Genetic Control of the Immune Response to Myoglobin. V. Analysis of the Cross-Reactivity of 12 Myoglobins with Sperm-Whale Myoglobin Antisera of Inbred Mouse Strains in Terms of Substitutions in the Antigenic Sites and in the Environmental Residues of the Sites

The determination of the entire antigenic structure of myoglobin has made it possible to focus attention on the molecular factors controlling and influencing immune recognition. Recently, using antisera raised against sperm-whale myoglobin (Mb) in six different host species and investigating their reactions with Mb from 15 species, we showed that the binding capacity of an antigenic site is influenced by substitutions within site residues as well as within the residues close (within 6.0 Å) to the sites. Based on these effects it was possible to correlate, at least in qualitative terms, the expected effects of the substitutions in each Mb and its observed cross-reaction with antisera to sperm-whale Mb. In the present work, these correlations were tested using sperm-whale Mb antibodies raised in four inbred mouse strains and in which the amount of antibodies directed to each antigenic site was determined. The amounts of ¹²⁵I-labelled antibodies that could be bound maximally by each of 12 Mb variants were determined. Because of genetic control, the response to some antigenic sites was not expressed and therefore permitted us to evaluate with a good degree of confidence the effects of amino acid replacements in various myoglobins upon the reactivity of the sites. The values of cross-reactions expected for each Mb variant from these considerations agreed well with the values found experimentally. The results confirm unambiguously that the major factors affecting the cross-reactions of proteins can be attributed to substitutions within the sites and within environmental residues of the sites.     

Genetic Control of the Immune Response to Myoglobin. VII. Antibody Responses to Myoglobin Variants Reveal that Gene Restriction of the Antibody Responses to Myoglobin Antigenic Sites is Dependent on the Chemical Properties of the Sites

Previously It has been shown that the immune responses to sperm-whale myoglobin are under H-2-linked Ir-gene control. More Importantly the responses to the synthetic antigenic sites are each under separate genetic control. In the present studies we investigated the genetic control of the antibody response to four different myoglobins of known structure, to determine whether this genetic control is influenced by changes in the properties of the sites. The results suggest that genetic control of the responses to individual antigenic sites on a protein antigen is not only determined by the genetic constitution of the host species but also by the chemical properties of the individual sites. It appears that the H-2 subregions mapping the responses to given antigenic sites can also recognize other sites, which were previously unrecognizable in a homologous protein, if their chemical properties are suitably altered.     

Genetic control of the immune response to myoglobin. VI. Inter-site influences in T-lymphocyte proliferative response from analysis of cross-reactions of ten myoglobins in terms of substitutions in the antigenic sites and in environmental residues of the sites

The recent determination of the antigenic structure of sperm-whale Mb⁴ and the findings that the same five antigenic sites are recognized by antibodies raised against this protein in several host species, including mouse, enabled us to focus attention on the molecular factors underlying immune recognition and responses. We have also shown that the sites recognized by mouse B-cells (antibodies) are also recognized by T-cells and that the responses to the sites are each under separate genetic control. Furthermore, we recently demonstrated that the binding capacity of an antigenic site in Mb variants is Influenced by substitutions in the antigenic sites and in residues close (within 7.0 Å) to the sites. The genetic exclusion of a response to some antigenic sites in given inbred mouse strains and the knowledge of the sites that suffer decreases or total losses in binding activity In several Mb variants affords a unique system of reduced complexity to study the molecular requirements for T-cell recognition of the Mb antigenic sites. In the present work the cross-reactions of ten myoglobins by proliferation of sperm-whale Mb-primed mouse T-cells were analyzed in terms of substitutions in the sites and in environmental residues of the sites. Help between sites 1 and 2 and between one or both of these and site 3 was observed in several mouse strains. A suppresslve Influence by site 5 on the overall proliferative response to Mb was observed in two strains (D2.GD and A.TTR4). It was concluded that the overall response to Mb is regulated by Inter-site Influences which can either be of a cooperative (help) nature or of a suppresslve nature.     

Original antigenic sin and the production of autoantibodies

Autoantibodies to sheep myoglobin have been raised by priming sheep with beef myoglobin and boosting with sheep myoglobin. The autoantibodies appear to be a subset of those produced when beef myoglobin is used for both priming and boosting. This subset of antibodies is presumably directed to the surface regions which are common to both myoglobins. The antibodies which bind to sheep myoglobin in the 2 types of antisera differ. Those elicited by boosting with beef myoglobin bind better to beef myoglobin than to sheep myoglobin, while those obtained by boosting with sheep myoglobin bind with equal avidity to the 2 myoglobins. It would seem therefore that the boosting immunogen determines which fraction of antibodies is selected from the antibody repertoire established by the priming immunogen. Our results also show that tolerance at the T-cell level can be circumvented by exposing the immune system to a protein closely related to a homologous self protein.     

Genetic control of the immune response to myoglobin. IX. Overcoming genetic control of antibody response to antigenic sites by increasing the dose of antigen used in immunization

Previously, it was reported that the immune response to myoglobin (Mg) was under genetic control, with the response to each site being under separate Ir-gene control. Here we have investigated the effect of antigen dose on the control of the antibody response to the five antigenic sites of sperm-whale Mb to determine whether or not the overcoming of genetic control by antigen dose has a uniform effect on all five antigenic sites. The antibody response to sperm whale myoglobin (Mb) and its five antigenic was measured in the following inbred strains of mice, C57BL/6J, AKR and SWR/J. These strains of mice are low responders to Mb following immunization with 50 micrograms, responding only to site 4. After immunization with 200 micrograms Mb: C57BL/6J mice are high responders to Mb and respond to antigenic sites 1, 3, 4 and 5; AKR mice are high responders to Mb and respond to antigenic sites 1 and 4; SWR/J mice are high responders to Mb and respond to all five antigenic sites. It was concluded that the genetic control of the immune response to Mb and its synthetic antigenic sites is dependent on antigen dose. Also, these studies have enabled us for the first time to separate the response to site 1 from the response to site 2 and thus have conclusively established that sites 1 and 2 are controlled by separate Ir-genes.     

GENETIC CONTROL OF THE IMMUNE RESPONSE TO MYOGLOBIN: VIII. CONTROL OF ANTIBODY AFFINITY

We have previously shown that the antibody response to mammalian myoglobins is under genetic control. In the present study we examined antibodies to sperm-whale, Atlantic bottlenosed dolphin, Black Sea dolphin, horse and badger myoglobins, raised in high responder strains of mice, to ascertain whether there is genetic control of antibody affinity to mammalian myoglobins. Using antisera of varying dilutions, the binding to ¹²⁵I-labelled homologous myoglobins was studied by inhibition with homologous myoglobin over a wide range of inhibitor concentration in a modified Farr assay. The results indicated that there are no large differences between high responder strains of mice in the affinity of antibodies to mammalian myoglobins.     

Original Antigenic Sin: Experiments with a defined antigen

The phenomenon referred to as ‘Original Antigenic Sin’ is the production of antibodies to a specific antigen elicited by a boosting injection of a related antigen. We have investigated this phenomenon using myoglobins of known amino acid sequence and structure. Our results indicated that in order to elicit an ‘Original Antigenic Sin’ response the boosting myoglobin should differ by less than 33–42% in overall sequence from the priming myoglobin. The antibodies in the antisera from rabbits primed with beef myoglobin and boosted with a different myoglobin from a different species are predominantly and perhaps exclusively directed towards antigenic regions common to the priming and boosting myoglobins but have a higher affinity for the priming myoglobin.     

GENETIC CONTROL OF THE IMMUNE RESPONSE TO MYOGLOBIN: IX. OVERCOMING GENETIC CONTROL OF ANTIBODY RESPONSE TO ANTIGENIC SITES BY INCREASING THE DOSE OF ANTIGEN USED IN IMMUNIZATION

Previously, it was reported that the immune response to myoglobin (Mb) was under genetic control, with the response to each site being under separate Ir-gene control. Here we have investigated the effect of antigen dose on the control of the antibody response to the five antigenic sites of sperm-whale Mb to determine whether or not the overcoming of genetic control by antigen dose has a uniform effect on all five antigenic sites. The antibody response to sperm whale myoglobin (Mb) and its five antigenic was measured in the following inbred strains of mice, C57BL/6J, AKR and SWR/J. These strains of mice are low responders to Mb following immunization with 50 μg, responding only to site 4. After immunization with 200 μg Mb:C57BL/6J mice are high responders to Mb and respond to antigenic sites 1, 3,4 and 5; AKR mice are high responders to Mb and respond to antigenic sites 1 and 4; SWR/J mice are high responders to Mb and respond to all five antigenic sites. It was concluded that the genetic control of the immune response to Mb and its synthetic antigenic sites is dependent on antigen dose. Also, these studies have enabled us for the first time to separate the response to site 1 from the response to site 2 and thus have conclusively established that sites 1 and 2 are controlled by separate Ir-genes.     

Use of immunoadsorbents for the study of antibody binding to sperm whale myoglobin and its synthetic antigenic sites.

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.     

Antibodies with preselected specificities to protein regions evoked by immunization with free synthetic peptides: dose response to myoglobin antigenic sites reveal an optimum dose for each antigenic site

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.     

Antibodies with Preselected Specificities to Protein Regions Evoked by Immunization with Free Synthetic Peptides: Dose Response to Myoglobin Antigenic Sites Reveal an Optimum Dose for Each Antigenic Site

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 μg to 100 μg. 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.     

Genetic control of the immune response to myoglobin. IV. Mouse antibodies in outbred and congenic strains against sperm-whale myoglobin recognize the same antigenic sites that are recognized by antibodies raised in other species

The determination of the entire antigenic structure of sperm-whale myoglobin (Mb) was initially performed with antisera raised in rabbits and goats. Subsequently, we demonstrated that the synthetic antigenic sites were effective in stimulating $\text{in vitro}$ mouse T-cell proliferation and that this proliferative response was under genetic control, with each antigenic site being controlled by a unique Ir gene. To determine unambiguously whether T-cells recognize the same molecular features as do B-cells, it was necessary to establish whether mouse antibodies are directed against these same sites. In the present work, using immunoadsorbent titration studies, these synthetic antigenic sites were shown to bind mouse ¹²⁵I-labelled antibodies against Mb. With each of three outbred mice and four congenic strains, the total amounts of antibodies bound by the five sites accounted quantitatively for the entire antibody response against Mb. Moreover, with the four congenic strains, only the sites that were active in T-cell proliferation bound significant amounts of antibodies. It was concluded that (at least for Mb) the molecular features recognized by B-cells are also recognized by T-cells. These studies also confirm that the antigeniclty of the sites Is Independent of the immunized species and is inherent in their three-dimensional locations.     

Structural and antigenic properties of merozoite surface protein 4 of Plasmodium falciparum

Merozoite surface protein 4 (MSP4) of Plasmodium falciparum is a glycosylphosphatidylinositol-anchored integral membrane protein of 272 residues that possesses a single epidermal growth factor (EGF)-like domain near the carboxyl terminus. We have expressed both full-length MSP4 and a number of fragments in Escherichia coli and have used these recombinant proteins to raise experimental antisera. All recombinant proteins elicited specific antibodies that reacted with parasite-derived MSP4 by immunoblotting. Antibody reactivity was highly dependent on the protein conformation. For example, reduction and alkylation of MSP4 almost completely abolished the reactivity of several antibody preparations, including specificities directed to regions of the protein that do not contain cysteine residues and are far removed from the cysteine-containing EGF-like domain. This indicated the presence of conformation-dependent epitopes in MSP4 and demonstrated that proper folding of the EGF-like domain influenced the antigenicity of the entire molecule. The recombinant proteins were used to map epitopes recognized by individuals living in areas where malaria is endemic, and at least four distinct regions are naturally antigenic during infection. Binding of human antibodies to the EGF-like domain was essentially abrogated after reduction of the recombinant protein, indicating the recognition of conformational epitopes by the human immune responses. This observation led us to examine the importance of conformation dependence in responses to other integral membrane proteins of asexual stages. We analyzed the natural immune responses to a subset of these antigens and demonstrated that there is diminished reactivity to several antigens after reduction. These studies demonstrate the importance of reduction-sensitive structures in the maintenance of the antigenicity of several asexual-stage antigens and in particular the importance of the EGF-like domain in the antigenicity of MSP4.     

T-Cell Dependency of the Antibody Response to Free Small Synthetic Peptides of a Protein: Demonstration With an Antigenic Site of Myoglobin

Recent studies from this laboratory have found that synthetic peptides of proteins, as small as 6 residues, when administered in their free form (i.e. without coupling to any carrier) elicit the formation of antibodies with submolecular binding specificities to preselected protein regions. These peptides could represent either the antigenic sites of the protein or surface regions that are not immunogenic when the intact protein is the antigen. In either case the antibodies bind specifically to the intact protein, exclusively at the region used in immunization. In the present study we immunized BALb/c. By J and nude (BALb/c derived) strain mice with either Mb or synthetic antigenic site 5 of Mb. Radio immune antibody binding studies showed that immunization of BALb/c. ByJ strain mice with either Mb or synthetic peptide resulted in the formation of antibodies that bound specifically to Mb, whereas nude mice did not produce such antibodies. These results indicate that the antibody response to both Mb and synthetic antigenic site is T-cell dependent.     

Genetic control of the immune response to myoglobin. XVI. Control of antibodies with preselected specificities following immunization with free synthetic peptides representing the antigenic sites or surface non-immunogenic locations in the protein

Previous studies in this laboratory have resulted in the determination of the antigenic structure of myoglobin. The present work was carried out to investigate the genetic control of the murine antibody response to myoglobin following immunization with free (i.e., not coupled to a carrier) synthetic antigenic sites or other peptides corresponding to surface regions of myoglobin that are not immunogenic when the native molecule is the immunizing antigen. Synthetic peptides corresponding to antigenic 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 regions, peptide 1-6 and peptide 121-127, were injected in complete Freund's adjuvant in different strains of mice. Serum antibodies specific for myoglobin were subsequently obtained and were measured by means of a radioimmune plate binding assay in which Mb was used as the solid phase antigen. It was found that the genetic control of the antibody response to myoglobin following immunization with the free synthetic peptides was different from the genetic control obtained following immunization with native myoglobin. The significance of this finding is discussed.     

Preparation of T-lymphocyte lines and clones with specificities to preselected protein sites by in vitro passage with free synthetic peptides: Demonstration with myoglobin sites

Recently, this laboratory has demonstrated that antibodies to preselected regions of a protein can be obtained by immunization with free small synthetic peptides (6–7 residues) without conjugation to a carrier. In the present work, we report the use of free synthetic peptides representing myoglobin (Mb) antigenic sites to prepare T-cell lines and clones of preselected specificities. Lymph node cells from mice primed $\text{in vivo}$ with sperm-whale Mb were periodically passaged $\text{in vitro}$ with synthetic peptide. After several passages, the peptide-driven long term T-cell cultures responded to the intact protein and exclusively to the peptide that was used to drive the cells. From these cultures, T-cell clones were prepared that responded only to the driving peptide and to the whole protein. The ability to prepare T-cell lines and T-cell clones with preselected submolecular specificities to a protein by driving cultures with desired synthetic peptides affords an important and simple tool for basic immunological investigations and for clinical applications.     

Molecular localization of the full profile of the continuous regions recognized by myoglobin-primed T-cells using synthetic overlapping peptides encompassing the entire molecule

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.     

Molecular Localization of the Full Profile of the Continuous Regions Recognized by Myoglobin Primed T-Cells Using Synthetic Overlapping Peptides Encompassing the Entire Molecule

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-2^d) 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.     

GENETIC CONTROL OF THE IMMUNE RESPONSE TO MYOGLOBIN: XVI. CONTROL OF ANTIBODIES WITH PRESELECTED SPECIFICITIES FOLLOWING IMMUNIZATION WITH FREE SYNTHETIC PEPTIDES REPRESENTING THE ANTIGENIC SITES OR SURFACE NON-IMMUNOGENIC LOCATIONS IN THE PROTEIN

Previous studies in this laboratory have resulted in the determination of the antigenic structure of myoglobin. The present work was carried out to investigate the genetic control of the murine antibody response to myoglobin following immunization with free (i.e., not coupled to a carrier) synthetic antigenic sites or other peptides corresponding to surface regions of myoglobin that are not immunogenic when the native molecule is the immunizing antigen. Synthetic peptides corresponding to antigenic 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 regions, peptide 1-6 and peptide 121-127, were injected in complete Freund's adjuvant in different strains of mice. Serum antibodies specific for myoglobin were subsequently obtained and were measured by means of a radioimmune plate binding assay in which Mb was used as the solid phase antigen. It was found that the genetic control of the antibody response to myoglobin following immunization with the free synthetic peptides was different from the genetic control obtained following immunization with native myoglobin. The significance of this finding is discussed.     

T cell recognition of myoglobin. Localization of the sites stimulating T cell proliferative responses by synthetic overlapping peptides encompassing the entire molecule

A comprehensive strategy for the systematic localization of all continuous antigenic sites within a protein has previously been introduced by this laboratory. The strategy consists of studying the immunochemical activity of a series of consecutive synthetic peptides that encompass the entire protein chain and that are uniform in size and in overlap at their N- and C-terminals with neighbouring peptides. By application of this strategy to sperm whale myoglobin, we have been able to delineate the continuous sites of T cell recognition of myoglobin in three high responder mouse strains. Thirteen 17-residue peptides that encompass the entire myoglobin chain and overlap by five residues at both ends were synthesized, purified and characterized. The peptides were examined in vitro for their ability to stimulate lymph node cells from myoglobin-primed DBA/2 (H-2d), BALB/c (H-2d) and SJL (H-2s) mice as well as long-term cultures of myoglobin-specific T cells. Several regions of the molecule (T sites) were found to stimulate myoglobin-primed lymph node cells and myoglobin-specific longterm T cell cultures. This strategy has enabled the localization of the full profile of dominant sites of T cell recognition in myoglobin for these mouse strains. Of these T sites, one region, residues 107-125, was clearly immunodominant in these strains and was found to coincide with the antigenic (i.e. antibody binding) site 4 of myoglobin. Also, other regions stimulated T cells and appeared to coincide with previously known antigenic sites. It is noteworthy that, in addition to sites recognized by both T and B cells, the protein has other sites which are recognized exclusively by T cells and to which no detectable antibody response is directed.