Interleukin-10-deficient mice develop chronic enterocolitis

Analysis of the cell culture fluid from two new human hepatoma-derived cell lines reveals that 17 of the major human plasma proteins are synthesized and secreted by these cells. One of these cell lines, Hep 3B, also produces the two major polypeptides of the hepatitis B virus surface antigen. When Hep 3B in injected into athymic mice, metastatic hepatocellular carcinomas appear. These cell lines provide experimental models for investigation of plasma protein biosynthesis and the relation of the hepatitis B viru genome to tumorigenicity.

https://science.sciencemag.org/content/sci/209/4455/497.full.pdf

Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen.

A procedure is described for rapid concurrent synthesis on solid supports of hundreds of peptides, of sufficient purity to react in an enzyme-linked immunosorbent assay. Interaction of synthesized peptides with antibodies is then easily detected without removing them from the support. In this manner an immunogenic epitope of the immunologically important coat protein of foot-and-mouth disease virus (type O1) is located with a resolution of seven amino acids, corresponding to amino acids 146-152 of that protein. Then, a complete replacement set of peptides in which all 20 amino acids were substituted in turn at every position within the epitope was synthesized, and the particular amino acids conferring specificity for the reaction with antibody were determined. It was found that the leucine residues at positions 148 and 151 were essential for reaction with antisera raised against intact virus. A lesser contribution was derived from the glutamine and alanine residues at positions 149 and 152, respectively. Aside from the practical significance for locating and examining epitopes at high resolution, these findings may lead to better understanding of the basis of antigen-antibody interaction and antibody specificity.

https://www.pnas.org/content/pnas/81/13/3998.full.pdf

Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid.

Publisher Summary Turns are a fundamental class of polypeptide structure and are defined as sites where the peptide chain reverses its overall direction. In the past 20 years, the peptide field has witnessed major development, stimulated by the discovery of a host of bioactive peptides. Turn structures have been proposed and implicated in the bioactivity of several of these naturally occurring peptides. In addition, many structural details of turns have been derived from conformational studies of model peptides. During this same period, more than 100 complete protein structures have been elucidated in single-crystal X-ray studies. These examples document the rich diversity of structural patterns in the chain folds of native proteins. Turns are intrinsically polar structures with backbone groups that pack together closely and side chains that project outward. Such an array of atoms may constitute a site for molecular recognition, and indeed, the literature abounds with suggestions that turns serve as loci for receptor binding, antibody recognition, and post-translational modification. In peptides, turns are the conformations of choice for simultaneously optimizing both backbone–chain compactness (intramolecular nonbonded contacts) and side-chain clustering (to facilitate intermolecular recognition). Presence of turns in bioactive conformations may in fact also reflect the lack of alternative conformational possibilities. The aim of this chapter is to examine structural and functional roles of turns in peptides and proteins.

Turns in peptides and proteins.

DNA recombinant technology has radically changed hepatitis B virus (HBV) virology. The genetic organization, transcription and replication of the virus are basically understood, structures of integrated HBV sequences in hepatocellular carcinoma have been characterized, and new vaccines produced by recombinant DNA technique are being developed.

The hepatitis B virus

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

A formalin-inactivated hepatitis B virus (HBV) vaccine has been produced in several laboratories (for review, see ref. 1). The sole source of material for these vaccines has been 22-nm lipoprotein particles composed of hepatitis B surface antigen (HBsAg) and derived from plasma of persons chronically infected with HBV. Such a source presents potential hazards in view of unknown factors that may be present in the plasma, and as high-risk populations are immunized, sources of plasma containing large quantities of HBsAg will become scarce. In addition, no tissue culture system has been developed for the propagation of HBV. The possibility of a synthetic peptide vaccine2,3 for HBV had been suggested2,3 following studies carried out with tobacco mosaic virus4,5 and MS-2 coliphage6. This possibility recently became a reality when the amino acid sequence for HBsAg was deduced from the nucleotide sequence of the cloned HBV genome (for a review, see ref. 7). More recently a portion of the major polypeptide derived from HBsAg, with a calculated molecular weight of 25,000 (P25), has been sequenced8. Lerner et al.9 have described a similar approach towards HBV synthetic peptides. Antibody to HBsAg (anti-HBs) was raised in rabbits inoculated with three or four doses of a series of peptides, each containing 14–15 amino acid residues, but only after covalent linkage of the peptides to a carrier protein. Activity was also found after multiple injections of a peptide containing 34 amino acids. We have now synthesized two cyclic peptides containing disulphide bonds, both unrelated to those prepared by Lerner, from a hydrophilic region of the major viral polypeptide, which elicited an antibody response in mice after a single injection without linkage to a protein carrier.

Antibody to hepatitis B surface antigen after a single inoculation of uncoupled synthetic HBsAg peptides

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.

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

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.

Epitopes associated with a synthetic hepatitis B surface antigen peptide.

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.

Irina Ionescu-Matiu

Papers

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

Antibody to hepatitis B surface antigen after a single inoculation of uncoupled synthetic HBsAg peptides

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

Epitopes associated with a synthetic hepatitis B surface antigen peptide.

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