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H M Geysen

Publications and source records attributed to H M Geysen.

At least 19 recordsLinked to original sources
PubMed1992

Altering the antigenicity of proteins.

To better understand the binding interaction between antigen and antibody we need to distinguish protein residues critical to the binding energy and mechanism from residues merely localized in the interface. By analyzing the binding of monoclonal antibodies to recombinant wild-type and mutant myohemerythrin (MHr) proteins, we were able to test the role of individual critical residues at the highly antigenic site MHr-(79-84), within the context of the folded protein. The results directly show the existence of antigenically critical residues, whose mutations significantly reduce antibody binding to the folded protein, thus verifying peptide-based assignments of these critical residues and demonstrating the ability of buried side chains to influence antigenicity. Taken together, these results (i) distinguish the antigenic surface from the solvent-exposed protein surface before binding, (ii) support a two-stage interaction mechanism allowing inducible changes in protein antigens by antibody binding, and (iii) show that protein antigenicity can be significantly reduced by alteration of single critical residues without destroying biological activity.

Amino Acid Sequence↗
PubMed1992

Evidence for immune selection of hepatitis C virus (HCV) putative envelope glycoprotein variants: potential role in chronic HCV infections.

E2/nonstructural protein 1, the putative envelope glycoprotein (gp72) of HCV, possesses an N-terminal hypervariable (E2 HV) domain from amino acids 384 to 414 of unknown significance. The high degree of amino acid sequence variation in the E2 HV domain appears to be comparable to that observed in the human immunodeficiency virus type 1 gp120 V3 domain. This observation and the observation that the HCV E2 HV domain lacks conserved secondary structure imply that, like the V3 loop of human immunodeficiency virus 1 gp120, the N-terminal E2 region may encode protective epitopes that are subject to immune selection. Antibody-epitope binding studies revealed five isolate-specific linear epitopes located in the E2 HV region. These results suggest that the E2 HV domain is a target for the human immune response and that, in addition to the three major groups of HCV, defined by nucleotide and amino acid sequence identity among HCV isolates, E2 HV-specific subgroups also exist. Analysis of the partial or complete E2 sequences of two individuals indicated that E2 HV variants can either coexist simultaneously in a single individual or that a particular variant may predominate during different episodes of disease. In the latter situation, we found one individual who developed antibodies to a subregion of the E2 HV domain (amino acids 396-407) specific to a variant that was predominant during one major episode of hepatitis but who lacked detectable antibodies to the corresponding region of a second variant that was predominant during a later episode of disease. The data suggest that the variability in the E2 HV domain may result from immune selection. The findings of this report could impact vaccine strategies and drug therapy programs designed to control and eliminate HCV.

Amino Acid Sequence↗
PubMed1992

Simultaneous multiple synthesis of peptide-carrier conjugates.

Recently, the multipin approach for simultaneous multiple peptide synthesis was applied to the analysis of T cell determinants by using a novel cleavage method (Maeji et al., 1990). A diketopiperazine forming linker allowed cleavage of peptides into aqueous buffer which, without further purification, could be used immediately in cell culture assays. Another potential application of the technique is the simultaneous cleavage and coupling of peptides to immunogenic carriers. Without further purification the resulting conjugates can be used for the production of antipeptide antisera. The choice of carrier and conjugation chemistry is not restricted as peptide/pin cleavage occurs in aqueous solution over a range of pH and ionic strength. The method was assessed using the 2,4-dinitrophenyl group as a model hapten, diphtheria toxoid as the carrier, and N-(epsilon-maleimidocaproyloxy)succinimide as the cross-linking reagent. The resulting DNP-DT conjugate was used to prepare high titered specific anti-DNP antisera in mice.

2,4-Dinitrophenol↗
PubMed1992

The specificity of recognition of a cytotoxic T lymphocyte epitope.

An Epstein-Barr virus (EBV)-specific CD8+ cytotoxic T lymphocyte (CTL) clone (LC13) was shown to recognize the minimal peptide determinant FLRGRAYGL from the EBNA 3 antigen of the BL74 strain of EBV. The equivalent epitope from the B95-8 strain (FLRGRAYGI) is not recognized when endogenously presented and the peptide is 15-fold less active than FLRGRAYGL. A replacement set of peptides was synthesized in which each residue within FLRGRAYGL was sequentially replaced with all other genetically coded amino acids. These peptides were tested for their ability to sensitize target cells to lysis by LC13. Of the 171 single-amino acid replacement peptides only 15 were more active than the peptide FLRGRAYGI. Five peptides had significantly greater activity than FLRGRAYGL and a peptide incorporating the most active of these single-amino acid substitutions (HIRGRAYSL) induced lysis at concentrations approximately 30-fold less than FLRGRAYGL. Simplified theoretical calculations based on this study suggest that CTL LC13 has a specificity for its target epitope of 1 in 4.7 x 10(10). This represents the first complete analysis of the role of single amino acids within a minimum epitope on the specificity of CTL recognition.

Amino Acid Sequence↗
PubMed1992

Subjugation of dominant immunogenic determinants within a chimeric peptide.

The mechanism of immunodominance was investigated using chimeric peptides from mouse myelin basic protein consisting of the immunodominant I-Au-restricted Ac1-11, attached by a peptide bond to I-Eu-restricted 35-47. Our results indicate that this chimeric peptide and certain of its derivatives were excellent immunogens both in vitro and in vivo. Notably, on immunization with Ac1-11:35-47 or Ac1-11 (Ala4):35-47, the proliferative T cell responses to each of its component peptides were almost completely "subjugated" in favor of neo-determinants that are I-Eu restricted. Furthermore, each of 11 hybridomas derived after immunization with Ac1-11:35-47 had specificity for junctional neo-determinants and none could be stimulated to produce interleukin-2 from Ac1-11 or 35-47. Subjugation of the immunogenicity of the original determinants occurred regardless of their dominance when separate. It did not appear to result from non-availability of the original determinants because the chimeric peptide was able to induce neonatal tolerance to each of its constituents. These results indicate that in an overlapping multideterminant array, the dominant determinant is unpredictable from historical data about any of the components. Determinant choice, at any stage of processing, may be governed by competitive aspects of determinant capture in an environment where all components--antigen, major histocompatibility complex and T cell receptor--are available.

Amino Acid Sequence↗
PubMed1991

Role of single amino acids in the recognition of a T cell epitope.

T cell epitopes can be defined by the use of synthetic peptides, which when added to APC efficiently mimic naturally processed Ag. Free peptide is thought to bind to cell-surface MHC glycoproteins and the TCR then recognizes the resulting complex. The specificity of a tetanus toxin-specific human Th cell clone was investigated using a complete replacement set of peptides in which every amino acid within the minimal T cell epitope was replaced by each of the 19 alternative genetically coded amino acids. Within the minimal epitope, found to be YSYFPSVI (tetanus toxin 593-600), a small number of substitutions could be made without significant loss of activity, defined as substitutions giving peptides whose activity fell within +/- 3 SD of the mean parent response. Y593 could be substituted with F, W, M, L, V, and I; S594 with G and T; Y595, F596, and P597 with no other amino acids; S598 with A; V599 with S, and I600 with L. Rank ordering of the substitutions allowed a precise description to be made of MHC and/or TCR interaction with each amino acid side chain within the epitope. Simplified theoretic calculations based on this study indicate that class II T cell recognition has a specificity greater than 1 in 10(8). Competition experiments indicate that Y595, F596, P597, and I600 are critical for binding of this epitope to its restricting element, HLA DR4Dw14.

Amino Acid Sequence↗
PubMed1991

Systematic fractionation of serum antibodies using multiple antigen homologous peptides as affinity ligands.

The fractionation of polyclonal antibodies on multiple peptide ligands is described. The method is an application of a procedure for the synthesis of large numbers of peptides on individual polyethylene pins (Geysen et al., 1987). In this application, each pin-bound peptide is used as an affinity support. Antibodies bound to the peptides are then eluted, using buffers of either high or low pH. Each eluted antibody is then tested for specific binding to peptides or proteins, using ELISA procedures. A rabbit antiserum raised to gonococcal pilin was fractionated on a complete set of octapeptides homologous with the sequence of the pilin protein. Antibodies eluted from some of the peptides bound to pilin in solution. In a second example three hyperimmune sera raised to three different potyviruses were fractionated on their respective homologous peptide sequences. Testing the eluted antibodies on the three virus coat proteins revealed peptides which bound cross-reacting antibodies. Thus the method can be used to confirm direct peptide binding evidence for sequential epitopes. These peptides can then be used in affinity chromatography to increase the specificity of polyclonal sera. This can be achieved either by elution of the specific antibody from the peptide or by removal of cross-reacting antibodies from the whole serum by absorption on peptide.

Amino Acid Sequence↗
PubMed1991

T cell determinant structure: cores and determinant envelopes in three mouse major histocompatibility complex haplotypes.

T lymphocytes recognize discrete regions on an antigen. The specificity of the T cell responses in three mouse strains of differing major histocompatibility complex (MHC) haplotype to a protein antigen, lysozyme, was analyzed using a series of peptides that walk the antigen in single amino acid steps. These peptide series were synthesized using the pin synthesis system, which was modified to allow the peptides to be cleaved from the pins into a physiological buffer free of toxic compounds. This methodology overcomes many of the problems associated with the production of peptides for screening proteins for antigenic determinants. The T cell determinants for the three strains were markedly different. This result points out the limitations of algorithms predicting determinants without reference to the MHC, and the importance of the empirical methodology. This analysis of the T cell response to lysozyme constitutes the most complete study of reactivity to a foreign protein to date and illustrates many important features of antigen recognition by T cells, e.g., presence of major and minor determinant regions. The outer boundaries of each immunogenic region, the determinant envelope, are difficult to define from recently immunized lymph nodes because of the heterogeneity in T cell recognition. However, core sequences common to all the immunogenic peptides in a continuous sequence can be easily defined.

Amino Acid Sequence↗
PubMed1991

Binding of peptides to proteins: an exercise in molecular design.

Peptides coupled to solid supports were systematically tested for binding activity with a polyreactive immunoglobulin light chain dimer by the methods of Geysen and colleagues. Once identified, peptides of progressively increasing affinity for the dimer were synthesized in milligram quantities and diffused into crystals of the protein. The three-dimensional structures of the peptide-protein complexes were determined by X-ray analysis and crystallographic refinement. Criteria for the design of ligands to fill the binding cavity in incremental stages could be formulated from the combined results of peptide scanning and crystallographic analyses. Histidine proved to be an important substituent in the binding series. It was possible to manipulate the properties of this amino acid residue to alter the structures and binding patterns of the ligands. For example, if two beta-alanine residues were added to the carboxyl end of a tetrapeptide ligand, the terminal carboxyl group formed an intramolecular ion pair with the imidazolium group (N-3) of histidine. This interaction was accompanied by cleavage of the intra molecular hydrogen bond between N-1 of histidine and the amide group of a glutamine side chain. The shape of the ligand shifted from a compact to an extended form and the mode of binding changed from a lock-and-key to an induced-fit type. The direction of entry of dipeptides of histidine and proline into the binding cavity (normally amino end first) could be reversed (carboxyl end first) by protonation of the histidine ring.

Amino Acid Sequence↗
PubMed1991

Framework peptides from kappa IIIb rheumatoid factor light chains with binding activity for aggregated IgG.

Most monoclonal human rheumatoid factors (RF) and some RF from rheumatoid patient's synovia are restricted in their light chains, using predominantly the kappa IIIb subfamily. Very few sequence differences are found between these light chains. Light chains with similar variable region framework sequences are also found in some mouse monoclonal RF derived from mice stimulated with lipopolysaccharide or secondarily immunized with protein antigens. There are two likely explanations for this restriction in framework sequences between the two species: (a) the sequences are important for the immunoregulation of RF production or (b) the sequences are concerned with the antibody binding specificity of the RF. We have examined overlapping octapeptides from the kappa IIIb light chain variable region and show that some framework peptides have the ability to bind aggregated IgG. Replacement of amino acids within the peak binding peptide have indicated the critical amino acids necessary for binding.

Amino Acid Sequence↗
PubMed1991

Subtype-specificity of antipeptide antibodies raised against unique sequences of human interferons-alpha.

A strategy for the production of human interferon-alpha (IFN-alpha) subtype-specific antibodies, based on immunizing rabbits with short unique synthetic peptides coupled to protein carriers, has been validated. These peptides correspond to amino acid residues 99-111 of IFN-alpha 1, 50-57 and 103-116 of IFN-alpha 2, and 37-50 of IFN-alpha 4. The antipeptide antibodies [anti-IFN alpha 1(99-111), anti-IFN alpha 2(50-57C), anti-IFN alpha 2(103-116) and anti-IFN alpha 4(C37-50)] were tested by ELISA and Western blotting for their reactivity with immunoaffinity-purified recombinant human IFN-alpha 1, -alpha 2b and -alpha 4a. The anti-IFN alpha 1(99-111) and anti-IFN alpha 2(50-57C) reacted with their corresponding IFN-alpha and did not crossreact with the other IFN subtypes. The anti-IFN alpha 2(103-116) reacted with IFN-alpha 2b and also crossreacted slightly with the other subtypes. The anti-IFN alpha 4(C37-50) reacted well with IFN-alpha 4a, crossreacted with significantly lower affinity with IFN-alpha 1 and did not bind IFN-alpha 2b. Residues 104-107 and 108-111 are the major components of the epitopes recognized by anti-IFN alpha 1(99-111) and anti-IFN alpha 2(103-116), respectively, as determined by ELISA against overlapping octapeptides.

Amino Acid Sequence↗
PubMed1991

Universal antibodies to human interferon-alpha subtypes--the production of antipeptide antibodies to conserved regions of interferon-alpha.

Antibodies to three conserved regions of all human interferon (IFN)-alpha 1 subtypes were raised by immunizing rabbits with short synthetic peptides coupled to a carrier. These peptides correspond to amino acid residues 29-36, 31-36, 126-131, 139-151, and 142-151 of the consensus sequence of IFN-alpha 1. The antibodies were tested for reactivity with IFN-alpha 1, -alpha 2a, -alpha 2b, -alpha 4a, and -alpha 14 and IFN-beta. Most antipeptide antibodies react only weakly with IFN-alpha subtypes; only the antibodies raised against the peptide corresponding to residues 142-151 conjugated to keyhole limpet hemocyanin using glutaraldehyde react appreciably with all IFN-alpha subtypes tested. These antipeptide antibodies are potentially universal antibodies to the human IFN-alpha 1 subtypes, since they exhibit similar binding affinity to all the IFN-alpha subtypes tested and do not react with IFN-beta.

Amino Acid Sequence↗
PubMed1991

Extension of a minimal T cell determinant allows relaxation of the requirement for particular residues within the determinant.

The determinant recognized by a class II restricted helper T cell clone raised against a peptide corresponding to the C-terminal 24 residues of the heavy chain of influenza virus hemagglutinin (HA) was examined in detail. The sequence 309VKQNTLKL316 was identified as the minimal determinant for T cell activation but its stimulatory capacity was augmented by extension at either end. Sets of peptide analogs, in which each residue within the minimal determinant was replaced in turn by every one of the other naturally occurring amino acids, revealed either an absolute requirement for the native residue or a very limited degree of replaceability, at seven of the eight positions. Only the N-terminal residue 309V could be replaced with almost any other amino acid without loss of reactivity; in fact, substitution at this position with residues containing bulky side groups enhanced the response. The reactivity of the clone with analogs of the longer peptide 307KYVKQNTLKL316, which induces maximal levels of stimulation, revealed a very different pattern of replaceability for certain residues; in particular, the requirement for a lysine at position 310 was no longer apparent. This study presents a complete analysis of the importance of each individual residue to the integrity of a T cell determinant and provides evidence that the critical requirement for a particular amino acid at a given location may be overridden by N-terminal extension of the minimal determinant. These findings indicate that, within different homologs of the native sequence, particular residues may assume quite different roles.

Amino Acid Sequence↗
PubMed1991

Fine specificities of monoclonal antibodies against the Plasmodium falciparum circumsporozoite protein: recognition of both repetitive and non-repetitive regions.

The fine specificities of 6 monoclonal antibodies (MoAbs) raised against the circumsporozoite (CS) protein of the human malaria parasite, Plasmodium falciparum, were defined by their binding to a series of overlapping octapeptides corresponding to the 7G8 variant of the CS protein. The precise specificities of the MoAbs to the immunodominant NANP repeat region were elucidated by their binding to all possible 4, 5, 6, 7 and 8 amino acid peptides in this region. All 6 MoAbs recognized the NANP repeats. In addition all MoAb bound to nonrepetitive sites with 4 of the 6 MoAbs recognizing known functional sites outside the repeat region including sites required for T cell recognition and hepatocyte invasion. Antibody pressure may therefore be responsible for generating the epitope variation observed at T cell sites. The multiple specificities for all the MoAbs suggests that the repeat region may act as an internal immunological 'smokescreen' by competing more effectively for antibody binding compared to single epitope copy functional sites located outside the repeat region.

Amino Acid Sequence↗
PubMed1990

Multi-pin peptide synthesis strategy for T cell determinant analysis.

Techniques to synthesize many peptides simultaneously exist, however their individual cleavage and subsequent purification constitutes a bottleneck to total throughput. Biological screening of peptides is generally carried out at physiological pH in aqueous solutions. However, peptides, unless individually purified are usually contaminated by residual compounds used in their preparation such as trifluoroacetic acid, organic solvents, scavengers etc. In testing with cellular systems, such as T cell determinant analysis, such contaminations must be rigorously excluded. We have extended the pin synthesis technique of synthesizing and screening large number of peptides (Geysen et al., 1984) to the analysis of T cell determinants. Peptides can be synthesized on polyethylene pins, the side chain protective groups removed and the peptides washed free of contaminants. A linker system stable under these conditions can then be triggered to cleave the peptides from the pins in an aqueous solution at neutral pH. This strategy enables the rapid mapping of T cell determinants. It is also applicable to other systems where large numbers of solution phase peptides are required, for example, in the study of hormone analogues.

Amino Acid Sequence↗
PubMed1990

Identification of B-epitopes in the human papillomavirus 18 E7 open reading frame protein.

A panel of murine mAb raised against a MS2 replicase/HPV 18 E7 fusion protein included 23 reactive by ELISA with HPV 18 E7 determinants. A total of 19 of the 23 recognized linear epitopes in the N-terminal region of the E7 molecule, while the other four were deduced by binding inhibition assays to recognize conformational determinants in this region. All tested antibodies precipitated a 14-kDa peptide doublet that corresponded with the predicted size of the E7 protein, from HeLa cells, but not from HPV 16 E7 containing CaSki cells. HPV 18 E7 protein was detected by immunolabeling with electron microscopy in both the nucleus and the cytoplasm of HeLa cells with the greater proportion occurring in the cytoplasm. No antibody reacted specifically by indirect immunofluorescence with HeLa cells. Weak cross-reactivity of some mAb with the E6 MS2-replicase fusion protein of HPV 16 was detected by ELISA, but no protein of the appropriate size was immunoprecipitated from CaSki cells. It is concluded that the B cell epitopes on the HPV 18 E7 transforming protein are located in the N-terminal region of the molecule and that some are weakly cross-reactive with HPV 16 E6 protein. E7 protein is either present in HeLa cells at a concentration too low to be detected by indirect immunofluorescence, or the N-terminal epitopes are masked by protein conformation or interaction with cellular or other viral components.

Amino Acid Sequence↗