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.     

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.     

Production of monoclonal antibodies to surface regions that are non-immunogenic in a protein using free synthetic peptide as immunogens: demonstration with sperm-whale myoglobin

Two synthetic peptides corresponding to surface regions of sperm whale myoglobin that are not antigenic in the native molecule were used in their free form (i.e. not coupled to a carrier) to immunize separate groups of Balb/cByJ mice. The synthetic peptides corresponded to regions 1-6 and 121-127. Serum samples obtained from each group of mice contained antibodies that bound specifically to myoglobin and exclusively to the immunizing peptide. Monoclonal antibodies to each of the two surface regions were subsequently obtained by hybridizing Fa/O mouse myeloma cells with spleen cells derived from each group of mice. These monoclonal antibodies were IgM (kappa). They expressed the same isotype as the antigen specific serum antibodies produced by the mice whose spleen cells were used for hybridization. Solid phase radioimmunoassay studies also indicated that each monoclonal antibody, like the immune serum of the parent animals, bound specifically to myoglobin and exclusively to the synthetic peptide used as an immunogen. These results suggested that the hybridoma antibodies expressed submolecular binding specificities that were the result of peptide immunization rather than hybrid selection and that monoclonal antibodies with preselected submolecular binding specificities to non-antigenic surface regions in a protein molecule can be produced by the techniques of somatic cell hybridization when the corresponding free synthetic peptides are used as immunogens.     

Antibodies with specificities to preselected protein regions evoked by free synthetic peptides representing protein antigenic sites or other surface locations: demonstration with myoglobin

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.     

Production of monoclonal antibodies with preselected submolecular binding specificities to protein antigenic sites: antibodies to sperm whale myoglobin sites

The five synthetic antigenic sites of sperm whale myoglobin were used in their free form (i.e. not coupled to any carrier) to immunize separate groups of BALB/cByJ mice. The synthetic peptides corresponded to: site 1, residues 15-22; site 2, residues 56-62; site 3, residues 94-99; site 4, residues 113-119; site 5, residues 145-151. Serum samples obtained from each group of mice contained antibodies that bound specifically to myoglobin and exclusively to the immunizing antigenic site. Monoclonal antibodies to each of the five antigenic sites were subsequently obtained by hybridizing Fa/O mouse myeloma cells with spleen cells derived from each group of mice. These monoclonal antibodies were either IgM(kappa) or IgGl(kappa). They expressed the same isotypes as the antigen specific serum antibodies produced by the mice whose spleen cells were used for hybridization. Solid phase radioimmunoassay studies also indicated that each monoclonal antibody, like the immune serum of the parent animals, bound specifically to myoglobin and exclusively to the synthetic peptide used as an immunogen. These results suggested that the hybridoma antibodies expressed submolecular binding specificities that were the result of peptide immunization rather than hybrid selection. This strongly supports our previous findings that it is possible to produce monoclonal antibodies with preselected submolecular binding specificities to continuous protein determinants by the techniques of somatic cell hybridization when the corresponding free synthetic determinants are used as immunogens.     

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. 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.     

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.     

T-lymphocyte recognition of sperm-whale myoglobin. Specificity of T-cell recognition following neonatal tolerance with either myoglobin or synthetic peptides of an antigenic site

Recently, in investigating the responses of T-cells from high responder mice that were primed with myoglobin (Mb) or with synthetic peptides containing antigenic site 5 and increasing in length stepwise by increments of two residues, we observed that T-cell recognition was highly dependent on conformation. In the present studies, tolerization experiments were carried out to further investigate this finding. Neonatal mice (BALB/cByJ) were either tolerized with Mb or with synthetic peptides of Mb containing antigenic site 5. Tolerization with Mb and subsequent immunization with Mb gave T-cells that did not proliferate in vitro to Mb or any of the peptides. T-cells from mice that were tolerized with a truncated peptide 139-153 (having deletions at Tyr-151 and Ala-144) and subsequently immunized with Mb proliferated in vitro to Mb and to peptides 132-153, 135-153 and 143-153. T-cells from mice that were tolerized with native Mb and subsequently immunized with peptides (which are unfolded in solution) did not proliferate in vitro to Mb, but responded well to the peptides. Conversely, tolerization with peptides had no effect on the recognition of, and the response to, native Mb by the T-cells, whereas the response to the peptides was completely removed. It was thus concluded that the recognition of protein antigens (or at least of Mb) by T-cells is (like the recognition by antibody) dependent on the conformation of the antigen.     

B-Cell Epitopes and Quantification of the ESAT-6 Protein of Mycobacterium tuberculosis

ESAT-6 is an important T-cell antigen recognized by protective T cells in animal models of infection with Mycobacterium tuberculosis. In an enzyme-linked immunosorbent assay (ELISA) with overlapping peptides spanning the sequence of ESAT-6, monoclonal antibody HYB76-8 reacted with two peptides in the N-terminal region of the molecule. Assays with synthetic truncated peptides allowed a precise mapping of the epitope to the residues EQQWNFAGIEAAA at positions 3 to 15. Hydrophilicity plots revealed one hydrophilic area at the N terminus and two additional areas further along the polypeptide chain. Antipeptide antibodies were generated by immunization with synthetic 8-mer peptides corresponding to these two regions coupled to keyhole limpet hemocyanin. Prolonged immunization with a 23-mer peptide (positions 40 to 62) resulted in the formation of antibodies reacting with the peptide as well as native ESAT-6. A double-antibody ELISA was then developed with monoclonal antibody HYB76-8 as a capture antibody, antigen for testing in the second layer, and antipeptide antibody in the third layer. The assay was suitable for quantification of ESAT-6 in M. tuberculosis antigen preparations, showing no reactivity with M. bovis BCG Tokyo culture fluid, used as a negative control, or with MPT64 or antigen 85B, previously shown to cross-react with HYB76-8. This capture ELISA permitted the identification of ESAT-6 expression from vaccinia virus constructs containing the esat-6 gene; this expression could not be identified by standard immunoblotting.     

The structural requirements for class II (I-Ad)-restricted T cell recognition of influenza hemagglutinin: B cell epitopes define T cell epitopes

A majority of I-Ad-restricted CD4+ clones elicited by influenza X31 (H3N2) virus infection, recognize a synthetic peptide of hemagglutinin (HA) corresponding to an antibody binding region of the HA1 subunit (site B: HA1 177-199). The structural requirements for class II-restricted T cell recognition were investigated by determining the proliferative responses of representative CD4+ clones to truncated HA1 peptides and synthetic peptide analogues. Two distinct T cell epitopes were identified and CD4+ clones, specific for either determinant, were sensitive to the same single amino acid substitutions in synthetic peptides at HA1 193 S----N or HA1 198 A----E, that had featured in antigenic drift and abrogated antibody binding to native HA. Competitive inhibition studies, between stimulatory HA1 peptides and non-stimulatory analogue peptides, for antigen presentation to CD4+ clones established that the 193 S----N and 198 A----E substitutions could affect either interaction with the T cell receptor or class II molecule, according to the specificity of the CD4+ clone examined. The structural requirements for class II-restricted T cell recognition of the linear sequence determinants of HA are, therefore, integrally linked to conformation-dependent antibody recognition of the native molecule.     

T- LYMPHOCYTE RECOGNITION OF SPERM-WHALE MYOGLOBIN. RECOGNITION OF SYNTHETIC PEPTIDES CARRYING ANTIGENIC SITE 5 BY MYOGLOBIN-PRIMED T-CELLS

Previous studies from this laboratory have resulted in the determination of the antigenic structure of sperm-whale myoglobin (Mb). In the present work, we have investigated the fine specificity requirements for T-cell recognition of one of the Mb antigenic sites (antigenic site 5). The antigenic site (peptide 145-153) and seven progressively longer peptides, increasing in length stepwise by two residues at a time, up to 22 residues in length (peptide 132-153), were synthesized. In addition, four truncated peptides were synthesized with intentional deletions at Tyr- 151 and Ala- 144. The T-cell recognition of these purified synthetic peptides was examined here in detail in three strains of mice (BALB/cByJ, B10.D2/n and SJL/J). Mb-primed mice afforded T-cells which proliferated to smaller peptides (two or four residues longer than the site; i.e. peptides 145-153 and 143-153) and more so to the longer peptides 135-153 and 132-153 and to Mb. No response was obtained to the truncated peptides, thus underscoring the fine specificity T-cells. No response was obtained also to intermediate-sized peptides. The latter result, due to an unfavourable mode of folding, suggested a conformational dependency in T-lymphocyte recognition.     

Antibodies to sperm-whale myoglobin evoked by free synthetic peptides of an antigenic 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.     

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.     

The antibody response to myoglobin--I. Systematic synthesis of myoglobin peptides reveals location and substructure of species-dependent continuous antigenic determinants

Sets of peptides representing all the possible hepta-, octa-, nona- and decapeptides of sperm whale myoglobin were synthesized. An ELISA method was used to detect the ability of antibodies, present in antisera raised against native sperm whale myoglobin, to bind to these peptides. Antisera made in two species were compared. It was found that the peptides recognized by the antibodies were a function of the species in which the antiserum was prepared and of the individual outbred member of that species. Peptides corresponding to surface epitopes of the native antigen were identified by reacting the antisera with native antigen prior to ELISA testing on peptides. More detailed analysis of one epitope revealed that, for some sera, a leucine residue which is facing inwards in the crystal structure is critical for the binding of antibody to the peptide. This suggests that binding between native antigen and antibody can require a restructuring of the native antigen.     

Production of monoclonal antibodies with pre-selected submolecular binding specificities to protein determinants: Demonstration with sperm whale myoglobin

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.     

GENETIC CONTROL OF THE IMMUNE RESPONSE TO MAMMALIAN CHYMOTRYPSINS IN MICE

The primary and secondary immune response to the antigen bovine pancreatic α-chymotrypsin was investigated in in inbred mice. It was found that strain differences in the immune response only became apparent after secondary immunization. The genetic control of the immune response was investigated in twelve different strains of mice, F₁, F₂ and F₁ backcross hybrids, following secondary immunization. A continous distribution for the mean antibody responsiveness was obtained. High responsiveness was associated with both the H-2 haplotype and three non-H-2 loci. Furthermore the F₁ hybrids produced a greater quantitative antibody response to chymotrypsin than either of the corresponding parental strains.     

Identification of Linear HLA Class II Amino Acid Sequences Recognized by Rabbit Antisera against Native Molecules

A rabbit was immunized with B lymphoblastoid cells, and subsets of the antibodies produced were isolated on affinity columns made from synthetic peptides corresponding to known amino acid sequences from the human class II antigens DQ and DP. Those peptides for which specific antibodies were isolated could be assumed to contribute to the antigenic properties of the intact antigen. The antibody subsets were tested for binding to synthetic peptides, to glycoprotein fractions isolated from cells with different DR and DQ specificities, and to the cells used for immunization of the rabbit. The isolation of those antibodies directed against well-defined amino acid stretches of the histocompatibility antigens is proof of the role of those regions in determining the antigenic properties of these molecules.     

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.