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.

Structural requirements for synthetic immunogens to induce measles virus specific CTL responses.

We have studied the immunogenicity of a synthetic peptide representing a cytotoxic T cell epitope (CTL) from the nucleoprotein of measles virus (MV). For the induction of peptide and MV-specific CTL responses after subcutaneous immunization, covalent linkage of the CTL epitope to a T-helper epitope was required. The presence of two copies of the T-helper epitope at the amino terminus of the CTL epitope (TT-CTL) resulted in the induction of strong CTL responses after administration in saline. In contrast, a chimeric peptide with one copy of the T-helper epitope at the amino terminus of the CTL epitope (T-CTL) was weakly immunogenic when given in saline. Analysis of the structure of the TT-CTL chimeric peptide by CD spectroscopy revealed an alpha-helical conformation, as compared to the random coil conformation favored by the T-CTL chimeric peptide. In addition, the CD spectra of the TT-CTL peptide in the presence of small unilamellar vesicules (SUV) revealed an increased helicity, as compared to the spectra of the T-CTL chimera in the presence of SUV. This suggests that the amphipathic character of the TT-CTL chimeric construct favors its interaction with the cell membrane of antigen presenting cells, therefore, facilitating its cytosolic delivery for class I presentation. These findings highlight the importance of antigen structure for the in vivo induction of CTL responses and may have implications for the design of synthetic peptide vaccines.

The effects of immunoglobulin isotype and antibody affinity on complement-mediated inhibition of immune precipitation and solubilization.

We have studied complement-mediated prevention of precipitation (PIP) and solubilization of immune complexes (IC) formed with DNP19-BSA and murine monoclonal antibodies (MCAs) of different isotypes and affinities. PIP was effective for IC formed with IgG1 and IgM antibodies, but not for IgA MCA. For IgG1 MCAs, affinity appeared to exert a minor effect on PIP, and IC formed in antibody excess or at equivalence were retained in solution more readily than those formed in antigen excess. For IgM MCAs affinity and antigen-antibody ratio did not affect PIP. As PIP did not occur in Mg-EGTA, it was concluded that PIP was entirely classical pathway dependent. Solubilization of IC containing IgG1 MCAs occurred more rapidly and to a greater extent with low affinity antibodies and an inverse relationship between affinity and the extent of solubilization was observed. Complexes formed with IgG1 MCAs were solubilized relatively poorly when formed in antigen excess. In contrast, affinity and antigen-antibody ratio did not influence the rate and extent of solubilization of IC containing IgM MCAs. IC formed with IgG2b were solubilized rapidly whereas those formed with IgG2a or IgA were solubilized poorly. The relative contributions of the classical and the alternative pathways to solubilization varied with each antibody and the effect of antigen-antibody ratio on these relative contributions was inconsistent.

The activation of C3 and C4 in human serum by immune complexes containing mouse monoclonal antibodies of different isotype and affinity: effects on solubilisation.

Radioimmunoassays for C3a and C4a have been used to measure the activation of complement in human serum by immune complexes containing DNP-BSA and each of 11 mouse anti-DNP monoclonal antibodies of varied isotype and affinity. When preformed complexes were added to serum, those containing IgG2 or IgM were potent activators of C4, whilst IgG1 complexes were less efficient. C3 activation in normal serum was similar for complexes containing IgG1, IgG2a, IgG2b or IgM. IgA complexes did not activate C3 or C4. Solubilisation of complexes was greatest for IgM and IgG2b and least for IgG2a and IgA. In serum containing Mg2+ EGTA C4 activation was abolished and the amount of C3 activation was lower for all IgG and IgM complexes. Antibodies of the same isotype did not necessarily activate complement to the same extent. Unexpectedly, three of the four IgMs activated C3 in EGTA. For IgMs, neither complement activation nor solubilisation correlated with affinity. For IgG1 antibodies, solubilisation was inversely proportional to affinity. C3 or C4 activation did not correlate with affinity.

Influence of the T-helper epitope on the titre and affinity of antibodies to B-cell epitopes after co-immunization.

We have assessed the influence of different T-helper cell epitopes on the level and affinity of antibody to B-cell epitopes induced following co-immunization with free peptides mimicking epitopes from measles and respiratory syncytial virus envelope proteins. The responses obtained following co-immunization have been compared to those obtained following immunization with chimeric synthetic peptide immunogens in which the epitopes were covalently coupled. The results show that covalent linkage of the B- and T-cell epitopes is not necessary for the generation of T-cell dependent antibody responses to non-immunogenic B-cell epitopes. In addition the induction of memory B-cells required adjuvant but subsequent stimulation of these memory cells did not. The responses obtained were non-MHC restricted since co-immunization resulted in the production of antibody responses to B-cell epitopes in a panel of five inbred mouse strains but there were differences in the ability of different T-cell epitopes to provide help for antibody production to the same B-cell epitope. The affinity of antibodies to the B-cell epitopes induced following immunization with chimeric T:B peptides was higher than that obtained following co-immunization. These results indicate the value of co-immunization for the induction of antibody responses to B-cell epitopes across MHC differences and suggest that this strategy may be of value in the development of synthetic peptide vaccines. However, modifications of the approach need to be developed to ensure the production of antibody of the highest possible affinity.

The effect of antibody isotype on the activation of C3 and C4 by immune complexes formed in the presence of serum: correlation with the prevention of immune precipitation.

Radioimmunoassays for C3a and C4a have been used to measure the activation of complement during the formation of immune complexes in human serum by the interaction of DNP-BSA and each of 11 mouse anti-DNP monoclonal antibodies of varied isotype and affinity. Those containing IgG2 or IgM were potent activators of C4, whilst IgG1 containing complexes were less efficient. C3 activation in normal serum was similar for complexes containing IgG1, IgG2a, IgG2b or IgM. IgA complexes did not activate C3 or C4. All complexes except those containing IgA precipitated more slowly in serum than in buffer. IgG2 antibodies were potent activators despite being very slow to precipitate in buffer. In serum containing EGTA activation of C4 was abolished and precipitation of complexes occurred at the same rate as in buffer. Nevertheless, C3 activation still occurred by the alternative pathway for all IgG and IgM complexes. Antibodies of the same isotype did not necessarily activate complement to the same extent. The ranking of the ability to activate complement was the same as that observed when performed complexes containing the same antibodies were added to serum. The levels of C4a generated were similar under both conditions but for most antibodies more C3a was generated by preformed complexes.

Dissociation of primary antigen-antibody bonds is essential for complement mediated solubilization of immune precipitates.

The role of dissociation of primary antigen-antibody bonds in the solubilization of immune complexes (IC) has been investigated using photo-affinity crosslinked IC comprising NAP15-BSA and murine monoclonal anti-DNP antibodies. Non-covalently linked IC were solubilized rapidly when incubated with normal human serum (NHS), whereas covalently-linked IC were solubilized poorly or not at all. The rate and extent of complement activation produced by incubating covalently-linked and non-covalently linked IC with NHS was similar as assessed by the production of the C1s:C1-inhibitor, C3:properdin and C5b-9 complexes and the anaphylatoxins C4a and C3a. Thus, the inability of serum to solubilize photo-affinity crosslinked IC must be due to failure of dissociation of primary antigen-antibody bonds.

Biodegradable microparticles as a delivery system for measles virus cytotoxic T cell epitopes.

Cytotoxic T-cell (CTL) responses are likely to be important for the clearance of a measles virus (MV) infection. To induce CTL responses. replicating vectors have generally been used but the use of such vectors in humans mav be problematic, and immunization with synthetic peptides may be more appropriate. We have investigated the potential of poly(lactide-co-glycolide)(PLG) microparticles as a delivery system for a CTL epitope representing residues 51-59 from MV nucleoprotein. After a single intraperitoneal injection in saline of the encapsulated epitope, CTL responses to the homologous peptide and MV were detected over a period of 4 months. Responses reached a maximum 30 days after priming and were maintained at high levels for 120 days. These responses were higher than those observed when the CTL epitope was administered in saline or as an emulsion in Incomplete Freund's Adjuvant. The pronounced immunostimulatory effect of microparticles, combined with their excellent tissue compatibility and biodegradability suggests that they represent a valuable delivery system for synthetic peptide immunogens.

Nasal delivery of epitope based vaccines.

Essentially all of the currently available vaccines are based on the use of inactivated or live-attenuated pathogens. However, these vaccines have several shortcomings, such as difficulties of in vitro culturing, biohazard risks, as well as loss of efficacy due to the genetic variations seen in many viruses. These problems may potentially be solved by immunising with epitope-based vaccines consisting of rationally designed protective epitopes, appropriately presented and easy to deliver, which are capable of stimulating effective B-cell, T-cell and cytotoxic immune responses whilst avoiding potentially hazardous and undesirable effects. Furthermore, the use of a mixture of defined epitopes could lead to an effective broad range immune response which has the potential to overcome both strain specificity of the pathogen and the MHC restriction of the host. Epitope-based vaccines can be designed to involve the use of synthetic materials that can be available in unlimited quantities and posing no biohazard. Other approaches include the use of naked DNA or recombinant viruses or bacteria expressing the epitopes. An important objective in the development of such vaccines is that they should be effective when delivered via the mucosal route and effective in the presence of maternal antibodies. In this review, we present examples of the use of various epitope-based vaccine constructs, focussing particularly upon their intranasal delivery to the immune system.

Engineering of a polymeric bacterial protein as a scaffold for the multiple display of peptides.

Protein assemblies with a high degree of repetitiveness and organization are known to induce strong immune responses. For that reason they have been postulated for the design of subunit vaccines by means of protein engineering. The enzyme lumazine synthase from Brucella spp. (BLS) is highly immunogenic, presumably owing to its homodecameric arrangement and remarkable thermodynamic stability. Structural analysis has shown that it is possible to insert foreign peptides at the ten amino terminus of BLS without disrupting its general folding. These peptides would be displayed to the immune system in a highly symmetric three-dimensional array. In the present work, BLS has been used as a protein carrier of foreign peptides. We have established a modular system to produce chimeric proteins decorated with ten copies of a desired peptide as long as 27 residues and have shown that their folding and stability is similar to that of the wild-type protein. The knowledge about the mechanisms of dissociation and unfolding of BLS allowed the engineering of polyvalent chimeras displaying different predefined peptides on the same molecular scaffold. Moreover, the reassembly of mixtures of chimeras at different steps of the unfolding process was used to control the stoichiometry and spatial arrangement for the simultaneous display of different peptides on BLS. This strategy would be useful for vaccine development and other biomedical applications.

Efficient binding to the MHC class I K(d) molecule of synthetic peptides in which the anchoring position 2 does not fit the consensus motif.

Peptides eluted from the MHC class I K(d) molecule are generally nonamers that display a strong preference for Tyr in position 2 and Ile or Leu in position 9. We investigated the binding ability of several synthetic peptides which did not fit this consensus motif. In our peptides, Tyr(2) was substituted by other amino acids, i.e. LeU, Ile or Met. These peptides were variants of the 252-260 K(d)-restricted peptide SYIPSAEKI derived from the Plasmodium berghei circumsporozoite protein. They bound to purified K(d) molecules in vitro with intermediate affinity. One of them was tested for in vivo stimulation of T cells and induced a cytotoxic response. These results demonstrate the importance of binding motif refinement to discover new binding characteristics and new ligands such as low-affinity peptides.

The influence of epitope density on the estimation of the affinity of antibody for complex antigens.

The influence of the epitope density of the antigen on antibody affinity values determined by fluid- and solid-phase immunoassays was assessed. The affinity of the interaction of a panel of monoclonal anti-DNP antibodies of different affinities (as determined by equilibrium dialysis) for DNP-protein conjugates of various hapten substitution ratios was used as the test system. The results obtained showed that the epitope density of the antigen markedly influences the observed affinity values obtained by both experimental approaches. However, the monoclonal antibodies were ranked in affinity terms by both assays in a similar order to that given by equilibrium dialysis. It is concluded that provided due care is exercised in choosing an appropriate epitope density for the test antigen, these methods can be used to provide rapid estimations of average antibody affinities.

Comparison of three methods used for the measurement of the avidity of antibody to DNA.

Three methods for determining the avidity of antibodies to single-stranded DNA (ssDNA) have been compared. None of the 3 methods was totally satisfactory but for theoretical and practical reasons the measurement of avidity by determination of dissociation rate is considered the most appropriate.

Measurement of antigen-antibody complexes in mouse sera by conglutinin, C1q and rheumatoid factor solid phase binding assays.

The application of three solid phase tests for the detection of antigen-antibody complexes in the sera of inbred and selectively-bred mice with experimentally induced chronic antigen-antibody complex disease is described. Two of the tests used--the C1q binding assay (C1qBA) and the conglutinin binding assay (KBA)--were previously described methods which we have modified for the detection of murine complexes. The third test is a new solid phase polyclonal rheumatoid factor binding assay (RFBA). The results demonstrate that the correlation of positivity with the three methods varied during the course of a chronic disease but in general results with the KBA showed a better correlation with the RFBA than with the C1qBA. Furthermore, the KBA and RFBA preferentially bound complexes of lower molecular weight than did the C1qBA. It is suggested that the parallel use of these three tests during the study of murine antigen-antibody complex disease will provide valuable information concerning the nature of the complexes involved; since the three tests, when run in parallel, will detect large complement fixing complexes (C1q), small complement fixing complexes (KBA) and small non-complement fixing complexes (RFBA).

An inhibition enzyme immunoassay for estimating relative antibody affinity and affinity heterogeneity.

A method to measure the relative affinity of antibodies using an inhibition enzyme immunoassay is described. It is validated using monoclonal antibodies of defined affinity characteristics and by comparison with conventional methods of affinity measurement. The method allows measurement of the relative affinity of low levels of antibody, and the calculation of an empirical estimate of the heterogeneity of affinity in antibody populations.

Production of anti-peptide specific antibody in mice following immunization with peptides conjugated to mannan.

In order to investigate the usefulness of polysaccharides as carriers for the induction of antibody to synthetic peptides, peptides representing residues 139-147 of the surface antigen of hepatitis B and residues 129-140 of the pre-S2 region of the protein were coupled to mannan and dextran via an aminocaproic spacer molecule. Of the two conjugates studied, only mannan was useful as a carrier for the efficient production of anti-peptide antibodies.

The measurement of antibody affinity: a comparison of five techniques utilizing a panel of monoclonal anti-DNP antibodies and the effect of high affinity antibody on the measurement of low affinity antibody.

The affinities of 9 IgG1 monoclonal anti-dinitrophenol (DNP) antibodies for 3H-epsilon-DNP-L-lysine, 125I-HOP-DNP-L-lysine and 125I-DNP-human serum albumin (HSA) were determined. 3H-DNP-lysine was used in equilibrium dialysis and ammonium sulphate globulin precipitation assays; 125I-HOP-DNP-lysine was used in equilibrium dialysis and polyethylene glycol precipitation; and 125I-DNP5-HSA in the polyethylene glycol precipitation assay for affinity. The ranking order of the monoclonal antibodies in terms of affinity by the assays was significantly correlated. Of particular importance was the observation that the simple and widely applicable globulin precipitation assay utilizing a protein antigen produced affinity values which showed concordance with the least equivocal but cumbersome assay, equilibrium dialysis. Mixing of antibodies of high and low affinity demonstrated that even a low proportion of high affinity antibody had marked effect on measurements of the amount and affinity of a predominantly low affinity antibody preparation.

HBsAg: anti-HBs immune complexes. A method for separating the constituent components and assessment of the affinity of the antibody.

A number of chemical disruption agents were assessed for their ability to dissociate HBsAg:anti-HBs immune complexes and to release both the antibody and antigen component in immunologically active forms. The most appropriate reagent was 0.1 M diethylamine which could elute up to 81% of anti-HBs antibody bound to solid-phase HBsAg and retained 93% of its antigen-combining activity. Complexes formed at various degrees of antigen excess and pre-exposed to 0.1 M diethylamine at room temperature for 18 h before ultracentrifugation on sucrose density gradients were effectively dissociated. The released antibody and antigen banded at their expected densities. However, the affinity of the isolated antibody for the detergent-solubilized polypeptide complex from purified HBsAg (gp30/p25) and cyclical peptides representing amino acids 124-137 and 139-147 of HBsAg were found to be considerably lower than that of the original pooled anti-HBs immunoglobulin used to form the immune complexes. These results suggest that the highest affinity antibody subpopulation may not be completely dissociated from the complex. Care should thus be exercised in the interpretation of the significance of the observed affinity of the antibody isolated by this and other similar dissociation procedures.

Mucosal immunization with a measles virus CTL epitope encapsulated in biodegradable PLG microparticles.

The immunogenicity of a cytotoxic T cell epitope (CTL) representing residues 52-60 from measles virus (MV) nucleoprotein, encapsulated in poly(lactide-co-glycolide) (PLG) microparticles was evaluated after mucosal immunization. After intranasal administration of the encapsulated CTL epitope linked at the carboxyl terminus of two copies of a T-helper epitope (TT-NP6), peptide-specific and MV-specific CTL responses were detected in splenocytes. However, these responses were lower than the responses observed when the TT-NP6 peptide was administered intranasally in saline or using CTB as an adjuvant. Intranasal coadministration of the encapsulated TT-NP6 peptide with CTB did not result in any significant potentiation of the CTL responses. The effectiveness of biodegradable PLG microparticles for mucosal delivery of CTL epitopes, combined with their excellent tissue compatibility and biodegradability suggests that they represent a valuable delivery system for synthetic immunogens. However, further work is needed to define the requirements for effective absorption by the nasal epithelium.

CTL responses induced by a single immunization with peptide encapsulated in biodegradable microparticles.

A synthetic peptide representing a measles virus (MV) cytotoxic T cell epitope (CTL) when encapsulated in poly (D,L-lactide co-glycolide) (PLG) 50:50 microparticles induced a strong CTL response after a single intraperitoneal immunization of mice which was greater than that following administration of the peptide in Freund's complete adjuvant. A 100 micrograms dose of encapsulated peptide was shown to be more effective for CTL priming than 50 and 25 micrograms doses. A vaccine formulation prepared by simply mixing empty 50:50 PLG microparticles with the peptide resulted in the induction of CTL responses comparable to those induced by the encapsulated peptide. Moreover, a CTL response against MV-infected target cells was observed. These findings highlight the potential immunostimulatory effect of PLG microparticles for the induction of MV and peptide-specific CTL responses.