How antigenic are antigenic peptides?

Most of a protein surface is potentially antigenic, consisting of numerous overlapping domains each complementary to antibody-combining sites. These domains may include peptide sequences that are demonstrably antigenic but only when antibodies from the appropriate host individuals and species are used. Methods for locating antigenic peptide sequences are described in which hydrophilic polyamide supports are used for peptide synthesis, then solid-phase radioimmunoassay with antisera and protein A. Most antigenic domains, however, comprise amino acid side chains contributed by two or more nearby polypeptide chains. Such domains can be identified by comparing the cross-reactivities of groups of very closely related proteins towards monoclonal antibodies raised to one of them. Such studies, using myoglobins, have identified a number of residues not previously shown to be antigenic and have provided a guide for the choice of synthetic peptides which are likely to carry several immunodominant side chains. One such peptide corresponding to residues (72–89) of beef myoglobin has been shown, using CD and antibodies to the parent protein, to have interesting conformational and antigenic properties. The peptide (25–55) is also antigenic.     

Mapping of linear B-cell epitopes of hepatitis B surface antigen

Eight monoclonal antibodies directed against the surface protein of hepatitis B virus (HBV) were tested using an epitope-mapping system (Pepscan) for characterizing antigenic domains. Four different amino acid sequences corresponding to linear epitopes were identified: one in pre-S1 corresponding to the sequence 29–36, two in pre-S2 corresponding to overlapping sequences 134–141 and 137–144, and one in the S region of the protein corresponding to the amino acid sequence 117–126.     

The antibody response to myoglobin is independent of the immunized species. Analysis in terms of replacements in the antigenic sites and in environmental residues of the cross-reactions of fifteen myoglobins with sperm-whale myoglobin antisera raised in different species.

The recent determination of the entire antigenic structure of sperm-whale myoglobin with rabbit and goat antisera has permitted the examination of whether the antigenic structure recognized by antibodies depends on the species in which the antisera are raised. Also, by knowledge of the antigenic structure, the molecular factors that determine and influence antigenicity can be better understood in terms of the effects of amino acid substitutions occurring in the antigenic sites and in the environmental residues of the sites. In the present work, the myoglobins from finback whale, killer whale, horse, chimpanzee, sheep, goat, bovine, echidna, viscacha, rabbit, dog, cape fox, mouse and chicken were examined for their ability to cross-react with antisera to sperm-whale myoglobin. By immunoadsorbent titration studies with radioiodinated antibodies, each of these myoglobins was able to bind antibodies to sperm-whale myoglobin raised in goat, rabbit, chicken, cat, pig and outbred mouse. It was found that the extent of cross-reaction of a given myoglobin was not dependent on the species in which the antisera were raised. This indicated that the antibody response to sperm-whale myoglobin (i.e. its antigenic structure) is independent of the species in which the antisera are raised and is not directed to regions of sequence differences between the injected myoglobin and the myoglobin of the immunized host. Indeed, in each antiserum from a given species examined, that antiserum reacted with the myoglobin of that species. The extent of this auto-reactivity for a given myoglobin was comparable with the general extent of cross-reactivity shown by that myoglobin with antisera raised in other species. The cross-reactivities and auto-reactivities (both of which are of similar extents for a given myoglobin) can be reasonably rationalized in terms of the effects of amino acid substitutions within the antigenic sites and within the residues close to these sites. These findings confirm that the antigenicity of the sites is inherent in their three-dimensional locations.     

Molecular cloning of bovine and chick nerve growth factor (NGF): delineation of conserved and unconserved domains and their relationship to the biological activity and antigenicity of NGF.

Previous experiments with purified mouse and bovine nerve growth factor (NGF) have shown that the biological activities of these two NGFs are identical, whereas the immunological cross-reactivity of antibodies produced against the two NGF molecules is very limited. This observation, together with the fact that antibodies to mouse NGF do not affect the development of sympathetic and sensory neurons in chick embryos, suggests that the domain of the NGF molecules responsible for the biological action has been highly conserved during evolution, whereas other domains determining the immunological properties were under less rigorous evolutionary constraint. The nucleotide sequences of bovine and chick NGF were determined from a cDNA clone prepared from mRNA of bovine seminal vesicles and from cloned chick genomic DNA, and the amino acid sequences deduced therefrom were compared with the available sequences of mouse and human NGF. All six cysteine residues were conserved in agreement with the previous finding that the biological activity of NGF is conformation-dependent requiring intact disulfide bridges. Amino acid changes are mainly confined to hydrophilic regions expected to be potential antigenic determinants, thus providing an explanation for the poor immunological cross-reactivities between the different NGFs. One single hydrophilic region is conserved in all NGFs and this region could be involved in the biological activity. The carboxy termini of bovine and chick NGF differ from that of mouse NGF, the changes in the amino acid sequences suggest that chick and bovine NGF are probably not processed by the gamma-subunit and that no 7S complex can be formed as in the mouse submandibular gland.     

Localization of antibody-binding sites by sequence analysis of cloned pilin genes from Neisseria gonorrhoeae

Immunological analysis of gonococcal pilin (the protein structural subunit of pili) has demonstrated the existence of cross-reacting and type-specific epitopes. The role in adhesion of the domains represented by these epitopes remains unclear. DNA sequencing of a series of pilin-expressing (pilE) genes from a number of otherwise isogenic pilus antigenic variants combined with previous immunological analysis of the corresponding encoded pilins has allowed us to correlate certain predicted amino acid sequences with monoclonal antibody reactivities. The putative epitopes for type-specific antibodies lie predominantly in hydrophilic domains that also contain beta turns. The epitopes for type-specific monoclonal antibodies were shown to depend on amino acid changes either in three separated blocks of amino acid sequence in the semi-variable (SV) region of pilin, or in discrete regions that lie in the disulphide loop in the hypervariable (HV) region of the polypeptide. In contrast, antibody SM1, which reacts with all gonococcal pili, recognizes a poorly immunogenic region of moderate hydrophilicity but low turn potential lying in a conserved portion of the pilin molecule. Our results confirm that antibodies directed against epitopes in both the SV and HV regions are able to inhibit adhesion.     

Effects of amino acid substitutions outside an antigenic site on protein binding to monoclonal antibodies of predetermined specificity obtained by peptide immunization: Demonstration with region 94–100 (antigenic site 3) of myoglobin

Amino acid substitutions outside protein antigenic sites are very frequently assumed to exert no effect on binding to antiprotein antibodies, especially if these are monoclonal antibodies (mAbs). In fact, a very popular method for localization of residues in protein antigenic sites is based on the interpretation that whenever a replacement causes a change in binding to antibody, then that residue will be located in the antigenic site. To test this assumption, mAbs of predetermined specificity were prepared by immunization with a free (i.e., without coupling to any carrier) synthetic peptide representing region 94–100 of sperm whale myoglobin (Mb). The cross-reactivities and relative affinities of three mAbs with eight Mb variants were studied. Five Mb variants which had no substitutions within the boundaries of the designed antigenic site exhibited remarkable, and in two cases almost complete, loss in cross-reactivity relative to the reference antigen, sperm whale Mb. Two myoglobins, each of which had one substitution within region 94–100, showed little or no reactivity with the three mAbs. It is concluded that substitutions outside an antigenic site can exert drastic effects on the reactivity of a protein with mAbs against the site and that caution should be exercised in interpreting cross-reactivity data of proteins to implicate residues directly in an antigenic site.     

Effects of amino acid substitutions outside an antigenic site on protein binding to monoclonal antibodies of predetermined specificity obtained by peptide immunization: demonstration with region 94-100 (antigenic site 3) of myoglobin.

Amino acid substitutions outside protein antigenic sites are very frequently assumed to exert no effect on binding to antiprotein antibodies, especially if these are monoclonal antibodies (mAbs). In fact, a very popular method for localization of residues in protein antigenic sites is based on the interpretation that whenever a replacement causes a change in binding to antibody, then that residue will be located in the antigenic site. To test this assumption, mAbs of predetermined specificity were prepared by immunization with a free (i.e., without coupling to any carrier) synthetic peptide representing region 94–100 of sperm whale myoglobin (Mb). The cross-reactivities and relative affinities of three mAbs with eight Mb variants were studied. Five Mb variants which had no substitutions within the boundaries of the designed antigenic site exhibited remarkable, and in two cases almost complete, loss in cross-reactivity relative to the reference antigen, sperm whale Mb. Two myoglobins, each of which had one substitution within region 94–100, showed little or no reactivity with the three mAbs. It is concluded that substitutions outside an antigenic site can exert drastic effects on the reactivity of a protein with mAbs against the site and that caution should be exercised in interpreting cross-reactivity data of proteins to implicate residues directly in an antigenic site.     

Identification of low density lipoprotein receptor binding domains of human apolipoprotein B-100: a proposed consensus LDL receptor binding sequence of apoB-100

The human liver apoB-100 gene cloned in the lambda gt-11 expression vector expresses fusion proteins reacting with apoB antibodies. A fusion protein induced from a apoB-lambda gt-11 clone reacted with apoB-100 monoclonal antibodies known to block the binding of LDL to the LDL receptor. The fusion protein contains an amino acid sequence domain enriched in positively charged residues which is complementary to the negatively charged amino acids present in the consensus LDL receptor binding domain. This sequence of apoB-100 is proposed as a binding domain for the interaction with the LDL receptor. Comparison of derived amino acid sequences from the entire structure of apoB-100 molecule revealed several similar domains enriched in positively charged amino acids. A consensus sequence of the potential LDL binding domain was identified which contained positively charged amino acids at positions 1, 5 and 8 and a loop of 8-11 amino acids followed by two adjacent positively charged amino acids. These results are interpreted as indicating that there are several potential LDL receptor binding domains in apoB-100.     

Antigenic structure of the flavivirus envelope protein E at the molecular level, using tick-borne encephalitis virus as a model.

A model of the tick-borne encephalitis virus envelope protein E is presented that contains information on the structural organization of this flavivirus protein and correlates epitopes and antigenic domains to defined sequence elements. It thus reveals details of the structural and functional characteristics of the corresponding protein domains. The localization of three antigenic domains (composed of 16 distinct epitopes) within the primary structure was performed by (i) amino-terminal sequencing of three immunoreactive fragments of protein E and (ii) sequencing the protein E-coding regions of seven antigenic variants of tick-borne encephalitis virus that had been selected in the presence of neutralizing monoclonal antibodies directed against the E protein. Further information about variable and conserved regions was obtained by a comparative computer analysis of flavivirus E protein amino acid sequences. The search for potential T-cell determinants revealed at least one sequence compatible with an amphipathic alpha-helix which is conserved in all flaviviruses sequenced so far. By combining these data with those on the location of disulfide bridges (T. Nowak and G. Wengler, Virology 156:127-137, 1987) and the structural characteristics of epitopes, such as dependency on conformation or on intact disulfide bridges or both, a model was established that goes beyond the location of epitopes in the primary sequence and reveals features of the folding of the polypeptide chain, including the generation of discontinuous protein domains.     

Mapping surface structures of the human insulin receptor with monoclonal antibodies: localization of main immunogenic regions to the receptor kinase domain

A panel of 37 monoclonal antibodies to the human insulin receptor has been used to characterize the receptor's major antigenic regions and their relationship to receptor functions. Three antibodies recognized extracellular surface structures, including the insulin binding site and a region not associated with insulin binding. The remaining 34 monoclonal antibodies were directed against the cytoplasmic domain of the receptor beta subunit. Competitive binding studies demonstrated that four antigenic regions (beta 1, beta 2, beta 3, and beta 4) are found on this domain. Sixteen of the antibodies were found to be directed against beta 1, nine against beta 2, seven against beta 3, and two against beta 4. Antibodies to all four regions inhibited the receptor-associated protein kinase activity to some extent, although antibodies directed against the beta 2 region completely inhibited the kinase activity of the receptor both in the autophosphorylation reaction and in the phosphorylation of an exogenous substrate, histone. Antibodies to the beta 2 region also did not recognize autophosphorylated receptor. In addition, antibodies to this same region recognized the receptor for insulin-like growth factor I (IGF-I) as well as the insulin receptor. In contrast, antibodies to other cytoplasmic regions did not recognize the IGF-I receptor as well as the insulin receptor. These results indicate that the major immunogenic regions of the insulin receptor are located on the cytoplasmic domain of the receptor beta subunit and are associated with the tyrosine-specific kinase activity of the receptor. In addition, these results suggest that a portion of the insulin receptor is highly homologous to that of the IGF-I receptor.     

Effects of amino acid substitutions outside an antigenic site on protein binding to monoclonal antibodies of predetermined specificity obtained by peptide immunization: Demonstration with region 113–120 (antigenic site 4) of myoglobin

Immunochemical cross-reactivity of protein variants has been very frequently used to map protein antigenic sites. The approach is based on the assumption that amino acid substitutions affecting the binding of a protein to its antibody, particularly when monoclonal antibodies (mAbs) are used, must be part of the antigenic site and not far from it. The assumption was investigated in this study by determining the effects of amino acid substitutions outside the antigenic site on the reactivity of six myglobin (Mb) variants with three mAbs of predetermined specificity prepared by immunization with a free synthetic peptide representing region 113–120 (antigenic site 4) of Mb. Two of the Mb variants used had no substitutions within residues 113–120 (the region to which the specificity of the mAbs is directed) and yet exhibited markedly decreased cross-reactions and binding affinities, relative to the reference antigen, sperm-whale Mb. The other three Mb variants possessed substitutions within, as well as outside, region 113–120 and showed very little cross-reactivities. The results of this study, particularly with the Mbs that have no substitutions within the indicated antigenic site, clearly show that substitutions outside the site, and which by design are not part of the site, can influence very markedly the reactivity of the protein variant with the anti-site mAbs. The approach can, therefore, lead to serious errors if used to identify residues of protein antigenic sites.     

Effects of amino acid substitutions outside an antigenic site on protein binding to monoclonal antibodies of predetermined specificity obtained by peptide immunization: Demonstration with region 145–151 (antigenic site 5) of myoglobin

Monoclonal antibodies of predetermined specificity were prepared by immunization with a free (i.e., without coupling to any protein carrier) synthetic peptide representing region 145–151 of sperm whale myoglobin (SpMb) and their cross-reactions with eight Mb variants were determined. Five Mbs—bottle-nose dolphin myoglobin (BdMb), pacific common dolphin myoglobin (PdMb), horse myoglobin (HsMb), dog myoglobin (DgMb), and badger myoglobin (BgMb)—have an identical sequence in that region. Nevertheless, these Mbs exhibited very different cross-reactivities. BdMb and PdMb exhibited cross-activities which were comparable to that of the reference antigen, SpMb; while the reactivity of HsMb was remarkedly decreased, DgMb and BgMb showed almost no cross-reactions with these mAbs. Since the region 145–151 has an identical sequence in all the five Mbs, it is concluded that the differences in their antigenic reactivities with anti-region 145–151 mAbs are due to the effects of amino acid substitutions outside the region 145–151. Another pair of myoglobins, echidna myoglobin (EdMb) and chicken myoglobin (ChMb), have the same sequence in that region, but reacted very differently with anti-region 145–151 mAbs. The reactivity and affinity of EdMb were substantially decreased while those of ChMb were almost completely absent, relative to SpMb. It is concluded, contrary to popular assumptions, that when an amino acid substitution influences the binding of a protein variant to a mAb, it is not necessary for that substitution to be an actual contact residue (i.e., a residue within the antigenic site where the mAb binds). Such effects, which are often very drastic, could be due to indirect influences of the substitution on the chemical and binding properties of the site residues. Furthermore, residues which had been postulated, on the basis of these assumptions, to constitute discontinuous antigenic sites in SpMb, were found [from the present studies and those recently reported with mAbs against the other four antigenic site of Mb (regions 15–22, 56–62, 94–100, and 113–120 of SpMb)] to merely be exerting indirect effects on the known five antigenic sites of Mb. The effects of substitutions, which can happen even in the absence of conformational changes, are determined by many factors, such as the chemical nature of the substitution, its environment, its distance from the site, and the nature of the site residue(s) being affected.     

Premature stop codons in the G glycoprotein of human respiratory syncytial viruses resistant to neutralization by monoclonal antibodies.

Mutants of human respiratory syncytial (RS) virus which escaped neutralization by monoclonal antibodies directed against the G glycoprotein were selected from the Long strain. Most mutants showed drastic antigenic changes, reflected in the lack of reactivity with several anti-G antibodies, including the one used for selection. Sequence analysis revealed the presence of in-frame premature stop codons in the mutated G genes which shortened the G polypeptide by between 11 and 42 amino acids. In contrast, two mutants selected with monoclonal antibody 25G contained two amino acid substitutions (Phe-265----Leu and Leu-274----Pro) and had lost only the capacity to bind the antibody used in their selection. These results demonstrate that the carboxy-terminal end of the G molecule is dispensable for infectivity in tissue culture and indicate the importance of this part of the G protein in determining its antigenicity.     

Polymorphism of murine major histocompatibility Class I antigen: assignment of putative allodeterminants to distinct positions of the amino acid sequence within the first external domain of the antigen

Forty-five new monoclonal antibodies reacting with the mouse H-2Dd antigen have been established. The specificities of 34 of these antibodies were mapped into the first external domain (N) of the Dd antigen by testing reactivities with the products of mosaic H-2 genes in which the coding sequences of the first and/or the second external domains of the H-2Dd genes were recombined in vitro with the remaining portion of the H-2Ld gene. These antibodies reacted with at least 13 distinct allodeterminants located in the N domain, composed of 91 amino acids, as judged from panel tests carried out on various H-2 haplotypes. To assign possible positions of antigenic determinants of these and other anti-H-2Dd antibodies, we compared primary sequences of seven H-2 antigens and searched for correspondence between the pattern of amino acid substitutions in the N domain, allowing 15 positions to be assigned for the antigenic sites. These putative antigenic determinants were assessed for possible relationships with several parameters of protein secondary structure postulated according to predictive methods. Many of these sites appear to be associated with greatest local hydrophilicity, known to correlate with sites of antibody binding in various proteins. We therefore propose that some of the correspondences found in this work represent structural correlates of allodeterminants.     

Multiple overlapping epitopes in the three antigenic regions of horse cytochrome c1

To gain a better understanding of the diversity of epitopes on a protein, the specificities of 103 monoclonal antibodies to a model antigen, horse cytochrome c(cyt c), were analyzed. The antibodies were generated in in vitro monoclonal, secondary antibody responses against horse cyt c coupled to hemocyanin in splenic fragment cultures. For this assay, horse cyt c-primed murine B lymphocytes were transferred to irradiated, hemocyanin-primed recipients. A panel of seven mammalian cyts c differing at one to six residues out of 104 and cyanogen bromide-cleaved fragments of horse cyt c containing residues 1-65, 1-80, and 66-104 was used to examine the specificities of the antibodies. Twenty-two distinct reactivity patterns were observed, even though the majority of the monoclonal antibodies were found to bind in the three previously identified antigenic regions of the molecule about residues 44-47, 60-62, and 89-92. The results indicate that each of the three antigenic regions consists of multiple overlapping epitopes. Few of the antibodies directed to any given antigenic region bound polypeptide fragments inclusive of the epitope sequences, demonstrating that some antibodies were more conformationally dependent than others. Only 13% of the antibodies bound to cyanogen bromide-cleaved polypeptide fragments that together encompassed the entire length of the protein. Considering the large number of antibodies analyzed and the reoccurrence of 13 of the 22 clonotypes in different lymphocyte donors, it is likely that the antibody specificities tabulated herein approach yet do not completely enumerate the total inventory of the horse cyt c-specific B cell repertoire. The remarkable diversity for epitope recognition within antigenic regions observed here is likely to pertain to protein antigens in general, and strongly supports the widely held notion that the entire surface of a protein is potentially antigenic. The restriction of the epitopes of horse cyt c to three antigenic regions where the amino acid sequences of the mammalian cyts c differ probably results from tolerance of the mice to their own cyt c.     

Effects of amino acid substitutions outside an antigenic site on protein binding to monoclonal antibodies of predetermined specificity obtained by peptide immunization: demonstration with region 56-62 (antigenic site 2) of myoglobin.

This work was carried out in order to study the effects of substitutions outside antigenic site 2 of sperm whale myoglobin (SpMb) on the reactivity of protein variants with antisite 2 monoclonal antibodies (mAbs). A synthetic peptide corresponding to region 56–62 (site 2) of SpMb was used as an immunogen in mice in its free form (i.e., without coupling to any carrier) to prepare a panel of mAbs whose predetermined specificity is directed, by design, against this region. The binding of three of these mAbs to eight Mbs from different species was studied. Myoglobins of Pacific common dolphin, finback whale, and horse, which have no substitutions within region 56–62 relative to SpMb, showed remarkable differences in their cross-reactivities and relative affinities with each of the mAbs. Myoglobins of badger, chicken, and dog, although they have an identical substitution within the site (Ala-57 to Gly), exhibited cross-reactivities with a given mAb that were affected differently. Echidna Mb, which has one replacement (Glu-59 to Ala) within region 56–62, displayed greatly reduced cross-reactivities and relative binding affinities. The results, especially those from Mbs that have no substitutions within the boundaries of site 2, clearly indicate that substitutions outside site 2 of Mb can exert drastic effects on the binding of the Mb variants with mAbs whose specificity was predesigned to be against the site. These indirect effects and their impact on site reactivity will completely explain previous findings on cross-reactivities of Mb variants with mAbs of unknown specificity and will rule out the postulations of discontinuous sites in Mb, which were based on the assumption that every substitution affecting reactivity is directly involved in binding to antibody.     

Isolation and characterization of three monoclonal antibodies to human serum low density lipoprotein apoprotein B

Human serum low density lipoprotein (LDL) is a large (Mr = 2-3 X 10(6), complex particle composed of lipid, protein and carbohydrate. We obtained about 40 mouse spleen-myeloma hybrid cell lines which produce antibodies against LDL. Three of them, SC2, SC3 and SC10, have been cloned and subcloned and their antibody products characterized. They recognize three non-overlapping epitopes in native LDL. Two of them, SC3 and SC10, also are capable of recognizing very low density lipoprotein, (VLDL), whereas SC2 reacts only weakly with VLDL. All three antigenic determinants remain intact, and accessible to antibodies on the LDL protein apo B, prepared by delipidation in a 'non-denaturing' detergent, sodium deoxycholate. However, apo B prepared by organic solvent, ether-ethanol, or sodium dodecyl sulfate (SDS) delipidation, while reacting strongly with SC10, is only poorly recognized by SC2 or SC3. Proteolysis of LDL with trypsin, chymotrypsin, Staphylococcus aureus protease, papain or thermolysin gives, in each case, several non-identical protein fragments which are separable by SDS-polyacrylamide gel electrophoresis. Upon immunoblotting, some of these fragments are now recognized by either SC3 or SC10 but not SC2, some are recognized by both SC3 and SC10, and others are immunologically unreactive. The protein bands that are separated by SDS gel electrophoresis are composed of several non-identical fragments and contain the antigenic sites to differing degrees. Some of the immunologically reactive fragments do not appear to contain carbohydrate. Reduction and carboxymethylation do not destroy the immunoreactivity of LDL toward any of the antibodies; however, modification of lysine residues by citraconic anhydride markedly diminishes the reactivity of LDL toward SC3. It is likely that the two antibodies SC3 and SC10 are directed against different linear amino acid sequences or very stable domains, whereas the third, SC2, is directed against a more fragile conformational domain of apo B.