Helical peptide arrays for lead identification and interaction site mapping

Libraries composed of linear and cyclic peptides cannot fully represent the higher order structures of most antigenic sites. To map the binding site of ligands or antibodies, a larger part of the three-dimensional space should be sampled. Because parallel synthesis of large arrays of peptides on hydrogels is restricted to relatively small peptides, a simple and robust homodimeric helical system was chosen for antigen presentation. First, it was established in an heterodimeric system that the 26-mer peptide could be synthesized and that the helical coiled-coil peptides interact in the hydrogel in a predictable manner. Next, libraries of homodimeric coiled coils were synthesized into which the epitope was grafted. Using dedicated helical dimeric and trimeric coiled-coil libraries, the epitopes of two anti-HIV-1 gp41 monoclonal antibodies known to interact with helical structures were mapped at high resolution. These mappings precisely reflect existing X-ray data, and the arrays can be applied to lead identification, epitope mapping, and systematic analysis of amino acid contribution to coiled-coil systems.     

Identification of peptide mimotopes for the fluorescein hapten binding of monoclonal antibody B13‐DE1

Using 6mer and 12mer phage peptide libraries three unique phage clones were identified which specifically bind to a monoclonal anti‐FITC antibody, B13‐DE1. The two 6mer and one 12mer peptide insert sequences are clearly related to each other and contain a high proportion of hydrophobic amino acids. The peptides are bound by the antibody combining site of B13‐DE1 probably in a similar manner to FITC and represent therefore true peptidic mimics of the fluorescein hapten. No reactivity of the peptides could be demonstrated with another monoclonal anti‐fluorescein antibody or with polyclonal anti‐fluorescein antibodies. Immunization of mice with the peptides resulted in the production of antibodies cross‐reacting with all peptides but not with fluorescein. The results show that phage peptide libraries can be used to isolate mimotope peptides which can mimic low molecular weight structures seen by a specific antibody and probably other recognition molecules. Copyright © 1999 John Wiley & Sons, Ltd.     

An oriented peptide array library (OPAL) strategy to study protein-protein interactions

One of the major questions in signal transduction is how the specificities of protein-protein interactions determine the assembly of distinct signaling complexes in response to stimuli. Several peptide library methods have been developed and widely used to study protein-protein interactions. These approaches primarily rely on peptide or DNA sequencing to identify the peptide or consensus motif for binding and may prove too costly or difficult to accommodate high throughput applications. We report here an oriented peptide array library (OPAL) approach that should facilitate high throughput proteomic analysis of protein-protein interactions. OPAL integrates the principles of both the oriented peptide libraries and array technologies. Hundreds of pools of oriented peptide libraries are synthesized as amino acid scan arrays. We demonstrate that these arrays can be used to map the specificities of a variety of interactions, including antibodies, protein domains such Src homology 2 domains, and protein kinases.     

Analysis on the molecular species and concentration of circulating ADAMTS13 in Blood

ADAMTS13 is the metalloprotease responsible for the proteolytic degradation of von Willebrand factor (VWF). A severe deficiency of this VWF-cleaving protease activity causes thrombotic thrombocytopenic purpura. This protease, comprising 1,427 amino acid residues, is composed of multiple domains, i.e., a preproregion, a metalloprotease domain, a disintegrin-like domain, a thrombospondin type-1 motif (Tsp1), a cysteine-rich domain, a spacer domain, seven Tsp1 repeats, and two CUB domains. We prepared one polyclonal and seven monoclonal antibodies recognizing distinct epitopes spanning the entire ADAMTS13 molecule. Of these antibodies, two of the monoclonal ones, which recognize the disintegrin-like and cysteine-rich/spacer domains, respectively, abolished the hydrolytic activity of ADAMTS13 toward both a synthetic substrate, FRETS-VWF73, and the natural substrate, VWF. In addition, these antibodies blocked the binding of ADAMTS13 to VWF. These results revealed that the region between the disintegrin-like and cysteine-rich/spacer domains interacts with VWF. Employing these established polyclonal and monoclonal antibodies, we examined the molecular species of ADAMTS13 circulating in the blood by immunoprecipitation followed by Western blot analysis, and estimated the plasma concentration of ADAMTS13 by enzyme-linked immunosorbent assay. These studies indicated that the major fraction of ADAMTS13 in blood plasma consisted of the full-length form. The concentration of ADAMTS13 in normal plasma was approximately 0.5-1 microg/ml.     

Purification of polyclonal anti-conformational antibodies for use in affinity selection from random peptide phage display libraries: A study using the hydatid vaccine EG95

The use of polyclonal antibodies to screen random peptide phage display libraries often results in the recognition of a large number of peptides that mimic linear epitopes on various proteins. There appears to be a bias in the use of this technology toward the selection of peptides that mimic linear epitopes. In many circumstances the correct folding of a protein immunogen is required for conferring protection. The use of random peptide phage display libraries to identify peptide mimics of conformational epitopes in these cases requires a strategy for overcoming this bias. Conformational epitopes on the hydatid vaccine EG95 have been shown to result in protective immunity in sheep, whereas linear epitopes are not protective. In this paper we describe a strategy that results in the purification of polyclonal antibodies directed against conformational epitopes while eliminating antibodies directed against linear epitopes. These affinity purified antibodies were then used to select a peptide from a random peptide phage display library that has the capacity to mimic conformational epitopes on EG95. This peptide was subsequently used to affinity purify monospecific antibodies against EG95.     

Selection of Peptide Mimics of HIV-1 Epitope Recognized by Neutralizing Antibody VRC01

The ability to induce anti-HIV-1 antibodies that can neutralize a broad spectrum of viral isolates from different subtypes seems to be a key requirement for development of an effective HIV-1 vaccine. The epitopes recognized by the most potent broadly neutralizing antibodies that have been characterized are largely discontinuous. Mimetics of such conformational epitopes could be potentially used as components of a synthetic immunogen that can elicit neutralizing antibodies. Here we used phage display technology to identify peptide motifs that mimic the epitope recognized by monoclonal antibody VRC01, which is able to neutralize up to 91% of circulating primary isolates. Three rounds of biopanning were performed against 2 different phage peptide libraries for this purpose. The binding specificity of selected phage clones to monoclonal antibody VRC01 was estimated using dot blot analysis. The putative peptide mimics exposed on the surface of selected phages were analyzed for conformational and linear homology to the surface of HIV-1 gp120 fragment using computational analysis. Corresponding peptides were synthesized and checked for their ability to interfere with neutralization activity of VRC01 in a competitive inhibition assay. One of the most common peptides selected from 12-mer phage library was found to partially mimic a CD4-binding loop fragment, whereas none of the circular C7C-mer peptides was able to mimic any HIV-1 domains. However, peptides identified from both the 12-mer and C7C-mer peptide libraries showed rescue of HIV-1 infectivity in the competitive inhibition assay. The identification of epitope mimics may lead to novel immunogens capable of inducing broadly reactive neutralizing antibodies.     

Two epitopes and one agretope map to a single HLA-A2 peptide recognized by H-2-restricted T cells

Mice immunized with syngeneic cells transfected with cloned genes coding for HLA class I molecules could recognize the human MHC Ag in the context of their own H-2 molecules. We obtained CTL clones from DBA/2 mice (H-2d) which had been immunized with P815 cells (a mastocytoma of DBA/2 origin) expressing either HLA-A2 or HLA-A3 or two different molecules containing recombined sequences of HLA-A2 and HLA-A3. Fourteen of these clones recognized a synthetic peptide corresponding to the region 170-185 of HLA-A2 in the context of H-2Kd. Moreover, from their activity on P815 cells expressing HLA-Cw3, two subpatterns could be distinguished: subpattern Cw3+, defined by those clones which lysed P815-Cw3, and subpattern Cw3- defined by those clones which did not lyse P815-Cw3. By testing the activity of clones of each subpattern on a series of modified synthetic peptides, we were able to define two epitopes on the same 170-185 peptide of HLA-A2. One of them was dependent on amino acids at positions 173 and 177, whereas the other was dependent on amino acid 177 alone. By using competition experiments, we were also able to define an agretopic region strongly dependent on the amino acid at position 178. Furthermore, experiments with L cells expressing molecules containing recombined sequences between H-2Kd and H-2Dd demonstrated the determinant role of residues 152, 155, and 156 from H-2Kd in the presentation to murine T cells of the 170-185 peptide of HLA-A2.     

Antigen fingerprinting of polyclonal antibodies raised in immunized chickens with tick total proteins: a reservoir for the discovery of novel antigens

Identification of tick-protective antigens remains the limiting step in vaccine development. The authors have generated several B cell epitope candidates by fingerprinting Rhipicephalus (Boophilus) microplus proteins that were characterized through bioselection of random peptide phage display libraries against polyclonal antibodies antitick proteins. From 280 clones selected and sequenced, 107 distinct reactive clones were validated by dot-blot assays. Eight consensus motifs were generated, and the most frequent ones were PXXKXH, NXXKXXL, and HTS (68.2%, 65%, and 42%, respectively). The consensus sequences identified potential vaccine targets by alignment with the protein database of R. microplus, which may have putative roles in the host response. Sequences that did not align with known proteins but shared extensive homology among each other were classified as conformational epitopes. Sequence alignments also recognized multiple targets, and the most predominant proteins were identified. Finally, immunized mice sera recognized tick proteins, demonstrating that functional epitope profiles can be identified through selection of phage-displayed peptide libraries with hyperimmune sera and revealing that the epitope-displaying phages can be used as potential vaccine immunogens.     

Primary sequence mapping of human apolipoprotein B-100 epitopes. Comparisons of trypsin accessibility and immunoreactivity and implication for apoB conformation

Differential trypsin-accessibility and monoclonal antibodies (Mabs) to human apolipoprotein (apo) B-100 are both important tools for probing apoB structure and conformation on low-density lipoproteins (LDL). In this study, we have mapped greater than 80% of the C-terminal region (720 residues) of LDL apoB-100 using trypsin digestion. Our results extend our previous data [Yang et al. (1986) Nature (Lond.) 323, 738-742] confirming that the C-terminal region of about 420 residues of apoB-100 is largely inaccessible to trypsin, whereas the part just preceding this region has interspersed trypsin-accessible and inaccessible peptides. We have determined the amino acid sequence of specific apoB-100 peptides containing epitopes recognized by four separate Mabs: two epitopes have been mapped to within 20 residues, one has been mapped to 36 residues, and the last to 80 residues. We used polyclonal antisera to identify 16 overlapping clones of varying lengths of apoB-100 cDNAs extending from the C-terminus of apoB-100 cloned in the expression vector, lambda gt11. These clones were then tested against individual Mabs. By nucleotide sequence analysis of overlapping clones that show differential reactivities to different Mabs, we have mapped the individual epitopes of each Mab to within about 50-150 amino acid residues predicted from the DNA sequences. Confirmation and further fine mapping were accomplished by competition for LDL binding using partially purified fusion proteins and chemically synthesized oligopeptides. Two epitopes (Mabs 7 and 22) were mapped to the C-terminal 20 amino acids of apoB-100, one (Mab 16) to residues 4154-4189, and another (Mab 20) to residues 3926-4005. Mab 16 precipitates more than 80% of LDL particles. Mab 20 precipitates only denatured apoB but not native LDL apoB [Milne et al. (1987) Mol. Immunol. 24, 435]. Mabs 7 and 22 are unique in that they precipitate LDL apoB modified by storage much better than freshly isolated LDL-apoB. Although epitope expression and trypsin-accessibility represent two useful probes for the study of protein conformation, there was no obvious correlation between these two parameters when applied to LDL apoB for the antibodies we have examined.     

A review of the utility of soluble peptide combinatorial libraries

This is paper reviews the preparation and use of soluble synthetic combinatorial libraries (SCLs) made up of millions of peptide and nonpeptide sequences for the identification of highly active individual compounds. First presented in 1991. SCLs have been prepared in a number of different lengths and formats, and are composed entirely of L -, D -, and unnatural amino acids. Also, existing peptide libraries have been chemically transformed to yield large diversities of nonpeptidic compounds. This review encompasses the published work from this laboratory using SCLs for the identification of antigenic sequences recognized by monoclonal antibodies, novel peptide agonists and antagonists to opioid receptors, new trypsin inhibitors, novel antibacterials, and compounds that inhibit melittin's hemolytic activity. SCLs offer a fundamental, practical advance in the study of interactions between peptide and nonpeptide sequences and their biochemical or pharmacological targets. © 1994 John Wiley & Sons, Inc.     

Analysis of the chicken fast myosin heavy chain family. Localization of isoform-specific antibody epitopes and regions of divergence.

cDNAs encoding the rod region of four different fast myosin heavy chains (MYCHs) in the chicken were identified, using anti-MYCH monoclonal antibodies, in two expression libraries prepared from 19-day embryonic and adult chicken muscle. These clones were used to determine the amino acid sequences that encompass the epitopes of five anti-MYHC monoclonal antibodies. Additionally, the amino acid sequences were compared to each other and to a full length embryonic MYHC. Although there is extensive homology in the chicken fast myosin rods, sequences within the hinge, within the central portion of the light meromyosin fragment, and at the carboxy terminus exhibit the largest number of amino acid substitutions. We propose that divergence within these subdomains may contribute to isoform-specific properties associated with skeletal myosin rods.     

Immunogenicity. I. Use of peptide libraries to identify epitopes that activate clonotypic CD4+ T cells and induce T cell responses to native peptide ligands

Recent studies have demonstrated the utility of synthetic combinatorial libraries for the rapid identification of peptide ligands that stimulate clonotypic populations of T cells. Here we screen a decapeptide combinatorial library arranged in a positional scanning format with two different clonotypic populations of CD4+ T cells to identify peptide epitopes that stimulate proliferative responses by these T cells in vitro. An extensive collection of mimic peptide sequences was synthesized and used to explore the fine specificity of TCR/peptide/MHC interactions. We also demonstrate that many of these deduced ligands are not only effective immunogens in vivo, but are capable of inducing T cell responses to the original native ligands used to generate the clones. These results have significant implications for considerations of T cell specificity and the design of peptide vaccines for infectious disease and cancer using clinically relevant T cell clones of unknown specificity.     

HIV p24-specific helper T cell clones from immunized primates recognize highly conserved regions of HIV-1.

We have investigated Th cell recognition of the HIV core protein p24 by using CD4+ T cell clones derived from cynomolgus macaques immunized with hybrid HIV p24: Ty virus-like particles (VLP). T cell lines from two immunized animals responded to p24: Ty-VLP, control Ty-VLP, purified p24, and whole inactivated HIV, indicating the presence of T cells specific for p24 as well as the Ty carrier protein. The HIV determinants recognized by the T cell lines were identified by using a series of overlapping peptides synthesized according to the sequence of p24. Both T cell lines recognized peptide 11 (amino acids 235-249) and peptide 14 (amino acids 265-279). In addition, one T cell line also responded to peptide 9 (amino acids 215-229). Definitive identification of two T cell epitopes on p24 was confirmed at the clonal level: from a total of four T cell clones generated from one of the T cell lines, two respond specifically to peptide 11 and two to peptide 14. The T cell clones were CD4+ and MHC class II-restricted and secreted IL-2 in response to stimulation with purified p24, inactivated HIV or a single synthetic peptide. The specificity of the Th clones for variant peptides demonstrated cross-reactivity with two simian immunodeficiency virus isolates, but only limited responses to HIV-2 sequences. However, the Th cell epitopes identified on p24 are highly conserved between 12 HIV-1 isolates and were recognized by both of the immunized primates. These sequences may therefore be useful for priming a broadly reactive immune response to HIV-1.     

Identification of phage-displayed peptides which bind to the human HnRNPA1 protein-specific monoclonal antibody.

We have screened a peptide phage display library to examine if monoclonal antibody-binding phages could be isolated from the library and thereby predict the antigenic epitopes of the antibodies from the isolated phages. The library was screened for high-avidity binding to monoclonal antibodies by an affinity purification technique called biopanning. Among the monoclonal antibodies examined, the human hnRNPA1 protein-specific monoclonal antibody 9H10 showed selective binding of phages. After two rounds of the biopanning, twelve clones of high-avidity-binding phages were chosen and their inserts were sequenced. Nucleotide sequence comparison of the 12 clones showed that there were 5 different species, with two species containing four members, implying that they were predominantly selected by the biopanning. The amino acid sequences of the inserts of the 12 clones were compared with that of the human hnRNPA1 protein in order to find the putative epitope of the human hnRNPA1 protein for 9H10. The C-terminal region of the human hnRNPA1 protein shows significant homology with the peptide sequences of the selected phage clones. These results show that this peptide phage display library can be useful in defining the epitope of some monoclonal antibodies.     

Phage presentation

There has recently been great interest in the use of the filamentous bacteriophage fd as a vehicle for the display of peptides and proteins. Phage libraries displaying random peptides up to 38 amino acids in length can be used (i) to select for ligands able to bind specific target molecules; (ii) to mimic non-proteinaceous ligands; and (iii) as a tool to map epitopes recognized by antibodies. The display of proteins or their functional domains provides a system for the analysis of structure-function relationships, and the potential to generate proteins with altered binding characteristics or novel catalytic properties. The display of short immunogenic determinants on fusion phage may provide a basis for the development of novel peptide vaccines, whilst the expression of libraries of antibody fragments may provide a method to by-pass hybridoma technology in the generation of monoclonal antibodies.     

How antigenic are antigenic peptides?

Most of a protein surface is potentially antigenic, consisting of numerous overlapping domains each complementary to antibody-combining sites. These domains may include peptide sequences that are demonstrably antigenic but only when antibodies from the appropriate host individuals and species are used. Methods for locating antigenic peptide sequences are described in which hydrophilic polyamide supports are used for peptide synthesis, then solid-phase radioimmunoassay with antisera and protein A. Most antigenic domains, however, comprise amino acid side chains contributed by two or more nearby polypeptide chains. Such domains can be identified by comparing the cross-reactivities of groups of very closely related proteins towards monoclonal antibodies raised to one of them. Such studies, using myoglobins, have identified a number of residues not previously shown to be antigenic and have provided a guide for the choice of synthetic peptides which are likely to carry several immunodominant side chains. One such peptide corresponding to residues (72–89) of beef myoglobin has been shown, using CD and antibodies to the parent protein, to have interesting conformational and antigenic properties. The peptide (25–55) is also antigenic.     

Identification of amino acids involved in recognition by dengue virus NS3-specific, HLA-DR15-restricted cytotoxic CD4+ T-cell clones.

The majority of T-cell clones derived from a donor who experienced dengue illness following receipt of a live experimental dengue virus type 3 (DEN3) vaccine cross-reacted with all four serotypes of dengue virus, but some were serotype specific or only partially cross-reactive. The nonstructural protein, NS3, was immuno-dominant in the CD4+ T-cell response of this donor. The epitopes of four NS3-specific T-cell clones were analyzed. JK15 and JK13 recognized only DEN3 NS3, while JK44 recognized DEN1, DEN2, and DEN3 NS3 and JK5 recognized DEN1, DEN3, and West Nile virus NS3. The epitopes recognized by these clones on the DEN3 NS3 protein were localized with recombinant vaccinia viruses expressing truncated regions of the NS3 gene, and then the minimal recognition sequence was mapped with synthetic peptides. Amino acids critical for T-cell recognition were assessed by using peptides with amino acid substitutions. One of the serotype-specific clones (JK13) and the subcomplex- and flavivirus-cross-reactive clone (JK5) recognized the same core epitope, WITDFVGKTVW. The amino acid at the sixth position of this epitope is critical for recognition by both clones. Sequence analysis of the T-cell receptors of these two clones showed that they utilize different VP chains. The core epitopes for the four HLA-DR15-restricted CD4+ CTL clones studied do not contain motifs similar to those proposed by previous studies on endogenous peptides eluted from HLA-DR15 molecules. However, the majority of these dengue virus NS3 core epitopes have a positive amino acid (K or R) at position 8 or 9. Our results indicate that a single epitope can induce T cells with different virus specificities despite the restriction of these T cells by the same HLA-DR15 allele. This finding suggests a previously unappreciated level of complexity for interactions between human T-cell receptors and viral epitopes with very similar sequences on infected cells.     

Human apolipoprotein B: identification of cDNA clones and characterization of mRNA.

Apolipoprotein B (apoB) is a major protein component of low density and very low density lipoproteins. Because of its large size and heterogeneity, molecular studies of apoB have been difficult, and its structure and regulation remain poorly understood. We now report the identification of human apoB cDNA clones by antibody screening of hepatoma libraries in the expression vector lambda gt11. Both oligo(dT) primed and random primed libraries were constructed and screened with polyclonal antibodies to intact apoB, as well as with antibodies raised against a synthetic peptide based on the limited amino acid sequence available for apoB. The identity of the clones was unambiguously established by comparisons of the cloned cDNA sequences with apoB amino acid sequences. The clones hybridize to an exceptionally large 20 kb mRNA that is present in liver and intestine but not other tissues examined, consistent with the distribution expected from protein biosynthetic studies. The properties of the mRNA have implications for the biogenesis of the multiple apoB molecular weight forms secreted by liver and intestine.