Synthetic Antigenic Sites Research Articles

Immunogenicity evaluation of a lipidic amino acid-based synthetic peptide vaccine for Chlamydia trachomatis.

Lipidic amino acid-based synthetic peptides derived from the variable domains (VD) of Chlamydia trachomatis outer membrane protein 1 were evaluated as potential candidate sequences in a vaccine. A peptide sequence designated P2 from the VD IV of serovar B contained a B cell epitope capable of eliciting antibodies binding to serovar B elementary bodies (EB) and a T helper site capable of presentation by multiple H-2 alleles. Polymerization of the P2 into polylysine to form lipid core peptides (LCP) significantly enhanced immunogenicity compared with P2 monomer alone. The LCP system incorporates lipidic amino acids into the polylysine system and enhances lipophobicity and membrane binding effects of the peptide. A second peptide sequence derived from the VD I of serovar C was cosynthesized with P2 into lipidic polylysine LCP and was designated LCP-H1. Antibodies to this construct reacted at high titer with EB of the three major trachoma causing C. trachomatis serovars A, B, and C. LCP-H1 was immunogenic among four of five murine H-2 alleles. Pepscan analysis showed that the fine specificity of antibodies generated to LCP-H1 were directed to the predetermined neutralizing epitope sequences. An in vitro HAK cell neutralization assay showed that LCP-H1 elicited neutralizing antibodies to serovars A, B, and C, but these were of low titer. Because LCP-H1 antibodies bound to the peptide sequence with 10-100-fold higher titer than to EB, the low neutralization titers most likely result from conformational differences between the synthetic peptide and antigenic sites on the native organism. Modification of LCP-H1 to incorporate a predefined conformation may result in improved antigenic properties.

Antigen Presentation by Non-Immune B-Cell Hybridoma Clones: Presentation of Synthetic Antigenic Sites Reveals Clones that Exhibit no Specificity and Clones that Present Only One Epitope

Recently, we reported the preparation and antigen-presenting properties of hybridoma B-cell clones obtained after fusing non-secreting, non-antigen presenting Balb/c 653-myeloma cells with non-immune SJL spleen cells. It was found that antigen presentation at the clonal level can be specific or non-specific, depending on the particular B-cell clone. In the present work, one specific and one general presenter B-cell clones were tested for their epitope presentation ability to SJL T-cells that were specific to lysozyme or myoglobin. B-cell clone A1G12, a general presenter which presented both lysozyme and myoglobin to their respective T-cell lines, was found to present all five myoglobin epitopes while clone A1L16, a lysozyme specific presenter presented only one of the three epitopes of lysozyme. The latter reveals a hitherto unknown submolecular specificity (to a given epitope within a protein) for antigen presenting cells at the clonal level. Therefore, the specificity of T-cell recognition does not only derive from the T-cell but may also be dependent on the epitope specificity of the antigen-presenting B-cell.

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.

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/0 mouse myeloma cells with spleen cells derived from each group of mice. These monoclonal antibodies were either IgM(κ) or IgGI(κ). 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 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.

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.

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 IgG 1 ( 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 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. 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 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.

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.

Immune recognition of serum albumin. 11. Mouse antibodies against bovine serum albumin recognize the same antigenic sites that are recognized by rabbit antibodies

In recent studies, we have localized and confirmed by synthetic peptides five major antigenic sites in each of bovine and human serum albumins which are recognized by rabbit antibodies against the respective protein. In order to determine whether the antigenic sites of albumin are peculiar to the protein or are dependent on the species of the immunized host, we have studied here the specificity of the antibody response in outbred mice against bovine serum albumin. By immunoadsorbent titration studies, the synthetic antigenic sites, previously localized with rabbit antisera against bovine serum albumin, were shown to bind considerable amounts of specific mouse 125I-labelled antibodies against bovine serum albumin. The amounts of mouse anti-bovine serum albumin antibodies bound by the adsorbents of the synthetic sites were comparable to the amounts of rabit anti-bovine serum albumin antibodies that could be bound. It was concluded that recognition of the antigenic sites of serum albumin is independent of the immunized species and is inherent in their structural and conformational uniqueness.

Immunochemistry of serum albumin. X. five major antigenic sites of human serum albumin are extrapolated from bovine albumin and confirmed by synthetic peptides

In a recent report from this laboratory, we have predicted and confirmed by synthesis the locations of five major antigenic sites of bovine serum albumin. In view of the high structural similarity between bovine and human serum albumins, analogous regions of human serum albumin are predicted here to comprise antigenic sites in this protein. Inimunochemlcal studies with antlsera to human albumin and the synthetic antigenic sites of bovine albumin verified this prediction and also identified the major structural locations responsible for the immunochemical cross-reaction between these two albumins.

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.

Evidence for cellular but not serologic cross-reactivity between keyhole limpet hemocyanin and sperm-whale myoglobin

The addition of keyhole limpet hemocyanin (KLH) to cultures of rabbit lymph node cells (LNC) primed with KLH and sperm-whale myoglobin (Mb) induced the synthesis of antibody to Mb as well as to KLH. Several mechanisms for this heterologous induction were considered. It was established that KLH does not nonspecifically activate rabbit T or B lymphocytes. It was also shown that KLH and Mb do not cross-react serologically by several sensitive and specific criteria. Therefore, it was surmised that heterologous induction of Mb antibody synthesis by KLH was due to cellular cross-reactivity between these proteins. Rabbits were primed by the injection of Mb-complete Freund's adjuvant (CFA), alum-Mb, or alum-KLH, and their LNC challenged with KLH, Mb, and synthetic antigenic sites of Mb. These experiments yielded much and diverse evidence for cellular cross-reactivity between KLH and Mb, and especially between KLH and the Mb peptides: KLH plus Mb-primed LNC evoked enhanced anti-KLH and anti-Mb syntheses. KLH plus KLH-sensitized LNC resulted in a lowered anti-Mb antibody response. Mb added to Mb-educated LNC either enhanced or inhibited the anti-KLH antibody response, depending on whether the priming adjuvant was CFA or alum. The addition of Mb to KLH-primed cells enhanced or inhibited the ensuing anti-Mb antibody synthesis; KLH did not affect or inhibit anti-KLH antibody synthesis. Addition of synthetic Mb antigenic sites to Mb-sensitized LNC elevated or suppressed anti-KLH antibody production, depending on the length of time between priming and in vitro challenge. A mixture of KLH and Mb peptide lowered the anti-Mb antibody response of Mb-educated LNC compared to KLH alone. A combination of KLH and Mb peptide also reduced the anti-KLH antibody synthesis of KLH-primed cells compared to KLH per se. The addition of KLH to Mb-sensitized LNC enhanced their uptake of tritiated thymidine, and their transport of tritiated cyclic AMP and protein synthesis. Added Mb induced the synthesis of protein and nonspecific IgG by KLH-primed LNC; Mb peptides evoked protein synthesis by these cells. It is postulated that cross-reactivity at the T-cell level is responsible for the induction of Mb antibody synthesis by adding KLH to either Mb-primed or KLH/Mb-primed LNC. The implications of these findings with respect to cellular and humoral immunity are discussed.

Genetic Control of Immune Response to Sperm Whale Myoglobin in Mice

Abstract The genetic control of T lymphocyte proliferative response to the five synthetic antigenic sites of myoglobin, two synthetic nonantigenic control peptides, and one “nonsense” peptide was determined in independent and recombinant strains of mice. In all the strains examined, the nonantigenic control peptides and the “nonsense” peptide did not invoke a response in myoglobin-primed mice. Further, when mice were not primed with whole myoglobin, no response was obtained with any of the antigenic sites. Haplotypes H-2d, H-2f, and H-2s are higher responders to sites 1 and 2, whereas haplotypes H-2d and H-2s are high responders to site 5. Response to site 3 may be controlled by a non-H-2-linked gene. Site 4 can stimulate H-2b and H-2k haplotypes that are nonresponders to the whole myoglobin. Studies with the recombinant strains suggested that Ir genes to sites 1 and 2 map in the I-A subregion and I-C subregion and were designated Ir-Mb-1,2(A) and Ir-Mb-1,2(C). Ir genes to sites 4 and 5 mapped only in the I-A subregion and were designated Ir-Mb-4(A) and Ir-Mb-5(A). These studies suggest that individual antigenic sites in a molecule are controlled by unique Ir genes.

Genetic Control of T-Lymphocyte Proliferative Response to Sperm-Whale Myoglobin and Its Synthetic Antigenic Sites in Mice

Abstract Previously we have reported the precise determination of the entire antigenic structure of sperm-whale myoglobin. This protein which is made up on a single polypeptide chain (153 residues, m.w. 17,816) has five antigenic sites located in exposed conformationally sensitive continuous portions of its polypeptide chain. The five antigenic sites occupy the following regions: Site 1: sequence 15–22; Site 2: sequence 56–62; Site 3: sequence 94–99; Site 4: sequence 113–119; Site 5: sequence 145–151. We investigated the genetic control of T lymphocyte proliferative response to myoglobin and its synthetic antigenic sites in mice (PETLES Assay). Two parts of the myoglobin molecule (sequences 1–6 and 121–127) that were not within antigenic sites were also synthesized and employed as controls. Also used as control was a synthetic ‘nonsense’ heptapeptide that did not resemble any part of myoglobin. Haplotypes H-2d, H-2f and H-2s gave substantial T lymphocyte proliferative response to whole myoglobin whereas haplotypes H-2d, H-2k, H-2p, H-2q and H-2r were low responders.