key: cord-0004974-rxg7wvr7 authors: Lenstra, J. A.; Kusters, J. G.; van der Zeijst, B. A. M. title: Mapping of viral epitopes with prokaryotic expression products date: 1990 journal: Arch Virol DOI: 10.1007/bf01310699 sha: 97c00a215be8409a293f1dc3a35d55b4770e35b3 doc_id: 4974 cord_uid: rxg7wvr7 Several systems are available for the expression of foreign gene sequences inEscherichia coli. We describe the use of prokaryotic expression products of viral gene fragments in order to identify the regions that specify the binding sites of antibodies. This approach is particulary successful if the antigenicity does not depend on the native protein, but only on the amino acid sequence, i.e., if the epitope is sequential. Combining prokaryotic expression with the use of synthetic peptides often permits a fast and accurate mapping of an epitope. The occurrence of immunodominant sequential epitopes on the surface of viruses seems to be a widespread phenomenon. As in most areas of biology and biochemistry, the study of viruses and their interactions with the immune system has been revolutionized by the recombinant-DNA technology. This review describes a new method for studying the antigenicity of viral proteins which involves the insertion of fragments from the gene coding for the protein into one of the many available expression vectors. By testing the antigenicity of the expression product, the protein segment involved in antibody binding can then be identified. After the initial studies of Rtither et al. [98] with lysozyme, this approach led to the localization of several B-cell and T-cell epitopes. Recombinant antigens are most suitable for identifying the same category of epitopes that are detected by the use of synthetic peptides, namely the so-called linear or sequential epitopes. Expression products of viral genes are also useful for serodiagnosis and for studying virus neutralization. The antigenicity of proteins has been a controversial issue for many years I-7, 10, 11, [129] [130] [131] . One source of confusion lies in the use of several terms that have overlapping meanings. Therefore, we will briefly define these terms as they are used in this review. An epitope is the part of the antigen that is involved in the binding of a particular antibody. This definition implies than an epitope is specified by the antibody; and that an antigen may have as many epitopes as there are different antibodies recognizing the antigen [130] . Further, it should be noted that an epitope also depends on the method of localization. For instance, the use of peptides cross-reacting with the antigen may delineate an epitope that corresponds to part of the antigen-antibody interface observed by X-ray crystallography E 131] . Not all residues within an epitope need to be essential for binding. A residue or a group of residues may be denoted as an antigenic determinant to indicate its measurable contribution to the binding of the antibody, rather than a passive presence in the epitope. Antigenic determinants can be defined by the analysis of escape mutants [84, 94, 137] or by the systematic replacement or deletion of residues [40, 73, 74] . Monoclonal antibodies that have identical, overlapping or adjacent epitopes will compete for binding to the antigen. We use the term antigenic site to denote the part of the antigen that is the target of a group of mutually competing antibodies. Although the whole surface of a protein may be potentially antigenic [10, 11] , the number of antigenic sites seems to be limited. Epitopes can be classified as sequential (or linear) or as conformational [ 106, 128] . Epitopes are sequential, if the antigenicity only depends on the primary structure of the protein antigen. Operationally, this is indicated if the antigenicity is retained after denaturation of the protein or if the epitopes can be mimicked by a peptide with the same sequence as the protein segment, but without any stable native-like conformation. Conversely, antibodies with conformational epitopes only bind to the antigen in its native conformation. The terms conformation-dependent or conformation-independent can be regarded as synonyms of conformational and sequential, respectively, 'conformation' referring to the native structure of the antigen. Presumably, any epitope will have a defined conformation when bound to the antibody. Conceivably, conformational epitopes depend on the same intramolecular interactions that stabilize the structure of the antigen, while the conformation of a sequential epitope is mainly stabilized by intermolecular interaction with the antibody. This agrees with the notion that sequential epitopes correspond to those parts of the surface that are relatively mobile [121, 129, 136] or even disordered [2] . An independent criterion distinguishes between continuous and discontinuous epitopes [7] . In most cases, conformational epitopes will be discontinuous, i.e., formed by residues that are not contiguous in the amino acid sequence but are Mapping of viral epitopes with prokaryotic expression products 3 brought together by the folding of the protein. However, continuous epitopes may also depend on the protein conformation [7] . Further, since replacement analysis [40, 73] has demonstrated that within an sequential epitope antigenic residues can be interspersed by non-antigenic ones, the distinction between continuous and discontinuous seems somewhat arbitrary [ 129] . Another reason for our preference of the original terms of Sela [106] is that a simple experiment-testing the effect of denaturation on antigenicity-~ecides on the classification. In order to be expressed as a functional gene in a prokaryotic cell, a coding sequence must be flanked by a number of signals [16, 65, 101] : a promotor, a transcription termination site, a ribosome binding site, an ATG start codon and a stop codon. Except for the start and stop codons, these signals are specific for the host cell. Normally, all these signals are provided by the expression vector. Three main factors determine the yield of an expression product: The strength of the promotor In most cases, a strong promotor, such as Ptac or ;~ PL, is preferred for a high level of gene expression. Further, to prevent continuous accumulation of the expression products leading to growth retardation or cell death, an inducible promotor is needed. These factors are the distance between the ribosome-binding site and the start codon, local mRNA secondary structure and other features of the sequence [104] . The problem of optimizing the initiation rate can be circumvented by fusing the foreign sequence to a well-expressed bacterial gene downstream of the bacterial start codon, leading to the synthesis of a hybrid protein. This is probably the most unpredictable and critical factor. Another advantage of fusing the sequence to a bacterial gene is that hybrid proteins are often relatively stable, particularly when the accumulated expression product precipitates inside the cell [65, 69, 115] . Many applications require posttranslational processing and correct folding of the polypeptide [12] . If eukaryotic processing such as phosphorylation or glycosylation is necessary for antigenic activity, prokaryotic expression systems are of no use at all. Intracellular precipitation competes with correct folding of the protein and necessitates the use of elaborate in vitro renaturation procedures [76] . P The sequence Ile-Glu-Gly-Arg at the C-terminus of the carrier protein allows specific cleavage by the blood coagulation protease Factor X q Excretion vector; signal sequence cleaved off after transport of product through inner membrane; outer membrane made permeable by the expression ofthe kil gene on the plasmid r Expression products of pGEX-2T and pGEX-3 X contain C-terminal of the carrier protein the recognition sequences of thrombin, and factor Xa, respectively, allowing specific cleavage of the hybrid protein Expression products reported to be both soluble and stable t A methionine C-terminal of the cheY protein allows specific cleavage of the hybrid protein by CNBr Table 1 . Indicated are the bacterial gene used to generate a hybrid protein (setting the compromise between solubility and degradation), the promotor and the unique restriction sites available for inserting the foreign sequence. The first expression system used to map viral antigenic determinants was the phage )~ Charon 16 [82] . This phage is comparable to )~ gt 11 [142] , one of the most popular expression vectors. In both phage systems, the foreign gene fragment is inserted in the EcoRI site near the 3' end of the lacZ gene. After adding the synthetic inducer isopropyl-[3-D-thiogalactopyranoside, the foreign sequence is expressed as part of a 13galactosidase hybrid protein. Direct immunoscreening allows the selection of recombinant plaques synthesizing an antigenic sequence. The main advantages of phage )~ systems are the high efficiency of transfection, the possibility of screening plaques at a high density and the availability of worked-out, reliable protocols. The main disadvantage of )~gt 11 is the availability of only an EcoRI site for insertion; this has been eliminated in a new variant )~ gt22 [47] . Like )~gt 11, several of the plasmid expression systems have been devised originally for the construction of cDNA expression libraries. Other systems have been constructed to investigate the products of open reading frames (pORF, pMR) or to produce native-like proteins. An advantage of plasmids is that the procedures for plating out, growing and DNA isolation are very simple. Furthermore, the new technique of electroporation allows efficiencies of transformation that are at least comparable to those of the packaging and transfection with )~ DNA. The most popular system for epitope mapping is the pEX system [116] . These plasmids contain the strong ~ P~ promotor, regulated by a temperaturesensitive repressor, and a polylinker region at the end of a cro-lacZ fusion gene, available in the three different readings frames. During the development of this vector [115] , it was found that insertions at the 3' end of the fusion gene gave more stable expression products than insertions at the 5' end. The pEX expression products are quite insoluble, ensuring that the product of virtually any foreign sequence is protected effectively against degradation. Further, lysis in SDS and transfer to nitrocellulose filters allow a direct immunoscreening of colonies. To increase the versatility of the pEX system, the plasmids pEX 11, pEX 12, and pEX 13 were constructed by incorporating a polylinker with 7 different sites [61 a]. Incorporation in the plasmid of the ci857 gene coding for the temperature-sensitive PR repressor yielded the pUEX plasmids, which can be propagated in normal host strains [13] . A prerequisite for epitope mapping via heterologous gene expression is the availability of recombinant DNA clones containing the relevant coding information. Different strategies have been used to generate subgenomic fragments Mapping of viral epitopes with prokaryotic expression products 9 which, after insertion in a expression vector, direct the synthesis of an antigenic expression product. Results can be obtained rapidly by using restriction enzymes, but the accuracy of localization in this case obviously depends on the presence of suitable cleavage sites. More accurate localization may be obtained by constructing a library of small DNase I fragments and selecting epitope-producing clones by immunoscreening of colonies [31, 25, 58, 66, 72, 82] . A third approach is the construction of a series of deletion clones with Bal31 [30, 100, 125] , exonuclease III [17, 52] or restriction enzymes [41, 132] . However, epitope delineation by progressive deletions from only one side may lead to erroneous interpretations. Since the antigenicity of the expression product is destroyed as soon as one essential antigenic determinant is deleted, it is this determinant that is mapped and not the complete epitope [17, 41, 125] . Finally, a delineation with a resolution of a single amino acid residue can be obtained by expressing synthetic oligonucleotides [20, 61] . Table 2 compiles the use of recombinant antigens for the mapping of viral epitopes. With only a few exceptions [17, 55, 80] fusion proteins were solubilized in buffers containing SDS and a reducing agent. Subsequently, the products are fractionated by gel electrophoresis, transferred to nitrocellulose and incubated with antibodies. However, this procedure is only suitable for antibodies that are capable of recognizing the viral protein after Western blotting [9, 17, 20, 31, 36, 55, 61, 66, 67, 111, 123] . Conversely, negative results have been reported with monoclonal antibodies that recognize denaturation-sensitive epitopes [ 17, 36, 41, 61, 66] . An interesting exception is the conformational site IV on the G2 protein of Rift Valley fever virus [55] , which could be localized within 20residues by immunoprecipitation of an expression product. To what extent is an expression product inside the E. coli cell or immobilized on a blotting membrane able to fold to a native-like structure? In most cases the antigenicity appears to depend only on a small subsequence that can be flanked by any bacterial or viral sequence. In such cases formation to a stable native conformation is not likely and the epitope is evidently sequential. Indeed, several epitopes could be delineated further by testing synthetic peptides [44, 52, 56, 58, 61, 67, 82, 88, 100, 132, 141] . So, we may consider a prokaryotic expression product as antigenically equivalent to denatured protein. This does not exclude a local native-like structure, but only in the afore-mentioned case [55] , this was substantiated by the negative effect of denaturation on antigenicity. a Species names denote polyclonal antisera b Numbers amino acid or codon numbers. In italics, sequences of residues that have been shown to contain antigenic determinants ° The reported epitopes boundary 608-625 is only based on an assumed epitope length of 6 and 9 residues d More accurate localizations by testing synthetic peptides e HBc, HBel, and HBe2 denote three antigenic sites on the core antigen HBcAg or on its antigenic variant HBeAg, both products of the C gene. HBc has been mapped with monoclonal antibodies, but is also the most immunodominant site recognized by human polyclonal antisera r Evidence cited that residues 2-77 contain essential determinants of site HBe2 g pBR322 derivative with a BglII cloning site near the 3' end of the trp gene h Localized more accurately by testing antibody binding of protein fragments or adenovirus-SV40 protein fragments i Exo III, exonuclease III k Affinity purification via adsorption to the expression products yielded two fractions that recognized the two different epitopes 1 Localized more accurately by PEPSCAN peptide analysis m Residue numbering according to [61] n Epitope sensitive to denaturation by boiling in SDS and dithiothreitol and localized by immunoprecipitation ° MAbs capable of blocking the interaction with the cell receptor CD4 P Same epitope localized with pepfides [43, 105] q Epitope localization confirmed by analysing escape mutants [84] r Similar to pEx30 and pEx31 As mentioned before, a sequential epitope may represent a component of the complete epitope as would be observed by X-ray crystallography. This is exemplified by two discontinuous epitopes of foot-and-mouth disease virus [87] . A similar situation may exist for site IV or D of transmissible gastroenteritis virus [88] . All epitopes discussed so far are the targets of the soluble immunoglobulins, which are relevant for the humoral immune response. The cellular response is mediated by the T-cell receptor of T-lymphocytes. According to the current consensus, T-cell antigens are processed inside the antigen-presenting cells. This generates antigen fragments, which are bound on the cell surface by class-I (for cytotoxic T-cells) or ctass-II (for helper T cells) major histocompatibility antigens [for a review, see 24] . As a consequence of this process, T-ceil epitopes are inherently sequential and can be mimicked by peptides [96, 97] or )~ gt 11 expression products [62] . In two recent reports, pEX expression products have been used to localize T-cell epitopes of a viral protein, the F protein of measles virus [27] or the E2 protein of Semliki Forest virus [114] . The suggestion that the antigenicity of bacterial expression products is almost exclusively limited to sequential epitopes implies that the same epitopes can also be mimicked by synthetic peptides. However, expression of gene fragments in E. coli should be considered as an approach complementary to the use of synthetic peptides, rather than as an alternative [61, 66] . Expression products can localize an antigenic sequence within 20 to 100 residues, depending on the available restriction sites and the mapping strategy. Within such a region, peptides can then be used for an exact localization. The combination of expression in pEX and PEPSCAN peptide synthesis has been applied successfully to measles virus [132] , different coronaviruses [61, 66, 88] and to a T-cell epitope of Mycobacteriurn tuberculosis [t26, 127] . A number of epitopes delineated by using expression products could not be detected by PEPSCAN analysis [61, 88] . This is most likely explained by the length of these epitopes: 11 and more than 17 residues for two epitopes of infectious bronchitis virus [61] and more than 21 residues for an epitope of feline infectious peritonitis virus [88] . Recently [87] , the use of combinations ofpeptides to delineate discontinuous epitopes of foot-and-mouth disease virus was reported. This method, if generally applicable, would be a useful alternative to the analysis of MAb-resistant or non-binding mutants, which in principle only gives information about antigenic determinants. A few reports [21, 34, 51, 70, 77, 123] describe the use of eukaryotic expression for epitope mapping. Three epitopes on the gD protein of herpes simplex virus I were sensitive to reduction and alkylation, but not to 0.1% SDS [21] , suggesting that these epitopes were partially conformational. However, other epitopes localized by eukaryotic expression could also be mimicked by prokaryotic products [77, 123] or peptides [28, 51] . It seems unlikely, therefore, that eukaryotic expression of gene fragments is a general method to localize conformational epitopes. Polyclonal antisera are likely to contain antibodies which, by their specificity for linear epitopes, recognize prokaryotic expression products. This then allows the use of such products for serodiagnosis. The sera of AIDS patients appeared to recognize the bacterial expression products of env [18, 19, 22, 50, 53, 139] , pol [83] , or gag [140] fragments from human immunodeficiency virus (HIV). In addition, differences between individual sera could be defined by expression of the HIF tat gene [3] or fragments of the env [139] Antisera raised against bacterial expression products that cross-react with the native antigen will have, like anti-peptide sera, a predetermined specificity. Studying the neutralization of viral infection by such antisera could be of relevance for vaccine development. Despite negative results with canine parvovirus [1123, infectious bursal disease virus [8] and bovine rotavirus [373, there are several reports about expression products that did induce in vitro neutralizing sera. Examples are the gp 120 sequence from HIV [90] , the VP7 sequence from simian rotavirus [6] , the VP7c sequence from bovine rotavirus [713, the major antigenic site on VP 1 from foot-and-mouth-disease virus [14, 15, 138] , the VP 1 regions 52-302 and 24-129 from poliovirus I [493, the VP2 sequence from infectious pancreatic necrosis virus [643, and the N-terminal gD sequence from herpex simplex virus I [56, 583. More spectacular is the induction of protective immunity. This was observed with a recombinant immunogen containing the core-antigen sequence of hepatitis B virus in one of two chimpanzees [783, with the G sequence from hematopoietic necrosis virus in fish [42] and with E2 sequences from Semtiki Forest virus in mice [46] . In the latter case, the sequences eliciting partial or complete protection were localized within residues 114-149 and 216-288, respectively. Remarkably, no in vitro neutralization was observed. The available information on the location of sequential epitopes allows a few generalization to be made. The distinction between antigenic sites, recognized by a group of mutually competing MAbs, and the epitopes of individual MAbs has now be substantiated [31, 61, 66, 88, 100] . The number of these sites found on a viral protein is usually limited. Often, one of the sites is immunodominant and is recognized by the majority of polyclonal antisera and/or monoclonal antibodies [43, 44, 61, 66, 84, 85, 94, 100, 105, 140] . All these sites appear to be sequential. Therefore, the preference of the immune system for certain sites may be explained by the location of regions that by their segmental mobility can conform to the paratopes of the antibody [136] . This does not exclude, however, the presence of conformationat sites on other parts of the accessible surface. So, the concept of an antigenic structure, specifying a limited number of antigenic sites [7] , can be reconciled partially with the notion that the whole surface of the protein is potentially antigenic [10, 11] . By their location, viral surface proteins are likely to be involved in molecular recognition processes and to interact with the host immune system. Conceivably, flexible regions on the surface are a typical feature of this category of proteins. Secretion of heterologous gene products to the culture medium of Escherichia coli The threedimensional structure of foot-and-mouth disease virus at 2.9 ~ resolution Synthesis of the complete trans-activation gene product of human T-lymphotropic virus type III in Escherichia coli: demonstration of immunogenicity in vivo and expression in vitro Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli Atg vectors' for regulated high-level expression of clones genes in Escherichia coli Synthesis of the outer-capsid glycoprotein of the simian rotavirus SA11 in Escherichia coti Antigenic structures of proteins Expression in Escherichia coli of cDNA fragments encoding the gene for the host-protective antigen Of infectious bursal disease virus Characterization and epitope mapping of a human monoclonal antibody reactive with the envelope glycoprotein of human immunodeficiency virus The antigenic structure of proteins: a reappraisal Intrinsic and extrinsic factors in protein antigenic structure Mapping of viral epitopes with prokaryotic expression products 17 ) pUEX, a bacterial expression vector related to pEX with universal host specificity Synthesis of fusion proteins containing antigenic determinants of foot-and-mouth disease virus Synthesis of fusion proteins with multiple copies of an antigenic determinant of foot-and-mouth disease virus Gene cloning, an introduction Expression of measles virus nucleoprotein in Escherichia coli: use of deletion mutants to locate the antigenic sites Serodiagnosis of antibodies to the human AIDS retrovirus with a bacterially synthesized env polypeptide Subregions of a conserved part of the HIV gp41 transmembrane wotein are differentially recognized by antibodies of infected individuals Location of a neutralizing epitope for the haemaggtutinin-neuraminidase glycoprotein of Newcastle disease virus Localization of discontinuous epitopes of herpes simplex virus glycoproteins D: use ofa nondenaturing ("native" gel) system of polyacrylamide gel electrophoresis coupled with Western blotting HLTV-III env gene products synthesized in E. coli are recognized by antibodies present in the sera of AIDS patients Versatile expression vectors for high-level synthesis of cloned gene products in Escherichia coli T-cell antigen receptor genes and T-cell recognition A survey of vectors for regulating expression of cloned DNA in E. coli Expression of the PDGF-related transforming protein of simian sarcoma virus in E. coli Measles virus-specific murine T cell clones: characterization of fine specificity and function Fine structure analysis of type-specific and type-common antigenic sites of herpes simplex virus glycoprotein D Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose-binding protein Analysis of HPV-1 E4 expression using epitope-defined antibodies Epitope mapping of the human immunodeficiency virus type 1 gp 120 with monoclonal antibodies Expression and secretion of foreign proteins in Escherichia coli A plasmid expression vector that permits stabilization of both mRNAs and proteins encoded by the cloned genes Localization of epitopes of herpes simplex virus type 1 glycoprotein D Use of a bacterial expression vector to map the Varicella-Zoster virus major glycoprotein gene, gC Location of antigenic sites of the S-glycoprotein of transmissible gastroenteritis virus and their conservation in coronaviruses Expression of bovine rotavirus neutralization antigen in Escherichia coli High-_level expression in Escherichia coli of a chemically synthesized gene for Rapid purification of a cloned gene product by genetic fusion and site-specific proteolysis Strategies for epitope mapping using peptide synthesis Mapping of antigenic domains of Sendai virus nucleocapsid protein expressed in Escheriehia coli Expression in Escherichia coli of an epitope of the glycoprotein of infectious hematopoietic necrosis virus protects against viral challenge Fine mapping ofan immunodominant domain in the transmembrane glycoprotein of human immunodeficiency virus Human immunodeficiency virus type 1 neutralization epitope with conserved architecture elicits early type-specific antibodies in experimentally infected chimpanzees Searching for clones with open reading frames Semliki Forest virus E2 envelope epitopes induce a nonneutralizing humoral response which protects mice against lethal challenge Lambda gt22, an improved lambda vector for the directional cloning of full-length cDNA pXmnATG: an E. coli vector for expression of unfused proteins Mapping of viral epitopes with prokaryotic expression products 19 Regions of poliovirus protein VP 1 produced in Escherichia coli induce neutralizing antibodies Comparison of Western blot (immunoblot) based on recombinant-derived p41 with conventional tests for serodiagnosis of human immunodeficiency virus infections Mapping of functional and antigenic domains of the ~4 protein of herpes simplex virus 1 Human antibodies react with an epitope of the human papillomavirus type 6b L 1 open reading frame which is distinct from the type-common epitope Human retroviral env and gag polypeptides: serologic assays to measure infection Construction of an excretion vector and extracellular production of human growth hormone from Escherichia eoli Use of bacterial expression cloning to define the amino acid sequences of antigenic determinants on the G2 glycoprotein of Rift Valley fever virus Immunological properties of an N-terminal fragment of herpes simplex virus type 1 glycoprotein D expressed in Escherichia coli Purification of fusion proteins expressed by pEX3 and a truncated pEX 3 derivative Recombinant fusion proteins of herpes simplex virus type 1 glycoprotein D. Academic thesis Immunoenzymatic detection of expressed gene fragments cloned in the lac Z gene of E. coli Conserved immunogenic region of a major core protein (p24) of human and simian immunodeficiency viruses Analysis of an immunodominant region of infectious bronchitits virus Improvement of the cloning linker of the bacterial expression vector pEX Mapping of T cell epitopes using recombinant antigens and synthetic peptides High-level expression in Escherichia coli of the carboxy-terminal sequences of the avian myelocytomatosis virus (MC29) vmyc protein Expression in Escherichia coli of the major outer capsid protein of infectious pancreatic necrosis virus Cloning and expression of viral antigens in Escherichia coli and other microorganisms Antigenicity of the peplomer protein of infectious bronchitis virus Amino acid sequence of a conserved neutralizing epitope of murine coronaviruses An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose-binding protein Purification of eukaryotic polypeptides synthesized in Escherichia coti Characterization of a human immunodeficiency virus neutralizing monoclonal antibody and mapping of the neutralizing epitope Expression of a major bovine rotavirus neutralization antigen (VP7c) in Escherichia coti Efficient mapping of protein antigenic determinants Antigenicity and immunogenicity of synthetic peptides of foot-and-mouth disease virus Specificity and function of the individual amino acids of an important determinant of human immunodeficiency virus type I that induces neutralizing activity Expression plasmid containing the ~PL promotor and ci857 repressor Protein folding intermediates and inclusion body formation Use of simian virus 40 large T-[3-galactosidase fusion proteins in an immunochemical analysis of simian virus 40 large T antigen Protective immunization against hepatitis B with an internal antigen of the virus Synthesis and sequence-specific proteolysis of hybrid proteins produced in Escherichia coli Analysis of functional domains on reovirus cell attachment protein sigma 1 using cloned S 1 gene deletion mutants Immobilization and purification of enzymes with staphylococcal protein A gene fusion vectors Method to map antigenic determinants recognized by monoclonal antibodies: localization of a determinant of virus neutralization on the feline leukemia envelope protein gp70 Recombinant polypeptides from the human immunodeficiency virus reverse transcriptase define three epitopes recognized by antibodies in sera from patients with acquired immunodeficiency syndrome Threedimensional structure of poliovirus serotype 1 neutralizing determinants A conserved region at the COOH terminus of human immunodeficiency virus gp 120 envelope protein contains an immunodominant epitope Production of oncogene-specific proteins and human T-cell leukemia (lymphotropic) retrovirus (HLTV-I) envelope protein in bacteria and its potential for use in human cancers and seroepidemiological surveys Neutralizing epitopes of type 0 foot-and-mouth disease virus. II. Mapping three oonformational sites with synthetic peptide reagents Linear neutralizing epitopes on the peplomer protein of coronaviruses HTLV-III/LAV-neutralizing antibodies to an E. col#produced fragment of the virus envelope New plasmid vectors for high level expression of eukaryotic fusion proteins in Escherichia coli Expression of an early Epstein-Barr virus antigen (EA-D) in E. coli The use of pKC30 and its derivatives for controlled expression of genes Structure of a human common cold virus and functional relationship to other picornaviruses The type-specific epitopes of the Epstein-Barr virus nuclear antigen 2 are near the carboxy terminus of the protein Peptides and the cellular immune response A sequence pattern common to T cell epitopes Exon cloning: immunoenzymatic identification of exons of the chicken lysozyme gene Easy identification of cDNA clones Antigenic determinants and functional domains in core antigen and e antigen from hepatitis B virus Molecular cloning. A laboratory manual High-level bacterial expression and purification of human T-lymphotropic virus type I (HTLV-1) transmembrane env protein Construction of a plasmid for expression or foreign epitopes as fusion with subunit B of Escherichia coli heat-labile enterotoxin Expression of eukaryotic genes in Escherichia coli with a synthetic two-cistron system B-and T-lymphocyte responses to an immunodominant epitope of human immunodeficiency virus Antigenicity: some molecular aspects High-level expression vectors to synthesize unfused proteins in Escherichia coll Expression, identification, and characterization of recombinant gene products in Escherichia coli A versatile phage lambda expression vector system for cloning in Escherichia coll A plasmid vector for cloning and expression of gene segments: expression of an HTLV-I envelope gene segment A neutralizing epitope on human rhinovirus type 2 includes amino acid residues between 153 and 164 of virus capsid protein VP2 Expression of canine parvovirus-13-galactosidase fusion proteins in Escherichia coti Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase Identification of a DTH inducing T-cell epitope on the E2 membrane protein of Semliki Forest virus Solubilization and immune-detection of 13-galactosidase hybrid proteins carrying foreign antigenic determinants Construction of a new family of high efficiency bacterial expression vectors: identification of cDNA clones coding for human liver proteins Topological mapping of complement C9 by recombinant DNA techniques suggests a novel mechanism for its insertion into target membranes High level expression of genes clones in kgt 11 Characterization of foot-andmouth disease virus gene products with antisera against bacterially synthesized fusion proteins A broad-host-range vector system for cloning and translational lacZ fusion analysis The atomic mobility component of protein antigenicity Identification of two epitopes in the carboxyterminal 15 amino acids of the E 1 glycoprotein of mouse hepatitis virus A59 by using hybrid proteins An immunodominant domain in adenovirus type 2 early region 1 A proteins Identification of a neutralizing epitope on glycoprotein gp58 of human cytomegalovirus Localization of a poliovirus type 1 neutralization epitope in viral capsid polypeptide VP 1 Efficient mapping and characterization of a T cell epitope by the simultaneous synthesis of multiple peptides Cloning of the mycobacterial epitope recognized by T lymphocytes in adjuvant arthritis Structure of viral antigens Antigenic cross-reactivity between proteins and peptides: new insights and applications Operational aspects ofepitope identification: structural features of proteins recognized by antibodies Structural and functional approaches to the study of protein antigenicity Epitope mapping of the fusion protein of measles virus An antigenic analysis of the adenovirus type 2 fibre polypeptide Open reading frame expression vectors: a general method for antigen production in Escherichia coil using protein fusions to [3-galactosidase Use of open reading frame expression vectors Correlation between segmental mobility and the location of antigenic determinants in proteins Structural identification of the antibodybinding sites of Hong Kong influenza haemagglutinin and their involvement in antigenic variation Bacterially expressed antigenic peptide from foot-and-mouth disease virus capsid elicits variable immunologic responses in animals Characterization of immunoreactive epitopes of the HIV-1 p41 envelope protein using fusion proteins synthesized in Escherichia coli Use of trpE/gag fusion proteins to characterize immunoreactive domains on the human immunodeficiency virus type 1 core protein A poliovirus type I neutralization epitope is located within amino acid residues 93-104 of viral capsid polypeptide VP1 Mapping of viral epitopes Efficient isolation of genes by using antibody probes Authors' address: Dr. J. A. Lenstra, Institute of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Rijksuniversiteit te Utrecht, P.O. Box 80165, NL-3508 TD Utrecht, The Netherlands.