Howard A. Fields

Linear B-cell epitopes of the NS3-NS4-NS5 proteins of the hepatitis C virusas modeled with synthetic peptides

A set of 150 synthetic peptides spanning the proteins NS3-NS4-NS5 of the hepatitis C virus (HCV) was synthesized and tested with a panel of 20 sera obtained from HCV-infected patients. Of 62 peptides prepared from the NS3 region, none exhibited strong antigenic reactivity. Rather, five peptides from this region demonstrated specific reactivity with only 5-10% of anti-HVC-positive sera. Nonetheless, it is well known that the NS3 region contains strong antigenic epitopes. These epitopes appear to be modeled in a functionally active manner with recombinant proteins and cannot be mimicked properly with short synthetic peptides. This finding suggests that the major NS3 antigenic epitopes are conformationally dependent. Seven of 20 peptides prepared from the NS4 region were immunoreactive. Five peptides from this region demonstrated very strong HCV-specific antigenic reactivity. Four of the five peptides belong to the recognized immunoreactive 5-1-1 region located inside the C100-3 antigen. One peptide demonstrating immunoreactivity with approximately 90% of anti-HCV-positive sera was found outside the C100-3 region at the C-terminal part of the NS4 protein. Of 68 peptides synthesized from the NS5 protein, 30 were immunoreactive. Six of the 30 demonstrated immunoreactivity with 35-50% of anti-HCV-positive sera. Thus, the NS4 and NS5 regions of the HCV polyprotein contain a large number of specific, broadly reactive, linear antigenic epitopes. The highly antigenic reactivity of the NS5 region suggests that this protein may have significant diagnostic potential.

Comparative characterization of antigenic epitopes in the immunodominant region of the protein encoded by open reading frame 3 in Burmese and Mexican strains of hepatitis E virus

To analyse the effect of strain-specific sequence variation on the antigenic properties of the protein encoded by the open reading frame 3 (ORF 3) of hepatitis E virus (HEV), two sets of short overlapping peptides spanning amino acids 91 to 123 of this protein from Burmese and Mexican strains were synthesized and tested with sera obtained from outbreaks of enterically transmitted non-A, non-B hepatitis in three different regions of the world (Mexico, Turkmenistan and Kenya). The data suggest strain-specific variation in the antigenic reactivity of the ORF 3 protein. The C-terminal region of this protein contains several antigenic epitopes located in the most variable positions. Individual sera were found to interact with different groups of epitopes from each set of peptides. The antigenic epitopes of the Mexican strain appear to be less conformation-dependent than those of the Burmese strain. The most immunoreactive epitope of the ORF 3 protein from the Mexican strain was localized at amino acid positions 95 to 101. The ORF 3 protein of the Burmese strain contains an immunodominant epitope at amino acid positions 112 to 117. Some of these short peptides may be useful for the development of a diagnostic assay to discriminate between the Burmese and Mexican strains.

Characterization of monoclonal antibodies and epitope mapping of the NS4 protein of hepatitis C virus.

Recombinant DNA containing sequences of HCV NS4 protein was expressed in Escherichia coli cells. Six hybridoma clones producing monoclonal antibodies (MAB) to recombinant NS4 protein (rNS4), aa 1677-1756, were developed. Mapping with a panel of 33 peptides and reciprocal competitive EIA have shown that MAB obtained revealed five antigen determinants, not described earlier: MAB 3F11 and 3F12-one genotype-independent epitope of NS4A (aa 1700-1707) common for genotypes 1, 2 and 3; MAB 1D11-genotype-independent epitope (aa 1713-1728) and MAB 1D3-genotype (subtype 1b)-specific epitope of NS4B (aa 1711-1731); MAB 6B11 and C1-two conformation-dependent determinants in 5-1-1 region. These data indicate that the 5-1-1 region of NS4 protein has a complex antigenic structure and contains at least eight epitopes, including five, revealed in the present work. MAB obtained recognized native viral protein in the cytoplasm of liver cells of patients with chronic hepatitis C. The positive rates of the immunostaining for NS4 antigen using MAB 6B11, 1D11 and 3F12 were 64, 59 and 50%, respectively. It was found that 6B11 MAB to a conformation-dependent epitope much more actively interacts with native NS4 than with the recombinant protein to which MAB was developed. The epitope recognized by 6B11 MAB is highly immunogenic since it induces the B-cell response in all patients investigated with identified anti-NS4 antibodies in blood serum. The MAB panel obtained in this study may become a useful tool for the diagnostic purposes, for the investigation of NS4B function and for the host-viral interactions at the cell level.

Development of sensitive immunoassays for detection of antibodies against hepatitis B surface antigen

Three micro solid phase immunoassays (a micro-SPRIA and two ELISA techniques) were developed and tested for the detection of anti-HBs antibodies. Two different crosslinkers (glutaraldehyde and N-succinimidyl 3-(2-pyridyldithio) propionate) were used to couple a goat anti-mouse IgG reagent to alkaline phosphatase for use as enzyme-labeled probes in the two ELISA tests. With the latter crosslinker, a defined conjugate with a 1:1 antibody-enzyme molar ratio was obtained. The sensitivities of micro-SPRIA and the two types of ELISA were compared to that of the commercial solid phase radioimmunoassay AUSAB test. All three microtests were significantly more sensitive than the AUSAB test. The ELISA using the glutaraldehyde cross-linked conjugate was 3-5 times less sensitive than micro-SPRIA, while the ELISA using the disulfide-linked conjugate was 2.6-4.0 times more sensitive than micro-SPRIA.

Purification of acute phase anti-hepatitis A virus (HAV) IgM and development of an IgM solid-phase radioimmunoassay for the detection of HAV

A simple two-step procedure for the purification of IgM from acute phase hepatitis A infected chimpanzee serum has been developed. The procedure involves the precipitation of the euglobulin fraction with boric acid and the fractionation of the resuspended material on Sephacryl S-300 (Pharmacia Fine Chemicals, Piscataway, NJ). The purified IgM was used to develop a solid-phase radioimmunoassay (SPRIA) in which IgM was used as capture antibody and iodinated IgM was used as a probe. This assay was compared with a conventional IgG-based SPRIA. The IgM-based assay resulted in a superior response (P/N) for the detection of antigen in crude preparations; however, endpoint analyses demonstrated similar sensitivities.

Artificial NS4 mosaic antigen of hepatitis C virus.

An artificial antigen composed of 17 small antigenic regions derived from the NS4‐protein of hepatitis C virus (HCV) genotypes 1 through 5 was designed and constructed. Eleven antigenic regions were derived from the 5‐1‐1 region, and 6 others were derived from the C‐terminus of the NS4‐protein of different genotypes. The gene encoding for this artificial antigen was assembled from synthetic oligonucleotides by a new approach designated as restriction enzyme‐assisted ligation (REAL). The full‐length synthetic gene was expressed in Escherichia coli as a fusion protein with glutathione S‐transferase. By the use of site‐specific antibodies raised against synthetic peptides, it was shown that all regions for which sequence‐specific antibodies were obtained were accessible to antibody binding. The diagnostic relevance of the NS4 artificial antigen was demonstrated by testing this antigen with 4 HCV seroconversion panels and a panel of previously tested and stored serum specimens. The artificial antigen was found to specifically detect anti‐NS4 antibodies in a number of specimens that were previously found to be anti‐NS4 negative. Furthermore, this antigen detected anti‐NS4 activity earlier in 2 of 4 seroconversion panels than did the antigen used in a commercially available supplemental assay. Equally important is the observation that the artificial NS4 antigen demonstrated equivalent anti‐NS4 immunoreactivity with serum specimens obtained from patients infected with different HCV genotypes, whereas the NS4 recombinant protein derived from genotype 1, used in the commercial supplemental test, was less immunoreactive with serum specimens containing HCV genotypes 2, 3, and 4. Collectively, these data support the significant diagnostic potential of the NS4 mosaic antigen. The strategy employed in this study may be applied to the design and construction of other artificial antigens with improved diagnostically pertinent properties. J. Med. Virol. 59:437–450 1999.

Enzyme potentiated radioimmunoassay (EPRIA): A sensitive third-generation test for the detection of hepatitis B surface antigen

A sensitive, specific immunoassay for detection of hepatitis B surface antigen (HBsAg) is described. The assay combined enzyme-linked immunosorbent assay and solid-phase radioimmunoassay and is termed enzyme potentiated radioimmunoassay (EPRIA). HBsAg was quantitated by enzymatic conversion of L[14C]glutamic acid to 14CO2 and gamma-aminobutyric acid by glutamate decarboxylase (GDC) conjugated wih goat anti-HGs IgG. Conjugation of IgG and GDC was by a thiol-disulfide bond exchange reaction after reacting N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) with each reagent. A positive/negative ratio of 2.2 was established as significant by examination of 40 normal sera negative for HBsAg. This value was the mean cpm plus 3 standard deviations. By an identical statistical analysis of sensitivity, EPRIA was found to be approximately 100-fold more sensitive than Ausria II (Abbott Laboratories, North Chicago, IL).

Artificial mosaic proteins as new immunodiagnostic reagents: the hepatitis E virus experience.

BACKGROUNDnNaturally occurring viral proteins derived from cell culture and recombinant proteins expressed in procaryotic systems have been used extensively as target proteins in the development of immunoassay methods for the detection of antibodies. However, immunoassays utilizing these proteins often yield false-positive reactions suggesting that it may be possible to identify and remove regions responsible for these non-specific reactions.nnnOBJECTIVEnIn this paper we describe a new strategy for the construction of immunoreactive recombinant proteins designed to improve immunoassay specificity.nnnSTUDY DESIGNnA synthetic gene encoding an artificial polypeptide composed of antigenic epitopes of the hepatitis E virus (HEV) proteins was constructed from short oligonucleotides by the polymerase chain reaction (PCR). The polypeptide comprises a mosaic of three antigenically dominant regions from the protein encoded by open reading frame 2 (ORF2), one antigenically active region from the protein encoded by ORF3 of the Burmese HEV strain, and one antigenically active region from the protein encoded by ORF3 of the Mexican strain. The mosaic protein was expressed in Escherichia coli as a chimera with glutathione-S-transferase or beta-galactosidase.nnnRESULTSnGuinea pig sera containing antibodies to the corresponding HEV synthetic peptides were used to demonstrate by immunoblot analysis and by enzyme immunoassay (EIA) the presence and accessibility of all HEV-specific antigenic epitopes designed into the mosaic protein. Both hybrid proteins were shown by immunoblot analysis using a panel of human anti-HEV-positive and -negative sera to be HEV-specific. A sensitive and specific EIA was developed to detect IgG anti-HEV activity in human sera. A neutralization test using individual synthetic peptides corresponding to the epitopes designed into the mosaic protein was also developed to confirm IgG anti-HEV activity by absorbing the specimen before retesting by EIA.nnnCONCLUSIONnAn artificial mosaic protein composed of short linear HEV-specific antigenic epitopes was constructed from synthetic oligonucleotides by PCR and used to develop a sensitive and specific EIA for the detection of anti-HEV activity in human sera.

Enzyme-antibody conjugation by a heterobifunctional reagent and its application in enzyme-linked immunosorbent assay (ELISA) for the detection of hepatitis B surface antigen.

The heterobifunctional reagent 3-(2-pyridyl-dithio)propionate (SPDP) was used to prepare defined conjugates composed of horseradish peroxidase (HRP) and goat anti-HBs IgG. The modification of HRP and IgG with SPDP was dependent on both the SPDP: protein molar ratio and the pH of the buffer. Conjugates were separated by a single affinity chromatographic step using concanavalin A-Sepharose 4B equilibrated with 0.1 M Tris-HCl buffer, pH 7.4, containing 0.5 M KCl and 1 mM EDTA. The conjugate was eluted with 10 or 100 mM alpha-methyl-D-mannoside and appropriate pools were made reflecting various HRP/IgG molar ratios. Each pool was examined for performance in an enzyme-linked immunosorbent assay for HBsAg. Conjugate composed of an HRP/IgG molar ratio of 2.5-4 yielded the greatest sensitivity.