Differential antigen presentation of hepatitis B surface antigen on cell membranes of responder and nonresponder mice.

In several systems it has been shown, that non-responsiveness to an antigen in mice of a particular haplotype is due to the lack of binding of an immunogenic peptide to class II molecules. Such studies have been done using detergent solubilized, affinity purified class II molecules. It has been reported, that the presence of certain phospholipids around class II molecules dramatically alters the extent of peptide binding to these molecules. It thus appears that the milieu in which the class II molecules are inserted influences to a considerable extent the level of peptide binding. Hence it is likely that the kinetics of binding of immunogenic peptides to class II on the cell surface, may be different from that of molecules inserted in detergent micelles. We therefore decided to test this by studying the binding of radiolabeled peptides to class II molecules on cell membranes. We report here a rapid and sensitive assay for peptide binding to murine class II molecules on cell membranes. Further, we have used this assay to study the nature of the interaction of immunogenic peptides and class II molecules on cell membranes of mice who are responders and non-responders to Hepatitis B surface Antigen (HBsAg). Interestingly, we find that immunogenic peptides bind in good correlation with their MHC restriction. We also observed that the HBsAg derived peptide which is unable to elicit a T cell response in the non-responder but fails to make a stable class II-peptide complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunological evaluation of three generations of anti-idiotype vaccine: study of B and T cell responses following priming with anti-idiotype, anti-idiotype peptide and its MAP structure.

A 15mer peptide (2F10 peptide) is capable of mimicking the group specific "a" determinant of human hepatitis B surface antigen (HBsAg), both at the B and T cell level. This peptide represents a sequence on the heavy-chain hypervariable region of a monoclonal "internal image" anti-idiotype (anti-id) 2F10 that has partial sequence homology to the "a" determinant epitope of HBsAg. In order to potentiate the immunological properties of 2F10 peptide, a synthetic polymer of the 2F10 peptide was constructed (2F10 MAP). In this study we present the immunological evaluation of three generations of anti-idiotype vaccines, namely the 2F10 anti-id, 2F10 peptide and 2F10 MAP. Our results indicate that there is significant anti-HBs production in mice immunized with 2F10 anti-id or 2F10 MAP, in comparison to mice immunized with the linear monomeric 2F10 peptide. In priming experiments we found that only 2F10 antibody or 2F10 MAP (both at a suboptimal dose), could effectively prime B cells in vivo which could be efficiently recalled by challenge with a suboptimal dose of HBsAg. Collectively our findings indicate that 2F10 MAP retains all the immunological properties of the intact anti-id, and is qualitatively similar and quantitatively superior to the linear monomeric 15mer 2F10 peptide. The 2F10 MAP is the smallest MAP structure composed of a naturally occurring contiguous sequence having both a B and T cell epitope capable of eliciting a response to the native antigen.

Toleration of amino acid substitutions within hepatitis B virus envelope protein epitopes established by peptide replacement set analysis. II. Region S(122-136).

The envelope of the hepatitis B virus (HBV) consists of three related proteins, designated S-, M- and L-protein, all of which share a common 226-amino acid residue sequence, corresponding to the S-protein that is sufficient for eliciting protective immunity against HBV. HBV variants, resulting from point mutations leading to replacements of amino acids within the S(122-160) segment of S-protein, have been recently recognized. In order to assure the continued success of vaccination against HBV and the adequacy of diagnostic tests for HBV envelope antigens and antibodies, it is necessary to understand the impact of amino acid replacements on the immunological recognition of S-protein at both the B- and T-cell levels. Immunologically tolerated and forbidden amino acid replacements within the S(139-147) segment of S-protein have already been discerned. The impact of amino acid substitutions within the S(122-136) segment on the immunological recognition of S-protein is analyzed in this report. Such replacements do not appreciably affect the binding of rabbit and goat anti-S antibodies to replacement set peptides, while decreased murine antibody binding was observed with some peptides having substitutions at residues 122, 123 and 133. On the other hand, amino acid substitutions within the (126-136) region, except those distinguishing serological subtypes of HBV from each other, abrogated murine T-cell proliferative responses to the peptides, while substitutions at residues 122, 123 and 125 had a lesser effect. Some of the peptides with amino acid substitutions peculiar to the variants had diminished stimulatory activity for T-cells from individuals vaccinated against hepatitis B. Amino acid substitutions in both the S(139-147) and S(122-136) segments of S-protein may potentially result in variant viruses escaping immunological surveillance based on current hepatitis B vaccines.

Toleration of amino acid substitutions within hepatitis B virus envelope protein epitopes established by peptide replacement set analysis. I. Region S(139-147).

B and T cell epitopes expressed on the surface of S-protein, a major constituent of the envelope of hepatitis B virus (HBV), are essential for eliciting protective immunity against HBV infection. A segment of the S-protein sequence encompassing residues S(139-147) is a portion of overlapping B and T cell epitopes. This sequence is conserved among distinct serological subtypes of HBV and has a 77.8% homology with an analogous sequence in S-proteins of nonhuman mammalian hepadnaviruses. Rare subtypes and variants of HBV having amino acid replacements within the S(139-147) sequence were discerned recently. The impact of amino acid replacements within this sequence on its immunological recognition at both the B and T cell levels was explored by peptide replacement set analysis. Results of the analysis permit discrimination between tolerated and forbidden amino acid replacements and provide a background for the development of reagents and immunogens specific for emerging HBV variants.

Recognition of several idiotopes on a monoclonal anti-hepatitis B virus surface antigen using monoclonal anti-idiotypic antibodies.

A monoclonal murine antibody H3F5 directed to the a determinant of hepatitis B surface antigen (HBsAg) was used to raise several monoclonal anti-idiotypes. Cross-blocking experiments between the anti-idiotypes and the patterns of inhibition produced by a number of other monoclonal anti-HBsAg, generated in the same fusion as H3F5, identified three idiotopes on H3F5 which were shared to varying degrees with the other anti-HBsAg monoclonals. One behaved as a dominant cross-reacting idiotope (CRI) in that it appeared strongly in 5/7 monoclonal idiotypes. The CRI could represent an important target for regulation by anti-idiotope. Monoclonal antibodies have many advantages over polyclonal sera in the detailed analysis of idiotope structures.

A surrogate hepatitis B virus antigenic epitope represented by a synthetic peptide and an internal image antiidiotype antibody.

The use of molecules that represent single, defined epitopes able to substitute for antigen (i.e. surrogate antigens) offers considerable advantages over the use of native antigen for the precise manipulation of the immune response. We have investigated the immunochemical characteristics of two types of surrogate hepatitis B surface antigen (HBsAg) epitopes: (a) linear and cyclical synthetic peptides representing amino acid residues 139-147, a hydrophilic region corresponding to part of the a determinant of the HBsAg, and (b) four monoclonal antiidiotypes raised against anti-HBs mAb, two of which behave as an internal image of an a determinant. Polyclonal anti-HBs antisera bound the monoclonal antiidiotypes with affinities of the order of 10(8)/M, and to the peptides with greater than 10-fold lower affinities. However, the levels of antibody in the polyclonal antisera for the peptides was greater than for the antiidiotypes. In inhibition RIA, the surrogate antigens show concordance in that the internal image antiidiotypes inhibit the binding of both monoclonal and polyclonal anti-HBs to the linear and cyclical 139-147 peptides. These results imply that surrogate antigens could indeed be useful as potential hepatitis vaccines, but while the antiidiotypes may stimulate B cells of higher affinity, they would react with a more restricted range of B cell reactivities than would the peptides. A future HBV vaccine may thus comprise a synthetic peptide such as cyclical 139-147 or a cluster of monoclonal internal image antiidiotypes.

Immunofluorescent technique for the detection of monoclonal internal image anti-idiotypic antibodies of hepatitis B surface antigen.

Monoclonal anti-idiotypic antibodies to HBsAg were screened by immunofluorescence for the presence of a subset behaving as the internal image of the original antigen. We describe the technique and the criteria fulfilled to establish that 2/6 monoclonals studied act as the internal images of the a determinant of hepatitis B surface antigen.

Monoclonal 'internal image' anti-idiotypic antibodies of hepatitis B surface antigen.

The hypervariable regions of the immunoglobulin molecule which function as the antigen-combining site are, themselves, capable of provoking an antibody response. These antigenic determinants on the immunoglobulin are termed the 'idiotype', and antibodies directed against them 'anti-idiotype'. In circumstances where there is a close complementarity of shape between antigen and idiotype, and subsequently between idiotype and anti-idiotype, it would be predicted that anti-idiotype would be like an 'internal image' of the antigen. Starting with a monoclonal antibody (idiotype) to the protective a determinant of the hepatitis B surface antigen (HBsAg), we have succeeded in raising two monoclonal anti-idiotypes which mimic HBsAg in their ability to bind polyclonal antibodies to HBsAg produced in a variety of species. These internal image anti-idiotypes may provide a strategy for immunization without the need for antigen.

Anti-idiotypes as surrogate antigens: structural considerations.

The immune response generates antibodies with combining sites complementary to the antigen. These molecules carry antigenic determinants which together define the idioype (Id) of that antibody. An anti-Id response generates some antibodies which complements the antigen-binding surface of the first population of antibodies, and thus represent, in Jerne's words(1), an 'internal image' of the antigen. In this journal recently, Erlanger(2) discussed the difficulties in accepting this concept caused by the persistent notion of the antibody combining site as a groove or cleft. Here Ivan Roitt and his colleagues return to this discussion and define three distinct sets of structural circumstances in which an anti-Id may substitute for an antigen.

Affinity, cross-reactivity and biological effectiveness of rabbit antibodies against a synthetic 37 amino acid C-terminal peptide of human chorionic gonadotrophin.

As a part of a programme to develop a fertility regulating vaccine, antibodies specific for human chorionic gonadotrophin were raised by immunizing rabbits with a synthetic 37 amino acid C-terminal peptide of beta hCG conjugated to tetanus toxoid as carrier, and using Bordetella pertussis or Freund's complete adjuvant as adjuvants. Lack of cross-reactivity of the antibodies with human luteinizing hormone was determined by direct binding in a radioimmunoassay and by immunofluorescence on adult human pituitary sections. Not only did the antibodies bind to native hormone in a radioimmunoassay but they also neutralized the biological activity of hCG as measured by an in vivo bioassay. Rabbits which had been injected with the conjugate with B. pertussis as adjuvant made antibodies of comparable affinity to those animals which had been immunized with the antigen in Freund's complete adjuvant, though the latter group did produce more antibodies.

Characterisation of the immunological response in marmoset monkeys immunised against hCG beta-subunit and its relationship with their subsequent fertility.

Female marmoset monkeys that are actively immunised against hCG beta-subunit remain infertile while antibody titres are high. With declining antibody levels the animals experience recurrent abortions that occur progressively later as the levels continue to wane. After booster immunisations the animals become infertile once more. The affinity and total binding sites of antibodies to beta-hCG were monitored after booster injections in marmosets and the values were correlated with subsequent reproductive events. The relationship between antibody amount and affinity varied considerably and the affinity was the important factor in producing the biological effects, pregnancies being often associated with a fall in antibody affinity. The antisera were also biologically active in inhibiting hCG-induced ovulation and increase in uterine weight in mice. There was no apparent cross-reaction between the antisera and human luteinizing hormone as tested by indirect immunofluorescence on adult human pituitary sections.

Immunological control of fertility: measurement of affinity of antibodies to human chorionic gonadotrophin.

Four baboons were primed with diazotized beta human chorionic gonadotrophin and boosted with diazotized C-terminal beta human chorionic gonadotrophin peptide, and the changes in antibody amount and affinity determined using a double isotope modified Farr assay, using labelled human chorionic gonadotrophin as the antigen. The degree of cross-reaction with human luteinizing hormone was also determined. Although appreciable reactivity with luteinizing hormone was observed soon after immunization, this declined rapidly during the response. At the time intervals studied, there was a progressive increase in affinity of antibodies to human chorionic gonadotrophin until day 248 after priming. At day 313, in two of the animals, there was a decrease in affinity from 1.04 X 10(11) to 6.80 X 10(10) and 1.05 X 10(11) to 4.93 X 10(10) l/mol, whereas in the other two baboons there was a further increase in antibody affinity. At corresponding time intervals, there was a steady decrease in values of total antibody binding sites. To determine the overall effect of the maturation of affinity with a decrease in antibody amount on biological efficacy, the theoretical amount of chorionic gonadotrophin that would be neutralized was calculated. We computed that in all instances, over 99% of a peak concentration of chorionic gonadotrophin that could be in circulation in a pregnant baboon would be neutralized. This was in excellent agreement with results of mating experiments in these baboons. In over forty cycles studied, none of the matings resulted in a sustained pregnancy.

Modification of the Farr assay using ethanol-ammonium acetate precipitation and its application to the measurement of affinity of anti-HCG produced in several species.

A double isotope modified Farr assay was used to determine the total binding sites and affinity of antibodies to human chorionic gonadotrophin. Precipitation of the antigen--antibody complex at equilibrium with ammonium sulphate gave very high levels of nonspecific binding. Good discrimination over background was observed using a specific anti-immunoglobulin serum. However since we were interested in measuring the affinity of antibodies raised in several animal species it was more appropriate to use a single nonspecies precipitating reagent. We found that the use of a mixture of ethanol-ammonium acetate gave very low levels of non-specific binding in baboons, marmosets, rabbits and mice.