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S J Rodda

Publications and source records attributed to S J Rodda.

At least 19 recordsLinked to original sources
PubMed1994

Scanning for T helper epitopes with human PBMC using pools of short synthetic peptides.

Major T helper epitopes of medically important antigens can be located by measuring the proliferative responses of human peripheral blood mononuclear cells (PBMC) to pools of short synthetic peptides. The length and endings of the peptides used were shown to be critical for success in identifying Th cell epitopes. Many epitopes would be missed if either long (31mers) or short (less than 12mers) peptides were used. Pools of 14 and 16mers were more efficient than 12mers spanning the same region, however, for a promiscuous Th cell epitope of tetanus toxin (tt 947-967), two of three donors tested did not respond to 18mers or shorter peptides spanning this region. Although peptides with either unblocked or blocked ends were stimulatory, peptides with blocked ends were generally more efficient. The peptide concentration and number of available APC were also found affect the efficiency of the proliferation assay as a measure of peptide recognition by Th cells. Two screenings of the entire set of tetanus toxin peptide pools using different samples of PBMC from the same donor identified common major stimulatory regions. Thus, PBMC and peptide pools can be used for the reproducible identification of Th cell epitopes. After immunization with tetanus toxoid (TT), peptide-responsive cells increased in frequency in parallel to the increase in TT responsive cells, indicating that the peptide-responsive cells were primed by TT.

Amino Acid Sequence↗
PubMed1993

Mapping the major human T helper epitopes of tetanus toxin. The emerging picture.

Progress on the mapping of Th epitopes of tetanus toxin (tt) has been slow due to reliance on studies of clones. In this paper, human Th cell epitopes of tt were mapped using proliferation tests on PBMC in response to synthetic peptides. PBMC from nine donors were tested over the entire set of tt homologous overlapping dodecapeptides. The 1304 peptides were initially tested as 66 pools, each containing an average of 20 peptides. PBMC from individual donors responded to as few as 1 and as many as 17 of the 66 peptide pools. The sequences responsible for proliferation were identified for the two most frequently recognized pools, and for another two pools within a major immunodominant region. Three new epitope sequences were mapped in detail and based on their recognition by most individuals are likely to be promiscuous. A cocktail of peptides including the newly identified Th cell epitopes was able to induce proliferation in PBMC from 24 of 31 tetanus toxoid (TT)-responsive donors. This cocktail is a chemically defined reagent that can be used to quantitate in vitro Ag-specific Th cells in PBMC from most subjects, and may thus be useful for serial measurements of specific immunity such as in subjects undergoing immunotherapy or immunosuppressive treatment.

Amino Acid Sequence↗
PubMed1993

Multipin peptide synthesis at the micromole scale using 2-hydroxyethyl methacrylate grafted polyethylene supports.

The multipin peptide synthesis procedure has been adapted to allow the synthesis of peptides at micromole loadings. The original solid pin support was replaced with a detachable crown-shaped polyethylene support with an increased surface area. In addition, the polyethylene crowns were radiation-grafted with 2-hydroxyethyl methacrylate monomer instead of acrylic acid to yield hydroxy functionalized supports with a larger concentration of polymer and hence a larger peptide capacity. Fmoc-beta-Alanine was directly esterified to the HEMA hydroxy groups with subsequent addition of a diketopiperazine-forming handle for peptide attachment. Peptides varying in length from 10 to 25 residues were assembled at a number of loadings from 1.0 to 2.2 mumol. Purity of peptides at all loadings was equal to, and in some instances superior to, that achieved on conventional solid-phase supports.

Amino Acid Sequence↗
PubMed1993

Epitope mapping of the Sta58 major outer membrane protein of Rickettsia tsutsugamushi.

Binding studies of 160 overlapping, synthetic octapeptides from the hydrophilic regions of the Sta58 major outer membrane protein of Rickettsia tsutsugamushi with sera from patients with scrub typhus revealed 15 immunodominant peptides which are recognized by all the sera tested. Further analysis of the specificity of peptide binding with five of these peptides indicated that the peptides showed significantly stronger binding to scrub typhus patients' sera than they did to sera from patients with other febrile illnesses common in the region, i.e., malaria, dengue fever, typhoid fever, and leptospirosis. The main antibody class binding to these peptides appears to be immunoglobulin M, and there appears to be little correlation between reactivity with peptides and antibody titers measured by the indirect immunoperoxidase test.

Amino Acid Sequence↗
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↗
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↗
PubMed1988

Cognitive features of continuous antigenic determinants.

We sought to identify the features controlling the specificity of antibody recognition and thus gain insights into molecular recognition between proteins in general. A total of 103 epitopes within 63 well-defined antigenic peptides homologous with the relevant antigen sequence were identified. The contribution of each amino acid residue to the antibody binding activity of each epitope was investigated by ELISA testing of complete sets of peptide analogs containing single amino acid replacements. The data are summarized in a replaceability matrix. Some of the high frequency replaceabilities were expected, such as aspartate for glutamate, serine for threonine, etc., but unexpected relationships were also found, such as a high degree of acceptability of methionine as a replacement. Replaceability with a residue of opposite charge was rare. Glycine and tyrosine were frequently of low acceptability, except for glycine as a replacement for alanine. It was found that on average only about four to five amino acid residues in epitopes were required to determine specificity and provide binding energy. Specificity and binding energy were attributed to amino acid side chains rather than main chain atoms. Propensity factors for occurrence of amino acids in antigenic determinants were calculated. The prominence of certain hydrophobic residues as residues critical to recognition by antibody suggests that the molecular surface of an antigen in its combined form with antibody is altered from that occurring in the absence of antibody. Thus, antigenicity is not a static surface phenomenon but depends on the ability of the antigen to undergo rearrangement, supporting the induced fit concept.

Amino Acids↗
PubMed1987

Strategies for epitope analysis using peptide synthesis.

A recently developed approach to the synthesis and ELISA screening of large numbers of peptides is described. The method has created the opportunity to tackle questions about the sites and specificity of antigenic determinants which were formerly thought to be too difficult to answer. The various strategies for application of this method are described along with examples of their successful use. They include a procedure for locating all the continuous antigenic peptides of a protein antigen, and the identification of non-replaceable amino acid residues within an antigenic peptide. An approach to the determination of amino acid residues involved in the epitope for any monoclonal antibody is also described. These strategies open up the prospect of rapid mapping of the antigenic properties of hitherto poorly understood antigens.

Antibody Affinity↗
PubMed1987

Chemistry of antibody binding to a protein.

The chemistry of antibody recognition was studied by mapping the antigenicity of the protein myohemerythrin with peptide homologs of the protein sequence. The results suggest that the entire protein surface is antigenic, but the probability of there being antibodies to a given site is influenced by local stereochemistry. Although accessible to an antibody binding domain, the least reactive positions cluster in the most tightly packed and least mobile regions and are closely associated with narrow, concave grooves in the molecular surface containing bound water molecules. The most frequently recognized sites form three-dimensional superassemblies characterized by high local mobility, convex surface shape, and often by negative electrostatic potential.

Antibodies↗
PubMed1987

Mechanisms of antibody binding to a protein.

The mechanisms of antibody binding to a protein were studied by an analysis of specific amino acid residues critical to nine antigenic sites on myohemerythrin. Rabbit antisera to the whole protein were assayed for binding to more than 1500 distinct peptide analogs differing from the protein sequence by single amino acid replacements. The results, combined with information from the three-dimensional crystallographic structure, were used to evaluate probable mechanisms of antibody binding at individual sites. The data from all sites examined indicate that initial binding to solvent-exposed amino acid residues may promote local side-chain displacements and thereby allow the participation of other, previously buried, residues.

Animals↗
PubMed1986

The delineation of peptides able to mimic assembled epitopes.

Present methods allow a detailed study of the immune system's recognition of sequential epitopes. The results so far suggest that peptides homologous with these epitopes may not fulfil the early promise of synthetic vaccines. A procedure is described which now allows the study and evaluation of assembled epitopes. Using a monoclonal antibody which had been shown both to strongly neutralize foot-and-mouth disease virus, and to bind to a discontinuous epitope, peptides mimicking this epitope were determined a priori. An iterative procedure based on the progressive identification of amino acids in a random mixture of antibody-binding octapeptides was used. Strongly binding peptides consisted of the two elements W-Q-M (Trp-Gln-Met) and H-S (His-Ser) separated by a spacer. It was also shown that element W-Q-M was best composed of D-isomers, and the element H-S of L-isomers. Comparison of the sequence of these peptides with that of the immunologically important coat protein of the virus leads to the prediction that the epitope recognized by this monoclonal antibody consists of the residues occurring at positions 29-30, 54-55 and 88. Application of this general approach will answer questions about the nature of discontinuous epitopes and the stereochemical requirements for antigen-antibody interactions, as well as defining useful peptide immunogens.

Amino Acid Sequence↗
PubMed1986

A priori delineation of a peptide which mimics a discontinuous antigenic determinant.

A technique was developed for identifying peptides with high affinity for a given antibody. By testing a monoclonal antibody directed against a discontinuous antigenic determinant on foot-and-mouth disease virus, peptides mimicking the determinant were identified even though the tertiary structure of the proteins comprising the virus capsid is unknown. The allowable variations in spacing and stereochemistry of the peptides shown to mimic this epitope suggest protein folding in which amino acid residues from three regions, distant from one another in the primary sequence, are brought into close proximity at this epitope. The technique has potential for identification of peptides which will bind with high affinity to receptors other than antibody molecules.

Amino Acids↗
PubMed1986

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

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

Animals↗