrophobic region of the NH2-terminal domain, and the TCR binding site is primarily in the major central groove of the COOH-terminal domain. These studies provide insight into the interactions necessary for superantigen-mediated disease in humans.

HBsAg levels reflect clinical stage and liver disease, and a combined quantification of HBsAg and HBV DNA may improve clinical staging.

Background Quantification of hepatitis B virus (HBV) DNA and surface antigen (HBsAg) serum levels have become increasingly important for the assessment of clinical stage and response to treatment for chronic hepatitis B. Effective immune clearance results in reduction of viremia by 4–5 log units and HBsAg levels by 2 log, but these processes are not well understood. Thus, it is uncertain to what extent mechanisms that inhibit transcription of the pregenomic RNA (pgRNA), an RNA intermediate, contribute to suppression of viremia. Likewise, it is unclear if transcriptional regulation is important for the excessive production of surface antigen (HBsAg) that is a hallmark of HBV infection. Methods HBV RNA and cccDNA were quantified in 19 liver biopsies from patients with chronic HBV infection, as well as in transfected Huh7.5 cells and in PLC/PRF/5 cells carrying integrated HBV genome. Results Patients negative for HBeAg had 2.15 log lower levels of cccDNA in liver tissue, 4.84 log lower serum levels of HBV DNA and 1.45 log lower serum levels of HBsAg, than HBeAg-positive patients. The pgRNA in liver tissue correlated strongly with cccDNA (R 2 = 0.87, p<0.0001) and HBV DNA levels in serum (R 2 = 0.81, p<0.0001), whereas S-RNA correlated strongly with cccDNA (R 2 = 0.65, p<0.0001) and HBsAg levels (R 2 = 0.57, p = 0.0003). The S-RNA/pgRNA ratio was higher in HBeAg-negative patients (ratio 40 vs. 3, p = 0.01) and in PLC/PRF/5 cells, and was in transfected Huh7.5 cells not influenced by mutations in the HBV core promoter. Conclusion The reduction of viremia that is observed after loss of HBeAg was mainly explained by reduced cccDNA load in the liver, whereas the contribution of down-regulation of pgRNA transcription was relatively small. Enhanced transcription of S-RNA does not explain excessive production of HBsAg.

The COBAS AMPLICOR hepatitis B virus assay targets a conserved region of the genome and is widely used to monitor treatment of hepatitis B in order to identify emerging resistance. However, the assay failed to recognize increasing viremia levels when YMDD mutations were paralleled by mutations in the segment targeted by the COBAS AMPLICOR probe.The aim of treating chronic hepatitis B virus (HBV) infection is to prevent the emergence of liver cirrhosis or hepatocellular carcinoma. Currently, treatment is given either as injections of pegylated interferon or as oral doses of antivirals, such as lamivudine or adefovir. Lamivudine therapy has the advantages of documented efficiency, lower cost, and minimal side effects but carries an annual 15 to 20% risk of emergence of resistant mutants, which may cause severe exacerbations of hepatitis. Lamivudine resistance is typically due to a mutation in the YMDD motif of the polymerase (M204I or M204V) (7) and is recognized as increases in HBV DNA levels (by more than 1 log).Here we describe a case where the COBAS AMPLICOR assay (Roche Diagnostics, Branchburg, NJ) failed to reveal resistance in a 56-year-old male Vietnamese patient with HBV cirrhosis.The patient had arrived in Sweden in 1999, and his chronic HBV infection was discovered in 2001 by a general practitioner (positive HBsAg and anti-HBc results obtained by use of AxSYM [Abbott, Illinois]). The patient was admitted to the infectious disease clinic, where he presented signs of active cirrhosis with a low platelet count (52 ϫ 10 9 /liter), increased prothrombin time (international normalized ratio, 1.7), and an aspartate aminotransferase level of 2.5 times the upper limit of normal. HBeAg (tested by use of AxSYM [Abbott]) was not detected in serum, but the HBV DNA level was high, above 10 million copies/ml, as detected by use of COBAS AMPLICOR. Hepatitis C antibodies (tested for with AxSYM [Abbott]) were not detected. The patient was put on long-term lamivudine therapy (100 mg daily), which resulted in a 3-log reduction in HBV DNA to around 20,000 copies/ml as measured by COBAS AMPLICOR after 3 months. Thereafter the HBV DNA levels did not decrease further but appeared to remain at levels between 20,000 and 50,000 copies/ml. The aminotransferase levels also remained moderately elevated. A sample drawn after 21 months of treatment was included (by chance) in a study comparing COBAS AMPLICOR and COBAS TaqMan (Roche) (4), which revealed a 2-log underestimation of viremia by COBAS AMPLICOR. This finding led us to further investigate the emergence of mutations in the YMDD motif as well as in the region targeted by the COBAS AMPLICOR assay.Viremia was measured by two different systems, the COBAS AMPLICOR Monitor (Roche) (5) and an in-house real-time PCR targeting a conserved region of the HBsAg gene. COBAS AMPLICOR testing was performed as instructed by the manufacturer. The S-region real-time PCR assay included DNA extraction by use of Magnapure LC (Roche) with a total nucleic acid isolation kit and 45 cycles of two-step...

BackgroundHepatitis B virus (HBV) DNA in serum of chronically infected patients declines by 3–4 log10 units at loss of HBe antigen (HBeAg) from serum. The mechanisms behind this decline, and the much smaller decline of surface antigen (HBsAg) levels, are still not well known. The aim of this study was to get a better understanding of this process by analysing both serum and intrahepatic markers of HBV replication.MethodsLevels of HBV DNA and HBsAg in serum, and covalently closed circular DNA (cccDNA), pregenomic RNA (pgRNA) and S-RNA and total intrahepatic HBV DNA (ihDNA) in liver biopsies from 84 chronically infected patients (16 positive and 68 negative for HBeAg) were analysed.ResultsLower HBV DNA levels within HBeAg-positive stage reflected lower levels of cccDNA and pgRNA with strong correlation. In HBeAg-negative patients, ihDNA levels were greater and HBV DNA levels in serum lower than expected from pgRNA levels. A lower HBV DNA/HBsAg ratio corresponded with lower pgRNA/cccDNA (p < 0.01) and higher S-RNA/cccDNA (p < 0.0001) ratios, suggesting that in HBeAg-negative patients transcription of pgRNA, but not of S-RNA, becomes suppressed.ConclusionsThe marked reduction of HBV DNA in serum after loss of HBeAg appears to be due to combined reduction of cccDNA, pgRNA and yet unidentified mechanisms downstream of reverse transcription. Such mechanisms include faster clearance of circulating virus or blocked secretion of virions, the latter supported by the observed relative increase of ihDNA in HBeAg-negative patients. The smaller reduction of S-RNA than of pgRNA partly explains why HBsAg remain high in the HBeAg-negative stage, supporting the possibility of HBsAg synthesis from integrated HBV DNA.