Short Papers Macromolecular self-association of a synthetic peptide derived from the hepatitis B surface antigen: construction of a quaternary epitope Kanury V.S. Rao *++, Subrat K. Panda* and Venkatasamy Manivel* A major impediment to the development ofpeptide vaccines has been the inability accurately to mimic conformationally constrained epitopes on the envelope proteins of pathogens 1-3. Th& limitation &further compounded by the fact that several viral envelope proteins ex&t either as covalently or non-covalently associated homo-oligomers in the native state 4. Evidence & now accumulatin 9 to indicate that, at least in some instances, these homo-oligomers display antigenic determinants that are not present in the dissoc&ted monomer units 4. Clearly th& problem will have to be addressed if peptide-based vaccines are ever to become feasible alternatives. In th& report we demonstrate that an oligomer&ed synthetic peptide that was derived from the hepatitis B surface antigen a99regates in solution to form macromolecular structures. These aggregates appear to represent a 'native 'form of the group-specific determinant presented by the hepatitis B surface antigen. Keywords:Syntheticpeptide; self-association; hepatitis B surfaceantigen; quaternaryepitope
The major or S protein of the hepatitis B surface antigen (HBsAg) is extensively crosslinked via both intra- and inter-molecular disulphide bridging resulting in immunodominant epitopes that are predominantly conformational s-9. While the synthetic peptide methodology has been used in an attempt to delineate some of the determinants resident in the monomer sequence of the S proteinlO 15, the question of whether additional epitopes are encoded by the aggregate or oligomeric form of this protein has not been addressed. We recently showed that a synthetic peptide derived from residues 124 to 147 ofHBsAg (peptide OS[124-147]) spontaneously self-assemblesthrough disulphide-dependent oligomerization to reconstruct a conformational groupspecific determinant present on the native protein (unpublished results). Comparative analyses with partially and fully reduced analogues indicated that expression of the HBsAg-related epitope by peptide OS[124-147] was stringently dependent upon the integrity of the oligomer. We were interested in further refining the presentation of this epitope so as to simulate presentation in the native form as closely as possible. The latter may be visualized as resulting from clusters of the S protein aggregates distributed across the viral envelope. Although methodologies are currently available *Virology Group, International Centre for Genetic Engineering and Biotechnology, Nil Campus, Shaheed Jeet Singh Marg, New Delhi-110 067, India. tDepartment of Pathology, All India Institute of Medical Sciences, New Delhi-110 029, India. tTo whom correspondence should be addressed. (Received 25 July 1991 ; accepted 25 September 1991) 0264-410x/92/040204-05 © 1992 Butterworth-Heinemann Ltd 204 Vaccine, Vol. 10, Issue 4, 1992
to achieve macromolecular clustering of immunogensl 6-i 8, we first decided to investigate whether peptide OS[124-147] per se has any intrinsic tendencies for self-association. The results of an experiment where this peptide was centrifuged through a 10-30% sucrose gradient, with a pre-S1 derived linear synthetic peptide run in parallel as a control, are shown in Figure I. A comparison of the peptide distribution among the various fractions of the gradient gave essentially similar profiles for both peptides except that there appeared to be some additional accumulation of peptide OS[124-147] in the fraction corresponding to 16% sucrose (Figure lc and d). However, when we assayed for antigenic activity using polyclonal anti-OS [ 124-147] and anti-HBsAg antisera we observed that fractions corresponding to higher sucrose content displayed significant reactivity. For the control peptide, antigenicity and peptide distribution profiles were nearly superimposable. While there was no significant amount of ninhydrin-detectable peptide OS [ 124-147] present in fractions containing 25-30% sucrose, nevertheless there was strong recognition by both anti-peptide and anti-HBsAg antisera. It must be noted here that the sensitivity of our assay for detecting peptide was of the order of 2 micrograms. Thus comparing the relative distribution of peptide OS[124 147] across the gradient with respect to the distribution of antigenic activity we infer that the minor component of peptide that sediments between 25-30% sucrose represents the most native constituent of the HBsAgrelated antigenicity of peptide OS [ 124-147]. The results depicted in Figure I indicate that, in
Short paper: K.V.S. Rao et al.
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The major or S protein of the hepatitis B surface antigen (HBsAg) is extensively crosslinked via both intra- and inter-molecular disulphide bridging resulting in immunodominant epitopes that are predominantly conformational s-9. While the synthetic peptide methodology has been used in an attempt to delineate some of the determinants resident in the monomer sequence of the S proteinlO 15, the question of whether additional epitopes are encoded by the aggregate or oligomeric form of this protein has not been addressed. We recently showed that a synthetic peptide derived from residues 124 to 147 ofHBsAg (peptide OS[124-147]) spontaneously self-assemblesthrough disulphide-dependent oligomerization to reconstruct a conformational groupspecific determinant present on the native protein (unpublished results). Comparative analyses with partially and fully reduced analogues indicated that expression of the HBsAg-related epitope by peptide OS[124-147] was stringently dependent upon the integrity of the oligomer. We were interested in further refining the presentation of this epitope so as to simulate presentation in the native form as closely as possible. The latter may be visualized as resulting from clusters of the S protein aggregates distributed across the viral envelope. Although methodologies are currently available *Virology Group, International Centre for Genetic Engineering and Biotechnology, Nil Campus, Shaheed Jeet Singh Marg, New Delhi-110 067, India. tDepartment of Pathology, All India Institute of Medical Sciences, New Delhi-110 029, India. tTo whom correspondence should be addressed. (Received 25 July 1991 ; accepted 25 September 1991) 0264-410x/92/040204-05 © 1992 Butterworth-Heinemann Ltd 204 Vaccine, Vol. 10, Issue 4, 1992
to achieve macromolecular clustering of immunogensl 6-i 8, we first decided to investigate whether peptide OS[124-147] per se has any intrinsic tendencies for self-association. The results of an experiment where this peptide was centrifuged through a 10-30% sucrose gradient, with a pre-S1 derived linear synthetic peptide run in parallel as a control, are shown in Figure I. A comparison of the peptide distribution among the various fractions of the gradient gave essentially similar profiles for both peptides except that there appeared to be some additional accumulation of peptide OS[124-147] in the fraction corresponding to 16% sucrose (Figure lc and d). However, when we assayed for antigenic activity using polyclonal anti-OS [ 124-147] and anti-HBsAg antisera we observed that fractions corresponding to higher sucrose content displayed significant reactivity. For the control peptide, antigenicity and peptide distribution profiles were nearly superimposable. While there was no significant amount of ninhydrin-detectable peptide OS [ 124-147] present in fractions containing 25-30% sucrose, nevertheless there was strong recognition by both anti-peptide and anti-HBsAg antisera. It must be noted here that the sensitivity of our assay for detecting peptide was of the order of 2 micrograms. Thus comparing the relative distribution of peptide OS[124 147] across the gradient with respect to the distribution of antigenic activity we infer that the minor component of peptide that sediments between 25-30% sucrose represents the most native constituent of the HBsAgrelated antigenicity of peptide OS [ 124-147]. The results depicted in Figure I indicate that, in
Short paper: K.V.S. Rao et al.
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