Neurodevelopmental follow-up of infants with birthweight less than 801 grams with intracranial hemorrhage.

The incidence and severity of intracranial hemorrhage (ICH) were determined in a group of surviving infants with birthweights less than 801 g born during the period of 1983 through 1985. Neurodevelopmental status was assessed at 2 and 3 years of age to determine the relationship between neonatal ICH and later outcome in these infants. Forty-four of 77 neonatal survivors (57%) had ICH. Infants with ICH were less mature than those without (25.7 weeks' vs 26.5 weeks' gestation, P less than .05). There were no other perinatal factors that significantly differed between groups. At both 2 and 3 years of age, there were no significant differences between groups of infants with no or mild ICH and those with severe ICH regarding frequency of developmental delay, cerebral palsy, or visual impairment. Persistent ventriculomegaly in infants with ICH was associated with the highest incidence of neurodevelopmental disabilities. For extremely low birthweight infants, the presence and severity of neonatal ICH, in itself, did not adequately predict neurodevelopmental outcome at 2 or 3 years of age. Among infants with ICH, ventriculomegaly persisting to hospital discharge may indicate the greatest risk for neurologic and visual disabilities.

Inhibition of a common human anti-hepatitis B surface antigen idiotype by a cyclic synthetic peptide.

A common human anti-hepatitis B surface antigen idiotype-anti-idiotype reaction was partially inhibited by a cyclic synthetic hepatitis B surface antigen peptide. Reduction of the intrachain disulfide bond and subsequent alkylation destroyed its inhibitory activity, suggesting that a conformation-dependent group alpha epitope was associated with this cyclic peptide.

Epitopes associated with a synthetic hepatitis B surface antigen peptide.

A synthetic peptide (SP1), corresponding to the amino acid residues 122 through 137 of the major polypeptide derived from hepatitis B surface antigen (HBsAg), subtype ayw, was analyzed for the presence of the major epitopes of HBsAg. Both a cyclic form, produced by introduction of an intrachain disulfide bond, and a linear form of the peptide were characterized. A panel of monoclonal antibodies with defined specificity for the cross-reactive group a antigenic determinant(s) and for the y and w subtype specificities was used for this analysis. The cyclic, but not the linear, form of SP1 reacted with five of 14 anti-a monoclonal antibodies, demonstrating that the cyclic peptide contains a conformation-dependent a epitope. Only one anti-a antibody was found to react with both cyclic and linear forms of SP1. Because SP1 failed to react with the remaining 8 anti-a monoclonal antibodies, it was concluded that the a antigenic reactivity associated with HBsAg contains an additional epitope(s) unrelated to that expressed on SP1. Both cyclic and linear SP1 reacted with three of three anti-y monoclonal antibodies, indicating that a sequential y epitope is also present on SP1; no w reactivity was detected. Analysis of the idiotypes associated with the monoclonal antibodies showed those that combined with cyclic SP1 also inhibited the binding of a common human anti-HBs (CHBs) idiotype with its rabbit anti-idiotype serum, whereas a monoclonal antibody that did not react with the cyclic SP1 epitope failed to inhibit the CHBs idiotype-anti-idiotype reaction. Thus, the conformational a epitope present on cyclic SP1 appears to contain the predominant epitope recognized by humans in response to a natural HBV infection.

Detection of interspecies idiotypic cross-reactions associated with antibodies to hepatitis B surface antigen.

A common idiotype was defined by a rabbit anti-idiotypic antiserum generated against human antibodies to hepatitis B surface antigen (anti-HBs). This idiotype was detected in anti-HBs from eight different individuals who had been previously infected with hepatitis B virus and is referred to as the CHBs idiotype. The CHBs idiotype was also identified in sera from rabbits, mice, guinea pigs, swine, goats and chimpanzees that had been immunized with hepatitis B surface antigen (HBsAg). Expression of the CHBs idiotype in sera from other species was associated with anti-HBs molecules. These results suggested that variable-region genes responsible for the CHBs idiotype have been conserved through long periods of evolution. It was noteworthy that the CHBs idiotype was not detected in the sera of a nonmammalian species, chickens, that had been successfully immunized with HBsAg.

Utilization of the biotin/avidin system to amplify the sensitivity of the enzyme-linked immunosorbent assay (ELISA).

The biotin/avidin system was incorporated into the enzyme-linked immunosorbent assay (ELISA) technique to increase the sensitivity of the standard ELISA for the detection of mouse antibody to hepatitis B surface antigen ((anti-HBs and HBsAg, respectively). Two biotin/avidin ELISA designs were studied. In both assays, 96-well polystyrene plates were coated with HBsAg, post-coated with 0.5% gelatin and incubated with dilutions of mouse anti-HBs. In the biotin/avidin (BA) ELISA, reagents were added to antibody reacted wells in the following sequence: biotinylated goat anti-mouse IgG (b-GAMG), avidin-alkaline phosphatase (Av-AP) and substrate. The order of reactants after mouse antibody in the biotin/avidin/biotin (BAB) ELISA was b-GAMG, avidin, biotinylated alkaline phosphatase (b-AP) and substrate. The sensitivities of BA ELISA, BAB ELISA and a standard ELISA using a glutaraldehyde conjugated goat anti-mouse enzyme were compared to AUSAB (a commercial radioimmunoassay) using a panel of 23 mouse anti-HBs sera. All 3 ELISAs were more sensitive than AUSAB; the standard ELISA, BAB ELISA and BA ELISA were respectively 50, 1173 and 4134 times more sensitive than AUSAB for detection of mouse anti-HBs activity.

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.

Comparative studies of the immunogenic activity of hepatitis B surface antigen (HBsAg) and HBsAg polypeptides.

Three hepatitis B surface antigen (HBsAg) preparations were compared: purified intact 22-nm HBsAg particles; HBsAg-derived, sodium dodecyl sulfate (SDS)-denatured P25 + GP30 polypeptide pool; and nondenatured P25 + GP30 micelles. The micelles had the same polypeptide composition as the P25 + GP30 pool. The immunogenicity in mice of each preparation, administered either in saline suspension or adsorbed to aluminum gel, was compared. The SDS-denatured polypeptides were less immunogenic than intact HBsAg particles, whereas the micelles were more immunogenic. High anti-HBs titers were observed in mice immunized with micelle preparations in either saline suspension or adsorbed to aluminum gel for as long as 200 days after a booster inoculation, administered 26 days after the primary dose.

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.

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.

Humoral and cellular immunity to hepatitis B virus-derived antigens: comparative activity of Freund complete adjuvant alum, and liposomes.

Complete Freud adjuvant, aluminum gel, and liposomes were compared for their ability to enhance the immunogenicity of an intact 22-nm HBsAg particle vaccine and an HBsAg-derived polypeptide vaccine in guinea pigs. Both humoral and cell-mediated immune responses were evaluated. The greatest immune response was obtained with complete Freund adjuvant, regardless of the antigen preparation. Aluminum gel appeared to be a better adjuvant for 22-nm HBsAg particles, but the liposomes rendered polypeptide preparations more immunogenic. The possibility that various proportions were entrapped in aqueous compartments instead of being inserted into the lipid bilayers of liposomes might account for this difference. The development of both humoral and cellular immunity was dependent upon the use of an adjuvant, because aqueous preparations had poor immunogenicity.

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.

[Prospects in the specific prevention of type B viral hepatitis]. / Perspective in profilaxia specifica a hepatitei virale de tip B

The present paper concerns the following more important problems: -- hepatitis virus type B: morphology, antigenic structure, virus -- host interaction; -- first attempts at vaccination in hepatitis B; -- vaccines prepared from purified viral subunits; -- study of the pathogenicity and population selected as candidates for testing the efficiency of the vaccine.