A semi-empirical method for prediction of antigenic determinants on protein antigens.

Analysis of data from experimentally determined antigenic sites on proteins has revealed that the hydrophobic residues Cys, Leu and Val, if they occur on the surface of a protein, are more likely to be a part of antigenic sites. A semi-empirical method which makes use of physicochemical properties of amino acid residues and their frequencies of occurrence in experimentally known segmental epitopes was developed to predict antigenic determinants on proteins. Application of this method to a large number of proteins has shown that our method can predict antigenic determinants with about 75% accuracy which is better than most of the known methods. This method is based on a single parameter and thus very simple to use.     

Screening of random peptide library of hemagglutinin from pandemic 2009 A(H1N1) influenza virus reveals unexpected antigenically important regions

The antigenic structure of the membrane protein hemagglutinin (HA) from the 2009 A(H1N1) influenza virus was dissected with a high-throughput screening method using complex antisera. The approach involves generating yeast cell libraries displaying a pool of random peptides of controllable lengths on the cell surface, followed by one round of fluorescence-activated cell sorting (FACS) against antisera from mouse, goat and human, respectively. The amino acid residue frequency appearing in the antigenic peptides at both the primary sequence and structural level was determined and used to identify "hot spots" or antigenically important regions. Unexpectedly, different antigenic structures were seen for different antisera. Moreover, five antigenic regions were identified, of which all but one are located in the conserved HA stem region that is responsible for membrane fusion. Our findings are corroborated by several recent studies on cross-neutralizing H1 subtype antibodies that recognize the HA stem region. The antigenic peptides identified may provide clues for creating peptide vaccines with better accessibility to memory B cells and better induction of cross-neutralizing antibodies than the whole HA protein. The scheme used in this study enables a direct mapping of the antigenic regions of viral proteins recognized by antisera, and may be useful for dissecting the antigenic structures of other viral proteins.     

Searching for local similarities between HIV-1 and human proteins. Application to vaccines

A method for identification of fragments with a high local similarity to human proteins within potentially immunopathogenic regions of HIV-1 proteins was developed. The method is based on the use of an original matrix of antigenic similarity of amino acids. The regions, whose fragments are frequent in human proteins, and regions, exhibiting a high similarity to the proteins responsible for important physiological functions, were identified in HIV-1 proteins. A possibility of participation of such regions in immunopathogenesis of HIV-infection due either to induction of cross-reacting effectors of immune system or through molecular mimicry of physiologically important human proteins, leading to an alteration of homeostasis of the organism, is discussed. Most of regions, identified in HIV-1 proteins, contain either T-cell (CD8+ CTL or CD4+ Th) or B-cell epitopes, or both of them simultaneously. The criteria for the design of safe polyepitopic antiviral vaccines which enable the exclusion of epitopes, having the (immuno)pathogenic potential, are discussed. According to these criteria, polyepitopic immunogens should be free of the virus protein regions, whose fragments are frequent in human proteins, as well as of regions exhibiting a pronounced local similarity to proteins that provide for important physiological functions.     

Molecular cartography of globular proteins with application to antigenic sites

A method, molecular cartography, is introduced as a way to quantitate the topographic structure of a protein surface. The method is applied to the problem of antigenic determinants, and it is used to examine local and global topography of reported antigenic regions on the surface of myoglobin and lysozyme. In nine antigenic sites taken from the literature and studied in detail, no local property was found in sites that was not also found in remaining regions of the surface. However, a strong correlation was found between antigenic sites and regions of the surface that are globally exposed. This finding suggests that global exposure of the protein surface may play a primary role in determining the antigenic structure of the protein. Molecular cartography may be useful in other instances of protein–protein interactions such as those between proteolytic enzymes and their substrates.     

Fluorescent staining of proteins transferred to nitrocellulose allowing for subsequent probing with antisera

A sensitive staining method for protein blots on nitrocellulose is described. It is based on the coupling of a fluorochrome, dichlorotriazynylaminofluorescein, to protein which yields products colorless in visible light but colored when protein blots are illuminated with long-range ultraviolet light. The coupling of a fluorochrome does not affect the antigenic properties of proteins and the stained blots can be subsequently probed with antisera. Thus, the method allows for the unambiguous identification of antigenic proteins transferred to nitrocellulose from sodium dodecyl sulfate-polyacrylamide gels.     

Searching for Local Similarities between HIV-1 and Human Proteins. Application to Vaccines

A method for identification of fragments with high local similarity to human proteins within potentially immunopathogenic regions of HIV-1 proteins was developed. The method is based on the use of an original matrix of antigenic similarity of amino acids. The regions whose fragments are frequent in human proteins, and regions exhibiting high similarity to the proteins responsible for important physiological functions, were identified in HIV-1 proteins. A possibility of participation of such regions in immunopathogenesis of HIV infection either through induction of cross-reacting effectors of the immune system or through molecular mimicry of physiologically important human proteins, leading to alteration of homeostasis of the organism, is discussed. Most of the regions identified in HIV-1 proteins contain either T-cell (CD8⁺ CTL or CD4⁺ Th) or B-cell epitopes, or both of them simultaneously. The criteria for the design of safe polyepitopic antiviral vaccines that would allow exclusion of epitopes with (immuno)pathogenic potential are discussed. According to these criteria, polyepitopic immunogens should be free of the virus protein regions whose fragments are frequent in human proteins, as well as of regions exhibiting pronounced local similarity to proteins that provide for important physiological functions.     

Use of a series of ompF-ompC chimeric proteins for locating antigenic determinants recognized by monoclonal antibodies against the ompC and ompF proteins of the Escherichia coli outer membrane

A method is presented for the efficient location of antigenic determinants using a series of chimeric proteins. By means of in vivo homologous recombination between the ompC and ompF genes coding for OmpC and OmpF, homologous proteins of the Escherichia coli outer membrane, a series of ompF-ompC chimeric genes was constructed (Nogami, T., Mizuno, T., & Mizushima, S. (1985) J. Bacteriol. 164, 797-801, and this work). The OmpF-OmpC chimeric proteins expressed by these genes were successfully used to locate antigenic determinants recognized by monoclonal antibodies, which specifically react with either the OmpC or OmpF protein. Interaction between monoclonal antibodies and the chimeric proteins was examined by means of either enzyme-linked immunosorbent assay or immunoblot analysis. The antigenic determinants recognized by three anti-OmpC antibodies and one anti-OmpF antibody were thus located. Finally, the polypeptides covering these regions were chemically synthesized for two of them and then tested as to their reactivity with the antibodies. The peptides reacted with the corresponding antibodies when the former were chemically coupled with bovine serum albumin. Most of the monoclonal antibodies isolated in this work were highly specific to the unfolded monomer of the protein against which the antibody was raised. But they did not react with the trimer, the native form. These results are discussed in relation to the structures and functions of the OmpC and OmpF proteins. The use of a series of monoclonal antibodies for studying the mechanism of protein translocation across the cytoplasmic membrane is also discussed.     

A simple method for predicting the functional differentiation of duplicate genes and its application to MIKC-type MADS-box genes

A simple statistical method for predicting the functional differentiation of duplicate genes was developed. This method is based on the premise that the extent of functional differentiation between duplicate genes is reflected in the difference in evolutionary rate because the functional change of genes is often caused by relaxation or intensification of functional constraints. With this idea in mind, we developed a window analysis of protein sequences to identify the protein regions in which the significant rate difference exists. We applied this method to MIKC-type MADS-box proteins that control flower development in plants. We examined 23 pairs of sequences of floral MADS-box proteins from petunia and found that the rate differences for 14 pairs are significant. The significant rate differences were observed mostly in the K domain, which is important for dimerization between MADS-box proteins. These results indicate that our statistical method may be useful for predicting protein regions that are likely to be functionally differentiated. These regions may be chosen for further experimental studies.     

Structurally inherent antigenic sites. Localization of the antigenic sites of the alpha-chain of human haemoglobin in three host species by a comprehensive synthetic approach.

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.     

Use of the averaged mutation rate in pieces of protein sequences to predict the location of antigenic determinants

Antigenic determinants in proteins show properties, which can be used to their prediction: high degree of accessibility, mobility, polarity etc. Furthermore, there is a remarkable correlation between the location of antigenic determinants and regions with high frequency of accepted mutations during the evolution. Consequently, it is possible to predict successfully the location of antigenic epitopes in proteins using the averaged mutation rate of protein sequence pieces.     

Analysis of autoantibody binding to different regions of the human La antigen expressed in recombinant fusion proteins

To determine the specificity of autoantibodies for various antigenic sites on a self-protein molecule, sera from 19 patients with anti-La antibodies were tested for their reactivity with molecularly cloned La protein fragments. By quantitative ELISA, anti-La sera from patients with various connective tissue diseases were shown to react with La fusion proteins containing different regions of the La molecule. Two recombinant La fragments containing the carboxyl three-fourths and the middle one-third of the La sequence, respectively, bound higher levels of anti-La antibodies than the two fragments representing the amino and carboxyl terminals. Purified bovine La protein effectively competed for the binding of human autoantibodies to three of the four recombinant La fusion proteins, suggesting similarity in antigenic presentation between the La epitopes in these fusion proteins and the native La molecule. Immunoadsorption experiments showed that most anti-bovine La protein antibodies were removed from a human serum by affinity chromatography by using the fusion protein containing the carboxyl three-fourths of the La sequence, thus supporting the results obtained by quantitative solid phase ELISA. These studies demonstrate that anti-La autoantibodies recognize three La fragments representing separate nonoverlapping regions of the La sequence and are compatible with a mechanism of autoantibody production based on an immune response to the entire self-protein molecule.     

Computer-assisted analysis of envelope protein sequences of seven human immunodeficiency virus isolates: prediction of antigenic epitopes in conserved and variable regions.

Independent isolates of human immunodeficiency virus (HIV) exhibit a striking genomic diversity, most of which is located in the viral envelope gene. Since this property of the HIV group of viruses may play an important role in the pathobiology of the virus, we analyzed the predicted amino acid sequences of the envelope proteins of seven different HIV strains, three of which represent sequential isolates from a single patient. By using a computer program that predicts the secondary protein structure and superimposes values for hydrophilicity, surface probability, and flexibility, we identified several potential antigenic epitopes in the envelope proteins of the seven different viruses. Interestingly, the majority of the predicted epitopes in the exterior envelope protein (gp120) were found in regions of high sequence variability which are interspersed with highly conserved regions among the independent viral isolates. A comparison of the sequential viral isolates revealed that changes concerning the secondary structure of the protein occurred only in regions which were predicted to be antigenic, predominantly in highly variable regions. The membrane-associated protein gp41 contains no highly variable regions; about 80% of the amino acids were found to be conserved, and only one hydrophilic area was identified as likely to be accessible to antibody recognition. These findings give insight into the secondary and possible tertiary structure of variant HIV envelope proteins and should facilitate experimental approaches directed toward the identification and fine mapping of HIV envelope proteins.     

Prospects and achievements of genetic engineering in development of antiviral vaccines

Proceeding from the known data various theoretical and experimental approaches to the construction of gene-engineering vaccines are considered. Gene-engineering subunit vaccines of the first generation are based on isolation of the genes coding for the synthesis of full length capsid proteins with the main antigenic determinants and their subsequent expression in suitable recipient cells. Initial idea of the microbiological synthesis as the main way for production of any antiviral vaccines was not confirmed by the later development. Now for this type of vaccines eucaryotic systems are widely employed using the animal virus vectors and the animal cell cultures. Gene-engineering subunit vaccine of the second generation appears to be a chimeric protein with built-in antigenic determinants of different viruses and maximal immunogenicity in monomeric form. The last point reopens the perspective to use a microbiological synthesis for the production of antiviral vaccines. Besides that the chemically synthesized polypeptide antiviral vaccine will be used widely. In gene-engineering subunit vaccines of the third generation it is possible to use not the natural antigenic determinants which often are characterized by high level of the primary structure changes but artificial (non-natural) antigens, that are the capsid protein conservative regions which under natural conditions of infection or immunization do not induce the protective antiviral antibodies. The recombinant DNA technology in addition to subunit type vaccine allows to construct living vaccines which represent a DNA-containing attenuated virus with build-in natural or synthetic gene of the capsid or chimeric protein with antigenic determinants of another viral species.     

Identification of antigenic regions of duck hepatitis B virus core protein with antibodies elicited by DNA immunization and chronic infection

The induction of humoral response in ducks by DNA-based immunization against duck hepatitis B virus (DHBV) core protein (DHBc) was investigated. In addition, the amino acid specificity of the induced response was compared by using peptide scanning to that elicited either by protein immunization or during chronic DHBV infection. Immunization of ducks with a plasmid expressing DHBc protein led to the induction of a long-lasting antibody response able to specifically recognize viral protein in chronically infected duck livers. Peptide scanning analysis of anti-DHBc response induced during chronic DHBV infection allowed us to identify six major antigenic regions (AR1 to AR6). The reactivity spectrum of duck sera elicited by protein immunization appeared narrower and was restricted to only four of these antigenic regions in spite of higher anti-DHBc antibody titers. Interestingly, anti-DHBc antibodies induced by DNA-based immunization recognized five of six antigenic regions, and the epitope pattern was broader and more closely related to that observed in chronic viral infections. To gain more insight into the location of antigenic regions, we built a three-dimensional (3-D) model of DHBc protein based on human and duck core sequence alignment data and the HBc 3-D crystal structure. The results suggest that two identified antigenic regions (AR2, amino acids [aa] (64)T-P(84), and AR5, aa (183)A-R(210)) are located at positions on the protein surface equivalent to those of the two HBc major epitopes. Moreover, we identified another antigenic region (AR3, aa (99)I-I(112)) that was recognized by all sera from chronically infected, DNA- or protein-immunized ducks within the large 45-aa insertion in DHBc protein, suggesting that this region, which lacks HBc, is externally exposed.     

Immunochemical characterization of the adenine nucleotide translocator. Organ specificity and conformation specificity

Antibodies have been prepared against purified preparations of the heart and kidney nucleotide translocator in the 'c'-conformation. The results show organ-specific antigenic determinants on the translocator proteins isolated from heart, kidney and liver although a partial cross-reactivity between these three proteins was demonstrable. The organ specificity was observed both with the solubilized and with the membrane-bound translocator protein indicating organ-specific determinants on exposed regions of the carrier. An organ-specific inhibition of the nucleotide transport in heart mitochondria by the heart carboxyatractylate-protein antiserum leads to the conclusion that the organ specificity is at least partially conditioned by the binding site for the substrate and/or the closely linked gate of the carrier protein. Apart from the organ specificity the results also demonstrate a specificity of the antibodies for the translocational conformations of the carrier: the 'c'-conformation stabilized in the carboxyatractylate-protein complex and the 'm'-conformation present in the bongkrekate-protein complex. However, after denaturation of the carboxytraktylate-protein and bongkrekate-protein complexes the binding of the anti-(carboxyatractylate-protein) antiserum to both inhibitor-protein complexes was nearly identical. The conformation specificity was also expressed by the inhibition of the conformation transition from the 'c'- to the 'm'- state. This side-specific inhibition of the nucleotide transport and the identical binding activity of the carboxyatractylate-protein antiserum against the denatured carboxyatractylate-protein and bongkrekate-protein complexes suggested that the conformation-specific antigenic determinants are topographic surface regions which are determined by the chain folding.     

Antigenic structure of human haemoglobin. Localization of the antigenic sites of the beta-chain in three host species by synthetic overlapping peptides representing the entire chain.

A comprehensive synthetic approach is applied here to localize the continuous antigenic sites of the beta-chain of haemoglobin. The approach was based on the synthesis and purification of the following consecutive 15-residue peptides (each overlapping by five residues at both ends with the peptides preceding it and following it in the sequence): 1-15, 11-25 etc. Quantitative radiometric titrations of protein and peptide adsorbents were performed with 125I-labelled anti-haemoglobin antibodies from three different host species. The specificity of antibody binding to peptide adsorbents was confirmed by inhibition studies and by the binding specificity of antibodies isolated from peptide adsorbents. These studies established the full profile of antigenic beta-chain regions, which was found to be independent of the host species. Five major antigenic sites were localized, and their three-dimensional and structural characteristics are discussed in relation to the immune recognition of haemoglobin and other proteins.     

Prediction of loop regions in protein sequence

We suggest an algorithm that inputs a protein sequence and outputs a decomposition of the protein chain into a regular part including secondary structures and a nonregular part corresponding to loop regions. We have analyzed loop regions in a protein dataset of 3,769 globular domains and defined the optimal parameters for this prediction: the threshold between regular and nonregular regions and the optimal window size for averaging procedures using the scale of the expected number of contacts in a globular state and entropy scale as the number of degrees of freedom for the angles phi, psi, and chi for each amino acid. Comparison with known methods demonstrates that our method gives the same results as the well-known ALB method based on physical properties of amino acids (the percentage of true predictions is 64% against 66%), and worse prediction for regular and nonregular regions than PSIPRED (Protein Structure Prediction Server) without alignment of homologous proteins (the percentage of true predictions is 73%). The potential advantage of the suggested approach is that the predicted set of loops can be used to find patterns of rigid and flexible loops as possible candidates to play a structure/function role as well as a role of antigenic determinants.     

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

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

Antigenic diversity among Portuguese clinical isolates of Helicobacter pylori

Background: The human gastroduodenal pathogen, Helicobacter pylori, is characterized by an unusual extent of genetic heterogeneity. This dictates differences in the antigenic pattern of strains resulting in heterogeneous human humoral immune responses. Here, we examined the antigenic variability among a group of 10 strains isolated from Portuguese patients differing in age, gender, and H. pylori‐associated gastric diseases. Material and Methods: Immunoassays were performed on two‐dimensional electrophoresis gels obtained for the proteome of each strain, using a commercial pool of antibodies produced in rabbit, against the whole cell lysate of an Australian H. pylori strain. Relevant proteins were identified by mass spectrometry. Results: Immunoproteomes of the Portuguese strains showed no correlation between the number of antigenic proteins or the antigenic profile, and the disease to which each strain was associated. The Heat shock protein B was the unique immunoreactive protein common to all of them. Additionally, seven proteins were found to be antigenic in at least 80% of strains: enoyl‐(acyl‐carrier‐protein) reductase (NADH); Catalase; Flagellin A; 2 isoforms of alkyl hydroperoxide reductase; succinyl‐CoA transferase subunit B; and an unidentified protein. These proteins were present in the proteome of all tested strains, suggesting that differences in their antigenicity are related to antigenic variance. Conclusions: This study showed evidence of the variability of antigenic pattern among H. pylori strains. We believe that this fact contributes to the failure of anti‐H. pylori vaccines and the low accuracy of serological tests based on a low number of proteins or antigens of only one strain.     

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

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