Yanuario Sanchez

Cellular and humoral immunity in guinea pigs to two major polypeptides derived from hepatitis B surface antigen.

Guinea pigs immunized with hepatitis B surface antigen (HBsAg), types adw, adr and ayw, and with two major polypeptides derived from HBsAg/adw developed cell-mediated immunity as determined by the macrophage migration inhibition assay. Peritoneal exudate cells from animals immunized with a 22000- or a 25000-mol. wt. polypeptide derived from HBsAg/adw showed significant migration inhibition after challenge with either polypeptide or with purified HBsAg. Significant inhibition of macrophage migration was not observed when polypeptide-sensitized cells were challenged with normal human serum or with normal human liver extract. Similarly, a cell-mediated immune response was not observed in peritoneal exudate cells from animals sensitized to normal human serum or normal human liver extract which were challenged with either of the polypeptides. The humoral immune response to either of the polypeptides, as measured by radioimmunoassay, was substantially lower than that observed in animals immunized with intact particles. This apparent difference between cellular and humoral responses suggests that the macrophage migration assay is a sensitive indicator of the immunogenicity of the smaller mol. wt. HBsAg-derived polypeptides in guinea pigs.

Characterization of anti-hepatitis B surface antigen monoclonal antibodies.

17 monoclonal antibodies generated against purified hepatitis B surface antigen (HBsAg), subtype ayw, were characterized by solid-phase radioimmunoassays. Eleven of these antibodies had specificity against the group-specific alpha determinant of HBsAg, two demonstrated antibody activity against the w HBsAg subtype, one against human serum albumin, and three against human IgG. All monoclonal antibodies were of the IgG class.

A common human anti-hepatitis B surface antigen idiotype is associated with the group a conformation-dependent antigenic determinant.

Abstract Human antibodies that express a common anti-hepatitis B surface antigen idiotype were directed against the group-specific a determinant. The ability of hepatitis B surface antigen (HBsAg)-derived polypeptides to inhibit the idiotype-anti-idiotype reaction was dependent on conformation, since denatured HBsAg viral polypeptides virtually lost their inhibitor capacity when compared to native polypeptides. This report further characterizes a common idiotype associated with human antibodies to HBsAg.

Enhancement of viral hepatitis B antibody (anti-HBs) response to a synthetic cyclic peptide by priming with anti-idiotype antibodies

In vivo injections of anti-idiotype antibodies were used to prime the immune system of mice to hepatitis B surface antigen (HBsAg). Anti-idiotype reagents in conjunction with a cyclic synthetic peptide analogous to positions 122-137 of HBsAg induced an antibody response to HBsAg (anti-HBs) comparable to that obtained with a single injection of intact HBsAg particles. In addition, high anti-HBs titers were produced in mice injected with HBsAg following anti-idiotype priming. These data indicate that anti-idiotype antibodies may be useful in priming the immune system of a host to a potential infectious agent.

Antigenic relationship of a hepatitis B surface antigen-derived polypeptide and human serum albumin.

Four major polypeptides with mol. wt. of 22000, 25000, 52000 and 68000 were isolated from solubilized preparations of hepatitis type B surface antigen (HBsAg). These four populations, referred to as P22, P25, P52 and P68, respectively, were used to immunize guinea-pigs. Guinea-pigs were also inoculated with HBsAg and with purified human serum albumin (HuSA). These antisera were utilized to establish that intact HBsAg particles are associated with HuSA antigenic reactivity. HuSA antigenic determinants were associated with purified preparations of P68. HuSA antigenic activity was not detected with purified preparations of P22, P25 and P52 or with respective specific antisera to each of the above. However, purified P68 contained the antigenic determinants of both host protein and hepatitis B virus-specified protein origin.

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.

Immunogenicity of Conjugates and Micelles of Synthetic Hepatitis B Surface Antigen Peptides

A cyclic peptide containing the amino acid sequence 122 through 137 of the major hepatitis B surface antigen (HBsAg) polypeptide was synthesized. The immunogenicity of this synthetic peptide, aggregated in micelles or covalently coupled to tetanus toxoid, was assessed in mice. Antibodies against HBsAg (anti-HBs) were obtained with both preparations, administered either in saline suspension or adsorbed on aluminum gel. The peptide-tetanus toxoid conjugate was more immunogenic than the peptide micelles, producing high levels of specific anti-HBs.

Evidence for the presence of repeating antigenic determinants in the major and minor polypeptides derived from hepatitis B surface antigen

Abstract The immunogenicity of the four major and seven minor hepatitis B surface antigen (HBsAg)-derived polypeptides was evaluated in guinea pigs and compared to that of 22-nm HBsAg particles. Both humoral and cell-mediated immune responses were determined. All antisera reacted with homologous HBsAg/ ayw and P22/ ayw . However, the glycosylated polypeptides (P25, P27, P31, and P52) generally elicited the most intense immune responses. At least nine polypeptides induced antibodies which also reacted with heterologous HBsAg/ adw particles and P22/ adw. Whole HBsAg/ ayw particles induced transformation in lymphocytes obtained from guinea pigs immunized with the major or minor polypeptides. These data tend to demonstrate that all HBsAg-derived polypeptides contain in their structure the group-specific and at least one of the subtype-specific antigenic determinants, and these determinants are also exposed on the surface of the HBsAg particle. It seems possible that a limited nucleotide sequence confers the same virus specificities to most if not all of the constitutive proteins of HBsAg.

Hepatitis B Surface Antigen Polypeptide and Synthetic Peptide Vaccines

The hepatitis B vaccine recently licensed for use in the United States has undergone several field trials in high-risk populations and has proven to be immunogenic, efficient and safe. The hepatitis vaccine is unique in that the hepatitis B virus (HBV), the ultimate source of the vaccine, cannot be grown in tissue culture. The immunizing antigen, purified from plasma of healthy human carriers of HBV, consists of noninfectious 22 nm particles of hepatitis B surface antigen (HBsAg), produced as excess viral surface protein and released in the bloodstream. Although the HBsAg particles are highly purified and inactivated, there is some concern that the vaccine may contain traces of host material or infective adventitious agents, which may trigger undesirable effects in the recipients. Ideally, a hepatitis B vaccine candidate should have the following properties: 1) be free of normal human serum protein contaminants, 2) be free of potential residual infectious HBV, 3) be free of other viruses and nucleic acid, 4) have a high degree of immunogenicity when administered in conjunction with an adjuvant suitable for use in humans, 5) be independent of human HBV carriers as a source of immunogenic material, 6) have a reproducible composition, 7) cost less than the present vaccine, and 8) be available in unlimited supplies.