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Previously, we mapped the antibody (Ab) and T-cell recognition regions on the HC domain (residues 855-1296) of the 848-residue heavy (H) chain of botulinum neurotoxin A (BoNT/A). We have mapped here the HN-domain (residues 449-859) regions that bind protective anti-BoNT/A Abs raised in four different species. We synthesized, purified, and characterized 29 19-residue peptides that spanned the entire HN and overlapped consecutively by 5 residues, and also region L218-231 around the L-chain's substrate-binding site. Human, horse, mouse, and chicken anti-BoNT/A Abs did not bind to the L-peptide but recognized similar HN regions within peptides 519-537/533-551/547-565/561-579 (with slight left- or right-shifts), 743-761, 785-803, and 813-831/827-845 overlap. Recognition of other peptides that bound lower Ab levels showed similarities and also some differences. Peptide 463-481, strongly immunodominant with horse antisera, did not bind human, mouse, and chicken Abs. However, peptide 449-467 bound Abs in these three antisera, and the region may have shifted to the right (peptide 463-481) with horse Abs. The overlap 659-677/673-691 reacted strongly with human Abs whereas with mouse and chicken antisera, only peptide 673-691 showed low reactivity. Horse antisera had no detectable Ab binding to region(s) 659-691. The Ab-recognition regions on the H chain occupy surface locations in BoNT/A three-dimensional structure, but the great part of the surface is not immunogenic. Regions recognized by the protective antisera of the four different species are prime candidates for inclusion in synthetic vaccine designs.

Humoral responses to a protein require T-B cell communication for B cell activation by T cells. Previous studies from this laboratory have mapped the T and B cell recognition sites (epitopes) on sperm-whale myoglobin (Mb) and several other proteins. It was found that, five of six regions on Mb recognized by T cells are also recognized by B cells (i.e. antibodies). There is, however, one region (E6) residing within Mb residues 61-77, that is recognized only by T cells and to which no antibody (Ab) responses are detectable. To investigate the function of this exclusive T cell epitope, we established, from E6-primed BALB/c mice, an E6-specific T cell line (T(e6)) which comprised Th2-type cells. These T cells provided help in vitro to B cells from Mb-primed BALB/c mice and activated them to produce anti-Mb Abs of the IgM (58.2%) and IgG (41.8%) isotypes. The helper activity of T(e6) cells was dependent on the concentration of the challenging Ag (intact Mb or peptide E6) in culture. Action of soluble factors released from E6-activated T(e6) cells on B(mb) cells led to low production of anti-Mb Abs, suggesting that activation of the B cells was more dependent on their contact with T cells. Mapping of the epitope recognition of the anti-Mb Abs produced in vitro by B(mb) cells on activation by T(e6) revealed that this activation was not general to all antigenic regions recognized by anti-Mb Abs in BALB/c mice. E6-specific T cells caused in vitro activation and differentiation of B(mb) cells into plasma cells that secreted anti-Mb Abs directed, in decreasing order, against the following Mb regions: E4 (107-120) > E3 (87 - 100) > E1 (10 - 22). Little or no Ab responses could be detected against peptides E2 (50 - 62), E5 (141 - 153) and E6 (61 - 77). With B cells of peptide-primed BALB/c mice, T(e6) cells activated strongly E4-, E3- or E1, and only very slightly E2- or E6-, primed B cells to secrete Abs against the correlate peptide, but failed completely to activate E5-primed B cells. The results show that a protein T cell epitope, to which no Abs are detectable, plays an active role in B cell responses against other epitopes within the same protein.

We have previously mapped the T and B cell epitopes on the alpha-subunit of acetylcholine receptor (AChR) in human myasthenia gravis (MG) and in experimental autoimmune MG-susceptible (C57BL/6 (B6)) and nonsusceptible mouse strains. In addition to regions recognized by both T and B cells, the AChR alpha-subunit has regions that are recognized solely by T cells. An exclusive T cell epitope within residues alpha 146-162 of Torpedo californica (t), tAChR, plays an important role in experimental autoimmune MG pathogenesis in B6 mice. To study its function, we established, from tAChR-primed B6 mice, two t alpha 146-162-specific T cell lines (P14Th) which comprised Th2-type cells because they secreted IL-4 but not IL-2. P14Th did not recognize the corresponding region on mouse (m) AChR (m alpha 146-162). They caused in vitro differentiation of tAChR-primed B cells into plasma cells that secreted anti-AChR Abs directed, in decreasing order, against the following tAChR alpha regions: t alpha 122-138 > t alpha 134-150 > t alpha 45-60 > t alpha 170-186 > t alpha 56-71. Little or no Ab response could be detected against peptides t alpha 182-198 or t alpha 146-162 itself. The major enhancement was in the Abs against region t alpha 122-150 (spanning the t alpha 122-138/t alpha 134-150 overlap) that is involved in ACh binding. These Abs cross-reacted completely with m alpha 122-150, the corresponding region on mAChR. Therefore, t alpha 146-162-specific T cells, although unable to recognize m alpha 146-162, are nevertheless pathogenic because they help B cells responding to a tAChR region that is conserved in mAChR and involved in ACh binding. These Abs cross-react with the corresponding effector-binding region of mAChR, thereby disrupting the normal physiologic function of the mouse receptor.

Previous study has shown that reduced T cell response to peptide alpha 146-162 of Torpedo californica acetylcholine receptor (tAChR) in B6.C-H-2bm12 (bm12) mice, a mutant of C57BL/6 (B6) mice, correlated with its nonsusceptiblity to experimental autoimmune myasthenia gravis. There are three amino acid differences between the I-A beta b of the two strains (positions 67, 70, and 71). We synthesized peptides I-A beta b62-76 (peptide b6), I-A beta bm1262-76 (peptide bm), and three additional peptides, b6(67F), b6(70Q), and b6(71K), and determined their ability to bind peptide alpha 146-162 and the dissociation constants (Kd) of the binding. Peptide alpha 146-162 bound with a significantly higher affinity to peptide b6 than to peptides bm or b6(71K), suggesting that the lower affinity of peptide alpha 146-162 to I-Abm12 is a factor in the reduced response to this peptide by bm12 T cells. This was confirmed by measurement of the Kd values of the binding of peptide alpha 146-162 to the I-A molecules of B6 and bm12. Furthermore, APC of bm12 presented the peptide, or tAChR, poorly to peptide-specific or to tAChR-specific B6 T cells. The major effect is caused by the change of Thr-71 in I-A beta b to lysine in I-A beta bm12. However, APC of B6 also presented peptide alpha 146-162 much less efficiently to peptide-specific T cells of bm12. This demonstrated that these three amino acid changes also influence the T cell receptor recognition of peptide-MHC complex and that both B6 and bm12 T cells recognizing peptide alpha 146-162 or tAChR are under a high H-2 restriction.

A gene conversion event between Ebb and Abb in the B6.C-H-2bm12 (bm12) strain, which alters three amino acids in the C-terminal half of the first domain of Abb (Ile-67-->Phe; Arg-70-->Gln; Thr-71-->Lys) resulted in resistance to experimental autoimmune myasthenia gravis (EAMG) pathogenesis. To study the effect of bm12 mutation on the T-cell responses to epitopes of acetylcholine receptor (AChR)-alpha subunit, C57BL6 (B6) and bm12 mice were primed with Torpedo californica AChR, and the profiles of T-lymphocyte proliferation were determined with 18 synthetic overlapping peptides encompassing the entire extracellular portion of the AChR-alpha subunit. The proliferative responses of AChR-primed bm12 lymphocytes were markedly reduced to two (alpha 146-162 and alpha 182-198) of the three AChR peptides (alpha 111-126, alpha 146-162, and alpha 182-198) that are immunodominant in B6 mice. Thus, the Ab residues encompassing the region 67-71 determine the immunogenicity of two of the AChR-alpha subunit T-cell epitopes. To test the involvement of AChR-alpha chain epitopes within peptide alpha 146-162 in EAMG pathogenesis, B6 mice were neonatally tolerized with soluble peptide alpha 146-162, and subsequently immunized with AChR in complete Freund's adjuvant. Neonatal tolerance to AChR or to peptide alpha 146-162 reduced the incidence of clinical myasthenia gravis and suppressed serum anti-AChR antibodies. This indicates the involvement of T-cell epitopes within AChR-alpha subunit region alpha 146-162 in EAMG pathogenesis. Neonatal tolerance to peptide alpha 146-162 could have caused specific clonal deletion, and/or clonal anergy, and/or recruited suppressor cells to prevent clinical EAMG. Presumably, epitope(s) with AChR alpha 146-162, in the context of Ab encompassing region 67-71, stimulate specific T helper cells which interact with specific B cells to produce pathogenic antibodies, the primary culprit causing the end plate lesion in patients with myasthenia gravis.

Eighteen synthetic overlapping peptides encompassing the entire extracellular part (residues alpha 1-210) of the alpha-chain of human acetylcholine receptor (AChR) and a 19th peptide (residues alpha 262-276) corresponding to an extracellular connection between two transmembrane regions were prepared and used for the measurement, by solid-phase radioimmunoassay, of the binding of autoantibodies in plasma from myasthenia gravis (MG) patients. Autoantibodies were found to recognize only a limited number of the synthetic peptides. The regions recognized resided predominantly within the areas alpha 10-30, alpha 111-145 and alpha 175-198 and, less frequently, region alpha 45-77. Differences in the recognition profile of the peptides from patient to patient indicated that the autoantibody responses were under genetic control. However, by using a mixture of the appropriate peptides, it was possible to determine autoantibodies in all 15 myasthenia sera and to distinguish between these, normal human sera and other neurological or autoimmune diseases. The mapping of the continuous antigenic regions recognized by autoantibodies on the alpha-chain of human AChR has permitted a comparison of the regions recognized by autoantibodies and autoimmune T-cells from the same donor. It also provided a peptide-based direct antibody binding method for diagnosis of MG.

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 myoglobin (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.

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.

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.

Monoclonal antibodies (mAbs) of predetermined specificity were prepared by immunizing with a free (i.e., not conjugated to any carrier) synthetic peptide representing region 15-22 (site 1) of sperm whale myoglobin (SpMb). The cross-reactions of Mb variants with three mAbs were studied in order to determine whether such interactions are influenced by substitutions outside the site. Finback whale Mb, which has no substitutions within region 15-22, showed lower cross-reactivity and relative binding affinity than the reference antigen, SpMb. Bottle-nose Atlantic dolphin myoglobin (BdMb) and badger myoglobin (BgMb), although they have identical substitutions within region 15-22 (Ala-15 to Gly and Val-21 to Leu), showed very different binding properties. The cross-reaction of BdMb was quite comparable to that of SpMb, while that of BgMb was much lower. Since the two proteins have identical structures in regions 15-22, the differences in their cross-reactivities are readily attributed to the effects of substitutions outside this region. Another pair of myoglobins, horse myoglobins (HsMb) and chicken myoglobin (ChMb), also have two identical substitutions (Ala-15 to Gly and Val-21 to Ile) within region 15-22, but possessed different cross-reactivity. The results indicate that the reaction of mAbs, whose specificity is precisely known and predetermined by the immunizing free peptide, can be markedly affected by substitutions outside the indicated binding region on the protein.

It is generally assumed that alloreactivity and the response to foreign antigens are equivalent T-cell recognition events. We have addressed this issue by examining the antigen presentation of beef insulin by two DR1-restricted human T-clones. One of the clones was dual-reactive and exhibited alloreactivity. By employing 20 synthetic consecutive overlapping peptides representing the entire extracellular (residues 1-198) and intracellular (residues 222-273) parts of the DR2 beta molecule, the effects of each of these peptides on antigen presentation and alloreactivity were examined. The DR1-restricted response to beef insulin by both clones was inhibited by peptides from DR2 beta region 1-25. Other DR2 beta (residues 21-198 en 222-237) or control peptides had no effect. The DR3-associated alloresponse of the dual-reactive clone was not inhibited by any peptide. These observations suggest that recognition of antigen is fundamentally different from allorecognition.

Conditions for preparing immunoadsorbents of sperm-whale myoglobin and its five synthetic antigenic sites and for desorption of radiolabeled antibodies from the immunoadsorbents were studied. In immunoadsorbent titration studies, the sum of the amounts of antibodies bound in the plateau (maximum binding) by the adsorbents of the five sites accounted quantitatively for the entire (100%) antibody response to sperm-whale myoglobin.

Intensive research in the author's laboratory had culminated in the determination and synthesis of all the antigenic sites of myoglobin in 1975 and of lysozyme in 1978. Very recently most of the antigenic sites of serum albumin were also localized and synthesized. These investigations provided the first unique insight into the molecular features responsible for the immune recognition of protein antigens and of the factors which determine and regulate the antigenicity of the sites. But moreover, these studies have charted a multi-approach chemical strategy for investigation and synthetic duplication of protein binding sites. Furthermore, the concept of 'surface-simulation' synthesis, which we introduced and developed during our determination of the antigenic structure of lysozyme, has provided a remarkable dimension of unlimited versatility for the synthetic mimicking of any type of protein binding sites. In this concept, the spatially adjacent residues of a protein binding site are linked directly via peptide bonds with appropriate spacers into a single peptide which does not exist in the protein but mimicks a surface region of it. This has proved to be a powerful concept in protein molecular recognition and has opened up many untapped avenues in investigation, duplication and perhaps manipulation of a variety of protein activities. In fact, binding sites representing other protein activities (including antibody combining sites) have or are now being mimicked synthetically in our laboratory by the concept of surface-simulation synthesis.

By using the antigenic structure of lysozyme determined in this laboratory and the X-ray co-ordinates we have calculated the closest-atom distances between each of the residues in the three antigenic sites and all the other amino acids of the lysozyme molecule. These calculations enabled us to identify the nearest neighbours to each of the site residues. Thus the immediate environment of each site residue is described. For the three antigenic sites there is a total of 71 neighbouring residues. The effects of evolutionary amino acid substitutions in site-neighbouring residues on the binding capacity of protein binding sites in general and on protein antigenic sites in particular are discussed. These, together with the direct replacements in site residues, will acount for the major effects. However, the limitations of this treatment are stressed. The smaller effects on antigenic sites of replacements at once-removed and even at more distant locations, which, when they become cumulative, could be considerable, are brought to attention, together with any influences of conformational readjustments that can take place as a result of evolutionary amino acid replacements.

Recently, we have localized and confirmed by synthesis the regions within which reside five major antigenic sites of bovine serum albumin and proposed that the remaining sixth major antigenic site (antigenic site 4) was localized within subdomain 3 (fragment 115-184) of albumin. In the present work, antigenic site 4 was localized to fall around the disulfide bond 166-175 by synthesis and immunochemical reactivity of the region 162-179. The synthetic 18-residue peptide was shown to bind substantial amounts of antibodies from early (38-day) and late (398-day) anti-albumin antisera from two rabbits. As much as half the total anti-albumin antibodies could be bound by the peptide from the late antisera. It was concluded that antigenic site 4 resides within, but may not include all of, the region 162-179 of albumin.

By applying the concept of 'surface-simulation' synthesis to the combining site of the myeloma protein M-603 we were able to mimic synthetically its phosphorylcholine-binding characteristics. The synthetic surface-simulation peptide was found to bind to phosphorylcholine, whereas a control peptide that had the same amino acid composition but a different sequence showed little or no binding activity. The specificity of the binding was further confirmed by inhibition studies in which the surface-simulation peptide, but not the control peptide, inhibited the binding of 125I-labelled surface-simulation peptide to phosphorylcholine. Furthermore, the surface-simulation peptide was found to completely inhibit the binding of the native myeloma protein, M-603, to phosphorylcholine. The control peptide was unable to inhibit this binding. These findings suggest that surface-simulation synthesis can be effectively employed to mimic synthetically antibody combining sites, and may in the future be a valuable tool with which to manipulate the immune response to clinically important antigens.

In previous studies, six T sites within myoglobin (Mb) were localized. To define precisely the boundaries of the T sites, a new approach is introduced and applied here to the T site residing within residues 107-120 of Mb. Two sets of peptides were synthesized. One set represents a stepwise elongation by one-residue increments of the Mb sequence. The other set represents an identical stepwise addition of one-residue increments of the Mb sequence, but which were extended by additional unrelated (nonsense) residues to a uniform size of 14 residues. The longer peptides (nonsense-extended) usually gave higher proliferative responses than did their shorter counterparts having the same Mb region. Thus a minimum peptide size is required for optimal T-cell stimulation. The T site subtends, in three high-responder mouse strains, residues 109-119 or 110-120, depending on strain, and, in three low-responder strains, maps to residues 108-120. Thus, in this case, the T site coincides with the site of B-cell recognition and resides in a small discrete surface region of the protein chain.

By using the known antigenic structure of sperm-whale myoglobin previously determined in this laboratory and the X-ray co-ordinates for the myoglobin molecule, we have calculated the nearest-atom distances between each of the residues of the antigenic sites and all the other amino acids of the myoglobin molecule. These calculations have enabled us to identify the nearest-neighbour residues to each of the residues in the five antigenic sites, and which thus describe the immediate molecular environment of the sites. The influences of chemical changes or replacements in these environmental residues on the binding capacity of an antigenic site, when considered together with replacements directly in the antigenic sites, are expected to account for the major effects and will be extremely useful in explaining the cross-reactions of myoglobins from various species. However, it is stressed that the analysis has limitations due to the qualitative estimates of the effects, the influences of substitutions of once-removed or even at more distant locations (especially when they are cumulative) and finally the influences of any conformational re-adjustments when these occur as a result of the replacement(s).

We have been able to isolate clones of sperm whale muscle myoglobin (Mb)-reactive T cells from (C57BL/6 x A/J)F1 [(B6A)F1] mice. Four types of clones were isolated, distinguished by their patterns of recognition of Mb cyanogen bromide (CNBr) fragments and antigen presenting cell (APC) requirements. Individual T cell clones proliferated in response to one of three CNBr fragments of Mb. Dose-response curves of all clones were identical for native Mb and the appropriate fragment. T cell clones reactive to fragment 1-55 did not proliferate in response to peptide 15-22 (a peptide that binds to serum antibody directed against 1-55). These data support previous findings suggesting differences between antigen recognition by T and B cells, i.e., T cells may not recognize antigen in its native conformation and/or T and B cells may recognize distinct epitopes on the same antigen. Using T cell clones to analyze genetic control of responsiveness to Mb, we found that certain (B6A)F1 T cells recognize Mb presented by low responder strain APC. Thus, genetically determined low responsiveness in this case is probably not due to failure of APC function. We also found that responsiveness to certain Mb epitopes mapped to the I-A subregion whereas others mapped, via gene complementation, to the I-A and I-E subregions. We found no examples of responsiveness mapping to the I-C subregion and suggest an alternative explanation for previous reports mapping genetic control of responsiveness to certain Mb determinants to I-C.

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.

The antigenic structure of the alpha-chain of human haemoglobin was studied by a synthetic approach consisting of the synthesis of a series of consecutive overlapping peptides that together systematically represent the entire primary structure of the protein. This approach enabled the identification of a full profile of immunochemically active alpha-chain peptides and the localization of its major 'continuous' antigenic sites. Antibodies to haemoglobin raised in each of three different species (goat, rabbit and mouse) recognize similar sites on the alpha-chain. Further, the molecular locations of these sites coincide with alpha-chain regions extrapolated from antigenic sites of the conformationally similar myoglobin molecule. These findings support our earlier proposed concept of 'structurally inherent antigenic sites', namely that antigenicity is conferred on certain surface regions of proteins by virtue of their three-dimensional locations. Thus the antigenic sites of conformationally related proteins are likely to have similar molecular locations.

Reaction of overlapping synthetic peptides spanning the extracellular part (residues 1-210) of the alpha chain of the Torpedo californica acetylcholine receptor (an alpha 2 beta gamma delta pentamer) with anti-receptor antibodies produced the profiles of the continuous antigenic regions of the correlate segment. Essentially similar profiles were recognized by rabbit and outbred mouse antibodies against isolated receptor or mouse antibodies against membrane-bound receptor. The antigenic sites reside within eight continuous regions: residues 1-14, 25-36, 41-53, 63-75, 102-114, 128-138, 172-182, and 188-198. Five of these regions (the second and the fifth through the eighth) appeared to be immunodominant. Significantly, two of these antigenic regions (i.e., those residing within residues 128-138 and 188-198) coincided with known toxin-binding regions. The antigenic profile suggests that recognition is directed to the intact molecule and only very slightly to the processed (fragmented) protein.

The molecular localization of the full antigenic profile for T-cell recognition of a complex multi-determinant protein antigen has not to date been accomplished. Previously, this laboratory has introduced a comprehensive strategy for the systematic localization of all continuous antigenic sites within a protein. This strategy depends on the synthesis of a series of overlapping peptides that together account for the entire structure of a protein. Such a strategy has been applied, in this report, to the delineation of the continuous sites of T-cell recognition of sperm whale myoglobin. Thirteen peptides, accounting for the entire protein chain, were synthesized and subsequently examined in vitro for their ability to stimulate lymph node cells from myoglobin primed DBA/2 (H-2d) mice, a known high responder. This strategy has enabled for the first time the localization of the full profile of the protein regions which contain the sites of T-cell recognition. Three regions gave a high response (one being immunodominant and coinciding with antigenic, i.e. antibody binding, site 4 of myoglobin). At least three regions appear to coincide with previously known antigenic (antibody binding) sites. Noteworthy is the finding of regions that are recognized by T-cells but to which no detectable antibody response is directed.

A region of the alpha-subunit of the nicotinic acetylcholine receptor (AChR) of the Torpedo electric organ, containing residues 161-166, has been proposed to be a major antigenic site in the native AChR protein. We report the synthesis of a peptide corresponding to residues 159-169, which contains the proposed antigenic region. In quantitative radiometric titrations, radiolabelled anti-(native AChR) antibodies from three different species, rabbit, rat and dog, exhibited considerable binding (approx. 15% relative to native AChR) to Sepharose-immobilized peptide 159-169, but did not bind significantly to Sepharose-immobilized unrelated proteins or peptides. Specificity was further confirmed by the finding that no rabbit anti-AChR antibodies bound to the peptide after absorption with native AChR. These data indicate that the region 159-169 contains an antigenic site that is readily accessible in solubilized native Torpedo AChR.

The fragmentation of native bovine serum albumin by trypsin has been studied in aqueous solution under various conditions with regard to the yield and size of the fragments obtained. From a partial tryptic hydrolysate at pH 8.2 (40 degrees, 1 hour), a homogeneous fragment was isolated in high yield by gel filtration on Sephadex G-100, followed by chromatography on DEAE-cellulose. The molecular weight of the fragment by gel filtration on calibrated Sephadex G-100 columns and by sodium dodecyl sulfate electrophoresis was 22,500. After reduction of the disulfide bonds followed by alkylation of the resultant thiol groups with iodoacetamide, the fragment retained homogeneity by disc electrophoresis and its molecular weight remained unchanged, indicating that it was composed of a single polypeptide chain. From its amino acid composition, sequence of the first 20 residues, and actions of carboxypeptidases A or B, it was unequivocally assigned to positions 377-571 in albumin. The inhibitory activity of the fragment was 90 to 93% towards the immune reaction of the protein with the IgG fraction of the antisera. The IgGfraction accounted for 96% of the total antibody activity in the antisera. An immunoabsorbent of fragment 377-571 removed 89 to 95% of the antibody to albumin. A fluorescent derivative of the fragment, which retained full immunochemical activity, was found to bind 2 mol of antibody/mol of peptide. The disulfides in peptide 377-571 were essential for its immunochemical reaction because the latter was entirely abolished upon reduction and S-alkylation of the disulfides. Since this fragment comprised only a third of the albumin molecule, but accounted for 90 to 95% of its antigenic reactivity, the results indicated that native albumin carries identical repeating antigenic reactive sites.

Previous studies from this laboratory on the immunochemistry of specific chemical derivatives of native lysozyme and of the two disulfide peptide 62-68 (Cys 64-Cys 80) 74-97 (Cys 76-Cys 94) (i.e. (SS)2-peptide), have established an antigenic reactive site to comprise the spatially contiguous surface residues: Trp 72, Lys 97, Lys 96, Asn 93, Thr 89 and Asp 87. In the present work, the identity of the site was verified by an entirely different and novel approach. The aforementioned amino acids were linked directly into a single linear peptide with an intervening spacer where appropriate and substituting phenylalanine for tryptophan (i.e. Phe-Gly-Lys-Asn-Thr-Asp). This peptide (which does not exist in native lysozyme but simulates a surface region of the protein) possessed a remarkable inhibitory activity towards the reaction of lysozyme with its antisera. The immunochemical reactivity of the peptide was equal to the maximum expected reactivity of the site (i.e. a third of the total antigenic reactivity of lysozyme). These findings define quite conclusively and accurately the reactive site which is clearly composed of spatially adjacent residues that are distant in sequence reacting as if in direct linear linkage. The unequivocal establishment of this concept indicates that antigenic sites need not always be composed of residues in direct peptide linkage in the sequence. The nature of the site may depend on the protein. This unorthodox attack at the problem provides a novel and powerful approach for final delineation of the antigenic reactive sites (and perhaps other types of binding sites) in native proteins, following the completion of accurate narrowing down by chemical methods.

Recently, we reported the synthesis and immunochemistry of two peptides designed, by complementarity and surface-simulation synthesis, to mimic antibody-combining sites against two antigenic sites of lysozyme. In the present work antibodies were raised against one of these peptides, which is complementary to antigenic site 3 of lysozyme, to determine whether these antibodies will react with anti-lysozyme antibodies. Radioiodinated antipeptide antibodies were bound by immunoadsorbents of the immune IgG from two goats anti-lysozyme antisera but not by adsorbents of myoglobin, non-immune goat IgG or immune IgG of antisera against cytochrome c. The binding of anti-peptide antibodies to adsorbents of anti-lysozyme antibodies was fully inhibited by free lysozyme but not by bovine serum albumin, human hemoglobin A, horse cytochrome c or bovine ribonuclease A. Thus, antisera against an antibody-combining site can be raised by immunizing with a peptide which probably does not exist in the antibody but is designed by surface-simulation synthesis to mimic an antibody-combining site.

Previous studies from this laboratory have resulted in the determination of the entire antigenic structure of myoglobin (Mb). The present work was carried out to investigate the antigenicity of the synthetic antigenic sites of Mb in their free form (i.e. without coupling to a carrier) and the effect of peptide size on antigenicity. Site 5 (peptide 145-151) and the longer peptides 145-153, 143-153 and 132-153, each of which carries within it the region of site 5, were synthesized. Immunization of three different mouse strains with each of these peptides, in their free form, resulted in the formation of antibodies that bound specifically to intact Mb. The advantages and significance of these findings are discussed.

Two monoclonal antibodies with pre-selected submolecular binding specificities to sperm whale myoglobin (Mb) were obtained by hybridizing Fa/0 mouse myeloma cells with spleen cells derived from mice which had been immunized with free (not coupled to any carrier) Mb synthetic peptides 132-153 or 145-151 (antigenic site 5). Both monoclonal antibodies were IgG1 (k). Their homogeneity was confirmed by analytical isoelectric focusing electrophoresis. According to competitive inhibition studies in which Mb, the five synthetic antigenic sites of Mb, Mb peptide 132-153, bovine serum albumin (BSA), and lysozyme were used as inhibitors, the binding specificities of both monoclonal antibodies were restricted to determinants present in the peptides used for immunizations. The results of direct binding studies confirmed this conclusion and suggested that monoclonal antibodies with pre-selected submolecular binding specificities can be readily obtained by the techniques of somatic cell hybridization when the corresponding free synthetic determinants are used as immunogens.

The complete antigenic structure of sperm-whale myoglobin was previously determined in our laboratory. By structural analogy with myoglobin, two regions in human haemoglobin were predicted to comprise antigenic sites. One region was on the alpha-chain [alpha-(15-23)] and the other on the beta-chain [beta-(16-23)]. These two regions were synthesized, purified and characterized, and their immunochemistry was studied. Each peptide was able specifically to bind considerable amounts of haemoglobin antibodies. In a set of homologous proteins, barring any drastic conformational or electrostatic inductive effects exerted by the substitutions, and allowing for obstruction due to subunit interaction, the determination of the antigenic structure of one protein may serve as a useful starting model for the others.

Previous studies have resulted in the determination of the entire structure of myoglobin. The present work was carried out to investigate the antigenicity of the synthetic antigenic sites and other surface peptides of Mb in their free form (i.e. without coupling to a carrier). Site 1 (peptide 15-22), site 2 (peptide 56-62), site 3 (peptide 94-100), site 4 (peptide 113-120), site 5 (peptide 145-151) and two surface peptides, peptide 1-6 and peptide 121-127, were injected in complete Freund's adjuvant into Balb/c mice. Radioimmune antibody binding studies showed that immunization with each of these peptides, in their free form, resulted in the formation of antibodies that bound specifically to Mb and to the immunizing peptide. The advantage and significance of these findings are discussed.

Previously, we have shown by radioimmune antibody binding studies that immunization with each of the five synthetic antigenic sites of sperm-whale myoglobin (Mb), in their free form (i.e. not coupled to a carrier), resulted in the formation of antibodies that bound specifically to Mb and exclusively to the peptide that was used in immunization. In the present series of studies we have immunized separate groups of Balb/cByJ mice with different amounts of each of the free synthetic antigenic sites so as to determine the optimum immunizing dose for eliciting serum antibodies that bind specifically to Mb. The synthetic peptides corresponded to: Site 1 (peptide 15-22), Site 2 (peptide 56-62), Site 3 (peptide 94-100), Site 4 (peptide 113-120, and Site 5 (peptide 145-151). The dose range of each of the antigenic sites used in immunization was from 6 micrograms to 100 micrograms. Radioimmune antibody binding studies indicated that there is an optimal immunizing dose for each of the five antigenic sites which was smaller than anticipated. The significance of these findings is discussed.

Recently we have reported the isolation of the two subunits (alpha and beta) of ragweed antigen E (AgE) in their active form. In the present work we have compared the allergenic activity of the two subunits with native AgE by the radioallergosorbent test (RAST) and by inhibition of the RAST. Also, the amounts of IgE antibodies in 30 ragweed-sensitive sera bound to alpha and beta subunits as well as native AgE were measured by RAST. In all individuals the percentage of RAST binding was greatest to native AgE; however, varying patterns of reactivity to alpha and beta subunits were noted. In RAST inhibition experiments, free beta subunit was equally effective in inhibiting the binding of IgE antibody (from a pooled ragweed-sensitive sera) to immoblized alpha or beta subunit. Similarly, fluid-phase alpha subunit inhibited in a comparable manner the binding of IgE antibody to solid-phase alpha or beta subunits. In further RAST inhibition experiments 7 x 10(-13) moles of fluid-phase native AgE was required for 50% inhibition of the binding of IgE antibody to solid-phase native AgE. Larger molar amounts of fluid phase beta (3.5 x 10(-11) and alpha (3 x 10(-10) were needed for 50% inhibition of the binding of IgE antibody to solid-phase native AgE. AgE allergenicity as measured by RAST could not be totally reconstituted by recombination of equimolar quantities of alpha and beta subunits. Less than 1 x 10(-12) moles of fluid-phase native AgE inhibited the binding of IgE antibody to solid-phase alpha or beta, greater than 50%. Finally, the isolated chains are bioligcally active as demonstrated by their ability to provoke wheal-and-flare skin reactions in subjects allergic to ragweed pollen, and the activities of the chains were approximately equivalent on a weight basis to native AgE.

A comprehensive synthetic approach, previously developed in this laboratory, has been applied to screen the entire first domain (residues 1-95) of the HLA-DR2 beta chain for the full profile of continuous regions that are recognized by human allo-antisera and anti-DR or anti-beta chain antisera raised in other species. Nine consecutive peptides, that were 15 residues each and overlapped by 5 residues, covering the first domain of the DR2 beta chain were synthesized, purified and characterized. The antibody-binding activities of the peptides were determined by radioimmunosorbent titrations. This established the full profile of peptides having specific antibody-binding activity with these various antisera. Three continuous antigenic sites were localized in this domain by all of antisera tested. Peptide 81-95 appeared to be the most immunodominant (i.e. bound the highest amounts of antibodies) in most antisera tested. Although their immunodominance varied with the antisera and boundary shifts were present, the submolecular regions recognized on the first domain of DR2 beta appeared to be similar with antisera against the DR2 (alpha beta I) complex or against the isolated beta chain. Furthermore, recognition was independent of the host species from which the antisera were obtained.

Ten consecutive 15-residue peptides (except for peptide 181-198) that spanned the entire second domain of the HLA DR2 beta chain and overlapped by 5 residues were synthesized, purified and characterized. The antibody-binding activities of the peptides with human alloantisera and with antisera raised in other species were determined by radioimmunosorbent titrations. This established the full profile of peptides having specific antibody-binding activity with these various antisera. The continuous antigenic sites in this domain were localized within four regions. The localization of the antigenic regions on the second domain completes the description of the antigenic profile of the entire extracellular part of the beta chain of HLA DR2. Thus, the continuous antigenic sites on the extracellular part of the DR2 beta subunit reside within seven regions with an eighth site being recognized somewhat weakly with some antisera. As was found for the first domain, the immunodominance of a given site varied with the antisera and boundary shifts were present. The submolecular regions recognized on the second domain of the DR2 beta were independent of the host species from which the antisera were obtained.