Antigenic characterization of pre- and post-liver transplant hepatitis B surface antigen sequences from patients treated with hepatitis B immune globulin.

BACKGROUND/AIMS: The success of treatment with hepatitis B hyperimmune globulin in preventing recurrence of hepatitis B virus infection in patients undergoing orthotopic liver transplantation depends on maintaining levels of anti-HBs sufficient to neutralise hepatitis B virus and also on patient compliance. Breakthrough infections may occur, and these have been associated with the emergence of variants in HBsAg. METHODS: Three patients, two who relapsed and one who had no evidence of hepatitis B virus infection post-orthotopic liver transplantation were studied. Polymerase chain reaction and sequencing of pre- and post-orthotopic liver transplantation samples was followed by antigenic analysis of the in vitro expressed cloned sequences. RESULTS: In two patients who were treated with hyperimmune globulin, amino acid variation in the region of the immunodominant B cell epitopes of HBsAg occurred. Sequencing of clones revealed fluctuating variant sequences over time. One had clinical relapse and immune escape was evident on in vitro antigenic analysis. Patient two lost HBsAg reactivity post-orthotopic liver transplantation. There was loss of an antigenically critical cysteine molecule; sequencing of clones revealed that this was the dominant species. The third patient relapsed when protective levels of anti-HBs were not maintained; HBsAg showed no variation compared to a standard subtype sequence. CONCLUSION: These data provide strong experimental evidence of immune escape. It appears that hyperimmune globulin provides the selection pressure. In these patients, HBsAg negativity does not exclude infection of the transplanted liver.

Selection of hepatitis B surface "escape" mutants during passive immune prophylaxis following liver transplantation: potential impact of genetic changes on polymerase protein function.

CASE REPORT: A patient is described who developed hepatitis B virus (HBV) reinfection five months following liver transplantation. Failure of hepatitis B immunoglobulin prophylaxis was associated with the emergence of mutations. HBV gene sequencing identified nucleotide substitutions associated with amino acid changes, one within the major hydrophilic region (MHR) of the HBV surface antigen at amino acid position 144 and one outside the MHR. Because of the overlapping reading frames of surface and polymerase genes, the latter surface antigen change was associated with an amino acid change in the polymerase protein. The patient developed significant allograft hepatitis and was treated with lamivudine (3TC) 100 mg daily. Rapid decline of serum HBV DNA was observed with loss of HBV e antigen and HBV surface antigen from serum. There was normalisation of liver biochemistry, and liver immunohistochemistry showed a reduction in HBV core and disappearance of HBs antigen staining. CONCLUSION: Surface antigen encoding gene mutations associated with HBIg escape may be associated with alteration of the polymerase protein. The polymerase changes may affect sensitivity to antiviral treatment. Selection pressure on one HBV reading frame (for example, HBIg pressure on HBsAg, or nucleoside analogue pressure on polymerase protein) may alter the gene product of the overlapping frame. Such interactions are relevant to strategies employing passive immune prophylaxis and antiviral treatment.

Hepatitis C virus core protein interacts with a human DEAD box protein DDX3.

Several studies have implicated hepatitis C virus (HCV) core in influencing the expression of host genes. To identify cellular factors with a possible role in HCV replication and pathogenesis, we looked for cellular proteins that interact with the viral core protein. A human liver cDNA library was screened in a yeast two-hybrid assay to identify cellular proteins that bind to core. Several positive clones were isolated, one of which encoded the C-terminal 253 amino acids of a putative RNA helicase, a DEAD box protein designated DDX3. Bacterially expressed glutathione-S-transferase-DDX3 fusion protein specifically pulled down in vitro translated and radiolabeled HCV core, confirming a direct interaction. Immunofluorescent staining of HeLa cells with a polyclonal antiserum showed that DDX3 is located predominantly in nuclear speckles and at low levels throughout the cytoplasm. In cells infected with a recombinant vaccinia virus expressing HCV structural proteins (core, E1, and E2), DDX3 and core colocalized in distinct spots in the perinuclear region of the cytoplasm. The regions of the proteins involved in binding were found by deletion analysis to be the N-terminal 59 amino acid residues of core and a C-terminal RS-like domain of DDX3. The human DDX3 is a putative RNA helicase and a member of a highly conserved DEAD box subclass that includes murine PL10, Xenopus An3, and yeast Ded1 proteins. Their role in RNA metabolism or gene expression is unknown. The significance of core-helicase interaction in HCV replication and pathogenesis is discussed.

Core variability does not affect response to interferon alpha in HBeAg negative chronic hepatitis B.

BACKGROUND/AIMS: The pre-core stop codon variant (A 1896) of hepatitis B virus (HBV) has been associated with chronic active liver disease with acute exacerbations and a high relapse rate after an initial response to alpha-interferon (IFN-alpha) therapy. Poor sustained response has been correlated with a high prevalence of mutations in the core region, potentially enabling escape from the immune system. The aim of this study was to analyse the predictive factors of response to IFN-alpha in such patients. METHODS: We studied the baseline clinical, biochemical, histological, serological and virological parameters in 30 hepatitis B s antigen positive (HBsAg-positive)/hepatitis B e antigen negative (HBeAg-negative) Greek patients with chronic liver disease. The patients were selected from a cohort who received IFN-alpha for 24 weeks. These were divided into three groups of ten sequential patients: those with no response to IFN-alpha treatment, those who relapsed after an initial response, and those with a sustained response. Serum HBV DNA was measured by a liquid hybridisation method, and the anti-HBc IgM was quantitated by the IMx analyser. The amino-acid sequence of core protein residues 40-89, a region where a clustering of mutations has been detected previously in severe hepatitis, was compared with a sequence from an HBeAg positive patient with chronic liver disease. RESULTS: Multiple logistic regression analysis showed that the initial response to IFN-alpha could be predicted by pre-treatment absence of HBcAg staining in the liver and high ALT values, but no parameter could predict sustained response. The pre-treatment extent and pattern of aminoacid substitutions in the core region sequenced was similar in all groups studied and was not associated with IFN-alpha response. CONCLUSIONS: In HBsAg-positive/HBeAg-negative patients with chronic liver disease, response to IFN-alpha therapy was not correlated with genomic variability of the core region. Other parameters such as pre-treatment HBcAg positivity in the liver and alanine aminotransferase values indicative of disease activity before treatment were associated with initial IFN-alpha response.

Role of the carboxy terminus of herpes simplex virus type 1 DNA polymerase in its interaction with UL42.

Several recent reports implicate sequences at or near the C terminus of the catalytic subunit (POL) of herpes simplex virus type 1 (HSV-1) DNA polymerase in its interaction with the accessory protein UL42. We have investigated further the involvement of this region by three different approaches: anti-idiotype antibodies, a competition ELISA and inhibition of the interaction by peptides. Antibodies raised in rabbits to peptides corresponding to regions of POL all reacted in Western blots with POL. Surprisingly, the sera raised against C-terminal peptides (amino acids 1221 to 1235 and 1224 to 1235) also reacted with UL42. The UL42 reactivity was shown to be due to the presence of anti-idiotype antibodies, providing direct evidence for complementarity of the structure of the extreme C terminus of POL to a region of UL42. To measure the contribution of the C terminus of POL to UL42 binding we developed a competition ELISA using POL, a truncated polymerase lacking the carboxyl-terminal 27 amino acids (POLd1) and UL42. UL42 binding to immobilized POL was inhibited approximately four times more effectively by competition, in solution, with POL than with POLd1, indicating that the C-terminal 27 amino acids of POL are responsible for at least 75% of the binding energy. A peptide corresponding to these 27 amino acids (residues 1209 to 1235) inhibited both the POL-UL42 interaction and the stimulation of POL by UL42 and did so more effectively than peptides corresponding to amino acids just away from the C terminus (residues 1195 to 1223 and 1177 to 1195).

Inhibition of herpes simplex virus type 1 DNA polymerase activity by peptides from the UL42 accessory protein is largely nonspecific.

To identify regions in the UL42 protein of herpes simplex virus type 1 which affect viral DNA polymerase activity, a series of 96 overlapping pentadecapeptides spanning the entire 488 amino acids of the UL42 protein were synthesized and tested for their ability to inhibit polymerase activity on a primed single-stranded M13 DNA template. Two assays were used: formation of full-length double-stranded M13 molecules and rate of incorporation of deoxyribonucleoside triphosphates. Peptides from five noncontiguous regions of the UL42 protein were found to inhibit herpes simplex virus type 1 polymerase activity in both the presence and absence of UL42 protein. The most active peptides from each region correspond to amino acids 23 to 38 (peptide 6), 64 to 78 (peptide 14), 89 to 102 (peptide 19), 229 to 243 (peptide 47), and 279 to 293 (peptide 57). By two different methods (DNA mobility shift and DNA precipitation), peptides 14, 19, 47, and 57 were found to bind DNA; they most probably inhibit enzyme activity by this mechanism. Peptide 6 did not bind DNA and must act by some mechanism other than competing for DNA. The inhibitory peptides were also tested for activity against mammalian polymerase alpha and the Klenow fragment of Escherichia coli polymerase. Although some limited specificity was demonstrated (up to 10-fold for peptide 6), all the peptides showed significant activity against both polymerase alpha and E. coli polymerase.

Advantages of branched peptides in serodiagnosis. Detection of HIV-specific antibodies and the use of glycine spacers to increase sensitivity.

The reactivities of antibodies with branched and monomeric peptides were compared in ELISA assays. We found that lower amounts of antibodies could be detected with branched peptides than with monomeric peptides. This was observed with a monoclonal antibody and with antibodies in the sera of various HIV-positive individuals. To investigate the physical aspects of branched peptides important for the observed increase in sensitivity, glycine spacers of different lengths were introduced between the branched lysine core and the epitope reacting with the monoclonal antibody. The effect of the number of glycine residues, both on the sensitivity of antibody detection and on the amount of branched peptide needed to produce a given signal, was studied and the optimum was found at 4-5 residues. We discuss the basis for these findings and conclude that the routine use of branched peptides for serodiagnosis will give both greater sensitivity and appreciable cost savings.

Generation of anti-peptide and anti-protein sera. Effect of peptide presentation on immunogenicity.

The technique of Fmoc chemistry has been applied successfully to the synthesis of branched peptides. The immunogenicity of branched peptides has been compared quantitatively with those of protein-conjugated and resin-linked peptides. Six different peptide sequences were used to immunise rabbits and both antipeptide and anti-protein titres were determined for each serum. The data show that the titres of sera from rabbits immunised with branched peptides were higher than those of rabbits immunised with protein-conjugated peptides which in turn were higher than those immunised with resin-linked peptides. The effect was demonstrated with two strains of rabbits.

Isolation and characterization of a high molecular weight lymphocyte Fc gamma receptor-blocking factor associated with renal allograft survival.

We have confirmed our previous observation that improved human renal allograft survival is associated with the presence in pre-transplant serum of a high molecular weight lymphocyte Fc gamma receptor-blocking factor. Serum fractionation studies suggest that this factor is a complex protein consisting of IgG together with an IgG-binding protein which has an apparent molecular weight of approximately 60 kD.

Mapping of epitopes on the 65k DNA-binding protein of herpes simplex virus type 1.

Previously we have described the isolation of seven monoclonal antibodies (MAbs) and two polyvalent rabbit sera directed against the product of herpes simplex virus type 1 (HSV-1) gene UL42, a 65K DNA-binding protein (65KDBP) which is essential for HSV DNA replication and virus growth. We now report the synthesis of all 483 overlapping hexapeptides of this 488 amino acid protein and describe their use for the identification of epitopes recognized by these MAbs and polyvalent sera. MAb 6898, derived from one fusion, recognizes the peptides EDLDGA and DLDGAA which correspond to amino acids 363 to 369 of 65KDBP. MAbs Z4D4, Z6F3, Z1A8, Z10Cl, Z3H12 and Z1F11, derived from a second fusion, all recognize the peptides GDPEDL and DPEDLD which correspond to amino acids 360 to 366. As expected both polyvalent sera recognize several different epitopes.

Microalgae and cyanobacteria as a source of glycosidase inhibitors.

Culture filtrates and organic solvent extracts of over 500 freshwater and marine eukaryotic microalgae and cyanobacteria were screened for the presence of glycosidase inhibitors. Rapid colorimetric assays were used to detect inhibitors of alpha-glucosidase, alpha-amylase and beta-galactosidase. Inhibitors were found from 38 species. The results suggest that microalgae and cyanobacteria have potential as a source of glycosidase inhibitors which may have clinical applications.