Biomedical subjects

R Houghten

Publications and source records attributed to R Houghten.

At least 19 recordsLinked to original sources

PubMed2001

Enhanced immune activity of cytotoxic T-lymphocyte epitope analogs derived from positional scanning synthetic combinatorial libraries.

The pp65(495-503) cytotoxic T-lymphocyte (CTL) epitope from cytomegalovirus (CMV) is universally recognized among CMV+ individuals who express an allele of the human leukocyte antigen A (HLA-A*0201). The relative binding affinity of the epitope to HLA-A*0201 is moderate, and its increased activity might prove beneficial in its use as a CTL epitope vaccine. A new approach to enhance the activity of T-cell epitopes is the use of positional scanning synthetic combinatorial libraries (PS-SCLs). Using a nonamer PS-SCL, the pp65(495-503) epitope was modified after screening a CMV-specific T-cell clone (TCC) (3-3F4) from which the native peptide sequence was derived. Two peptides with amino acid substitutions at P1, P3, P7, and P8 are between 10(3) and 10(4) more active than the native epitope. Although the native CTL epitope terminates as a free acid, both tetrasubstituted peptides only function as CTL epitopes when the carboxyl terminus is amidated. Selective substitution of the native sequence based on PS-SCL screening results identified 3 amidated monosubstituted and disubstituted peptides that are better recognized than the native epitope by TCCs from a cohort expressing HLA-A*0201. In vitro stimulation of peripheral blood mononuclear cells with each of the peptide epitope analogs stimulated memory CTLs, which recognized CMV-infected targets among a high percentage of CMV+ individuals. Binding studies of peptide analogs with HLA-Ig (immunoglobulin) dimers and 2 different TCCs correlated with in vitro lysis results. These data suggest that increasing the activity of CTL epitopes while maintaining broad recognition is possible, which holds promise for vaccine development in infectious disease and cancer.

Antigens, Viral↗

PubMed2000

Contribution of individual amino acids within MHC molecule or antigenic peptide to TCR ligand potency.

The TCR recognition of peptides bound to MHC class II molecules is highly flexible in some T cells. Although progress has been made in understanding the interactions within the trimolecular complex, to what extent the individual components and their amino acid composition contribute to ligand recognition by individual T cells is not completely understood. We investigated how single amino acid residues influence Ag recognition of T cells by combining several experimental approaches. We defined TCR motifs for CD4+ T cells using peptide synthetic combinatorial libraries in the positional scanning format (PS-SCL) and single amino acid-modified peptide analogues. The similarity of the TCR motifs defined by both methods and the identification of stimulatory antigenic peptides by the PS-SCL approach argue for a contribution of each amino acid residue to the overall potency of the antigenic peptide ligand. In some instances, however, motifs are formed by adjacent amino acids, and their combined influence is superimposed on the overall contribution of each amino acid within the peptide epitope. In contrast to the flexibility of the TCR to interact with different peptides, recognition was very sensitive toward modifications of the MHC-restriction element. Exchanges of just one amino acid of the MHC molecule drastically reduced the number of peptides recognized. The results indicate that a specific MHC molecule not only selects certain peptides, but also is crucial for setting an affinity threshold for TCR recognition, which determines the flexibility in peptide recognition for a given TCR.

Amino Acids↗

PubMed2000

Minimal peptide length requirements for CD4(+) T cell clones--implications for molecular mimicry and T cell survival.

CD4(+) T lymphocytes usually recognize peptides of 12-16 amino acids in the context of HLA class II molecules. We have recently used synthetic peptide combinatorial libraries to dissect in detail antigen recognition by autoreactive CD4(+) T cell clones (TCC). The results of these studies demonstrated that antigen recognition by T cells is highly degenerate and that many cross-reactive ligands can be defined, some of which much more potent than the selecting autoantigen. Based on these observations, we examined the response of a myelin basic protein-specific HLA class II-restricted CD4(+) TCC to truncation variants of optimal ligands. Surprisingly, pentapeptides, tetrapeptides and even tripeptides derived from different segments of the optimal ligands were recognized by the TCC, and some were even more potent than the selecting autoantigen. In addition, these peptides enhanced the survival of the TCC at low concentration. The relevance of this finding was supported by the generation of pentapeptide-specific CD4(+) TCC from peripheral blood lymphocytes. These observations not only change existing views on the length requirements for activation of CD4(+) HLA class II-restricted T cells, but also extend our knowledge about the flexibility of TCR recognition and the potential for cross-reactivity in the immune system.

Antigen Presentation↗

PubMed1999

Identification of candidate T-cell epitopes and molecular mimics in chronic Lyme disease.

Elucidating the cellular immune response to infectious agents is a prerequisite for understanding disease pathogenesis and designing effective vaccines. In the identification of microbial T-cell epitopes, the availability of purified or recombinant bacterial proteins has been a chief limiting factor. In chronic infectious diseases such as Lyme disease, immune-mediated damage may add to the effects of direct infection by means of molecular mimicry to tissue autoantigens. Here, we describe a new method to effectively identify both microbial epitopes and candidate autoantigens. The approach combines data acquisition by positional scanning peptide combinatorial libraries and biometric data analysis by generation of scoring matrices. In a patient with chronic neuroborreliosis, we show that this strategy leads to the identification of potentially relevant T-cell targets derived from both Borrelia burgdorferi and the host. We also found that the antigen specificity of a single T-cell clone can be degenerate and yet the clone can preferentially recognize different peptides derived from the same organism, thus demonstrating that flexibility in T-cell recognition does not preclude specificity. This approach has potential applications in the identification of ligands in infectious diseases, tumors and autoimmune diseases.

Adult↗

PubMed1998

Predictable TCR antigen recognition based on peptide scans leads to the identification of agonist ligands with no sequence homology.

The potential of CD4+ T cells for cross-recognition of self and foreign Ags has important implications for the understanding of thymic selection, lymphocyte survival, and the occurrence of autoimmune diseases. Here, we define the extensive flexibility of Ag recognition for three human CD4+ autoreactive T cell clones (TCC) by using ligands with single and multiple amino acid (aa) substitutions. Our results demonstrate that the spectrum of tolerated ligands and the resulting stimulatory potency of peptides for a TCC can be predicted by the relative influence of each aa. Using this approach, we have identified stimulatory ligands not sharing a single aa in corresponding positions with the Ag used to establish the TCC. These results argue for an independent contribution of each aa in the peptide sequence to the affinity of the MHC/peptide complex to the TCR.

Amino Acid Sequence↗

PubMed1998

Probing degeneracy in T-cell recognition using peptide combinatorial libraries.

Recent studies have demonstrated flexibility of the T-cell receptor (TCR) with respect to recognition of peptide bound to self major histocompatibility complex (MHC). With the introduction of peptide combinatorial libraries, it has become possible to dissect the extent of degeneracy in T-cell recognition. On the basis of these novel findings, Bernhard Hemmer and colleagues propose a conceptual framework for lymphocyte selection and survival and the occurrence of autoimmunity.

Humans↗

PubMed1998

The use of soluble synthetic peptide combinatorial libraries to determine antigen recognition of T cells.

T cells identify by their T-cell receptor (TCR) short peptides in the context of major histocompatibility complex (MHC) molecules. The interaction of the trimolecular complex composed of the TCR and MHC bound peptide was extensively studied using substitution analogs of the original peptide ligands to define those residues important for T-cell recognition in the peptide chain. This approach has led to the observation that T-cell recognition is highly flexible and that many different peptides can be recognized by an individual TCR. Others and we have recently introduced synthetic peptide combinatorial libraries (SCL) to investigate T-cell recognition. Here we review the SCL-based approaches and describe our current techniques for mapping TCR motifs for CD4+ T cells. The implications of our findings for the understanding of T-cell recognition, as well as for future applications to study T-cell responses in infectious diseases, autoimmune disorders and cancer are discussed.

Amino Acid Sequence↗

PubMed1998

A linear hexapeptide somatostatin antagonist blocks somatostatin activity in vitro and influences growth hormone release in rats.

Somatostatin (SRIF) is the main inhibitory peptide regulating growth hormone (GH) secretion. It has been difficult to establish the role of endogenous SRIF release in the absence of pure SRIF antagonists. Although several SRIF antagonists have recently been described, none have been shown to possess in vivo activity in the absence of added SRIF. Here, an SRIF antagonist with no detectable agonist activity has been identified from a synthetic combinatorial hexapeptide library containing 6.4 x 10(7) unique peptides. Each peptide in the library is amino-terminally acetylated and carboxyl-terminally amidated and consists entirely of D-amino acids. A SRIF-responsive yeast growth assay was used as a primary screening tool, and cAMP accumulation, competitive binding, and microphysiometry also were used to confirm and further characterize SRIF antagonist activity. The hexapeptide library was screened in stepwise iterative fashion to identify AC-178,335, a pure SRIF antagonist of the sequence Ac-hfirwf-NH2. This D-hexapeptide bound SRIF receptor type 2 with an affinity constant (Ki) of 172 +/- 12 nM, blocked SRIF inhibition of adenylate cyclase in vitro (IC50 = 5.1 +/- 1.4 microM), and induced GH release when given alone (50 micrograms intravenously) to anesthetized rats with or without pretreatment with a long-acting SRIF agonist.

Animals↗

PubMed1995

HLA-A2-restricted cytotoxic T lymphocyte responses to multiple Plasmodium falciparum sporozoite surface protein 2 epitopes in sporozoite-immunized volunteers.

CD8+ CTL specific for the Plasmodium yoelii sporozoite surface protein 2 (PySSP2) protect mice against malaria. For this reason, vaccines designed to induce CTL against P. falciparum SSP2 (PfSSP2) are under development. Optimal development of PfSSP2 as a component of human malaria vaccines requires characterization of HLA class I-restricted CTL against this Ag. For this purpose, PBMC from four HLA-A2+ human volunteers immunized with P. falciparum irradiated sporozoites were stimulated with a recombinant vaccinia virus expressing PfSSP2 and with 35 PfSSP2-derived 15-amino acid peptides containing sequences conforming to HLA-A2 binding motifs. Ag-specific, genetically restricted, CD8+ T cell-dependent cytotoxic activity against autologous target cells transfected with the PfSSP2 gene was demonstrated in the four volunteers. Twelve of the 35 peptides sensitized HLA-A2-matched target cells for lysis by peptide-stimulated effectors. Three volunteers had CTL against 9 of the 12 peptides, and one had no peptide-specific CTL. HLA-A*0201 restriction was confirmed by demonstrating that effectors from the three responders could be stimulated with six different peptides to lyse HLA-A*0201+ T2 cells incubated with the homologous peptides. Peptide-specific, genetically restricted, CD8+ T cell-dependent cytotoxic activity was also demonstrated against two peptides using unstimulated PBMC as effectors. Available data indicate that the motif-bearing sequences in 6 of the 12 positive peptides are conserved among P. falciparum isolates and clones. Demonstration of HLA-A2-restricted CTL responses to multiple PfSSP2-derived peptides, and of circulating activated CTL against PfSSP2 in immune volunteers provide important information for optimal design and evaluation of vaccines containing this pre-erythrocytic stage Ag.

Amino Acid Sequence↗

PubMed1995

Identification of a nonameric H-2Kk-restricted CD8+ cytotoxic T lymphocyte epitope on the Plasmodium falciparum circumsporozoite protein.

Class I-restricted CD8+ cytotoxic T lymphocytes (CTL) against the circumsporozoite protein (CSP) protect mice against the rodent malaria parasite, Plasmodium yoelii, and vaccines designed to produce protective CTL against the P. falciparum CSP (PfCSP) are under development. Humans and B10.BR (H-2k) mice have been shown to have CD8+ CTL activity against a 23-amino-acid region of the PfCSP (residues 368 to 390 from the PfCSP 7G8 sequence) that is too long to bind directly to class I major histocompatibility complex molecules. To identify within this 23-amino-acid peptide a shorter peptide that binds to an H-2k class I major histocompatibility molecule, a primarily CD8+ (97.8%) T-cell line (PfCSP TCL.1) was produced by immunizing B10.BR mice with recombinant vaccinia virus expressing the PfCSP and stimulating in vitro spleen cells from these immunized mice with L cells transfected with the PfCSP gene (LPF cells). PfCSP TCL.1 lysed LPF cells and L cells pulsed with peptide PfCSP 7G8 368-390. When 15 overlapping nonamer peptides spanning the 368 to 390 sequence were tested, only one peptide, PfCSP 7G8 375-383 (Y E N D I E K K I), which includes an H-2Kk-binding motif, E at amino acid residue 2, and I at residue 9, sensitized targets for lysis by PfCSP TCL.1. Furthermore, a 10(3)- to 10(4)-fold lower concentration of the nonamer than that of the 23-amino-acid peptide was required to sensitize target cells for lysis by PfCSP TCL.1. Presentation by H-2Kk was demonstrated by using 3T3 fibroblast cells transfected with the murine H-2Kk or H-2Dk genes, and only the H-2Kk transfectants were lysed by PfCSP TCL.1 after incubation with peptide PfCSP 7G8 375-383. Binding to H-2Kk was confirmed by competitive inhibition of binding of labelled peptides to affinity-purified Kk molecules. Substitution of the anchor amino acid residue, E, at position 2 with A dramatically reduced binding to Kk and eliminated the capacity of the peptide to sensitize target cells for killing. Variation of non-anchor residues did not markedly reduce binding to Kk but in some cases eliminated the capacity of the peptide to sensitize targets for cytolysis by PfCSP TCL.1, presumably by eliminating T-cell receptor-binding sites. These data suggest that similar studies with human T cells will be required for optimal development of peptide-based vaccines designed to produce protective class I-restricted CD8+ CTL against the PfCSP in humans.

3T3 Cells↗

PubMed1994

Synthetic compound peptide simulating antigenicity of conformation-dependent autoepitope.

Proliferating cell nuclear antigen (PCNA), also known as auxiliary protein of DNA polymerase delta, is involved in DNA replication and repair. Certain patients with systemic lupus erythematosus (lupus) produce autoantibodies to PCNA and the autoantibody-defined epitopes of PCNA have been inferred to be conformation-dependent. Based on antigenic properties of continuous primary structure peptides, compound peptides composed of covalently linked discontinuous sequences were synthesized and used as immunogens in rabbits. One compound peptide induced an antibody response to a nuclear antigen which demonstrated a cell cycle-related pattern of nuclear immunofluorescence with maximum expression in the DNA synthesis phase of the cell cycle associated with other features which simulated the conformation-dependent properties of lupus defined auto-epitopes. Other rabbit antibodies raised against different compound peptides did not show such properties and recognized epitopes which were continuous primary sequence regions. These results show that from analysis of antigenic sites on a protein, it might be possible to construct synthetic compound peptides which can mimic conformation-dependent epitopes recognized by spontaneously occurring autoantibodies.

Amino Acid Sequence↗

PubMed1994

Crystal structure of deltakephalin: a delta-selective opioid peptide with a novel beta-bend-like conformation.

The solid-state structure of deltakephalin (Tyr-DThr-Gly-Phe-Leu-Thr) has been determined by single-crystal X-ray diffraction. Deltakephalin (DTLET) is a synthetic opioid peptide which differs from enkephalin in that a D-Thr has been substituted for Gly2 and a sixth residue, L-Thr, has been added. Clear colorless plates obtained using vapor diffusion and macro-seeding crystallization techniques were monoclinic; space group C2 with a = 27.389(5), b = 9.205(2), c = 16.788(2) A, beta = 98.87(2) degrees and V = 4181.4(14) A3. The asymmetric unit contained one molecule of DTLET and six molecules of water, giving a calculated density of 1.28 g cm-3. The crystal structure revealed that DTLET has a pseudo type I' beta-bend which is stabilized by an intramolecular side-chain to backbone hydrogen bond. This is the first reported observation of a pseudo beta-bend conformation in a solid-state structure of an enkephalin analog.

Crystallography, X-Ray↗

PubMed1994

An epitope in hepatitis C virus core region recognized by cytotoxic T cells in mice and humans.

Several cytotoxic T-lymphocyte (CTL) epitopes have been defined in hepatitis C virus (HCV) proteins. CTL may play an important role in the control of infection by HCV. Here, we identify a highly conserved antigenic site in the HCV core recognized by both murine and human CTL. Spleen cells from mice immunized with a recombinant vaccinia virus expressing the HCV core gene were restimulated in vitro with 11 peptides from the core protein. CTL from H-2d mice responded to a single 16-residue synthetic peptide (HCV 129-144). This conserved epitope was presented by a murine class I major histocompatibility molecule (H-2Dd) to conventional CD4- CD8+ CTL mapped by using transfectants expressing Dd, Ld, or Kd, but was not seen by CTL restricted by H-2b. The murine epitope was mapped to the decapeptide LMGYIPLVGA. The same 16-residue peptide was recognized by CTL from two HCV-seropositive patients but not by CTL from any seronegative donors. CTL from two HLA-A2-positive patients with acute and chronic hepatitides C recognized a 9-residue fragment (DLMGYIPLV) of the peptide presented by HLA-A2 and containing an HLA-A2-binding motif, extending only 1 residue beyond the murine epitope. Therefore, this conserved peptide, seen with murine CTL and human CTL with a very prevalent HLA class I molecule, may be a valuable component of an HCV vaccine against a broad range of HCV isolates. This study demonstrates that the screening for CTL epitopes in mice prior to human study may be useful.

Amino Acid Sequence↗

PubMed1993

Analysis of the epitopes of proliferating cell nuclear antigen recognized by monoclonal antibodies.

BACKGROUND: Monoclonal antibodies to proliferating cell nuclear antigen (PCNA) are increasingly used for evaluation of cell proliferation especially in diagnostic pathology. To understand the different staining characteristics of such antibodies, we found it important to map the reactive epitopes in detail. EXPERIMENTAL DESIGN: Overlapping, synthetic 15-mer peptides encoding the entire PCNA amino acid sequence were analyzed for reactivity with 6 anti-PCNA monoclonal antibodies using enzyme-linked immunosorbent assay and flow cytometric competition analysis. One immunodominant region was studied using a set of peptides with single amino acid substitutions. Further epitope localization was obtained by immunoblotting of fusion protein constructs. RESULTS: The epitopes recognized by the antibodies could be assigned to specific peptides. Five monoclonal antibodies (19A2, 19F4, TO17, TO30, PC10) reacted with the same protein region (aa 111-125) whereas one antibody (TOB7) recognized a separate region of the protein (aa 181-195). The reactivity pattern with 8 recombinant PCNA-fusion proteins agreed with the peptide data. The immunodominant aa 111-125 peptide reactive antibodies differed concerning immunofluorescence patterns. These differences could be attributed to epitope microheterogeneity within this peptide as determined by reactivity with Ala-substituted peptides. The 19A2, 19F4 and PC10 antibodies showed nuclear fluorescence and similar but not identical peptide recognition patterns. Compared with 19A2 and 19F4, the PC10 antibody was directed against a simpler epitope. TO17 and TO30 gave cytoplasmic filamentous staining and their epitopes were different from those defined by the other monoclonal antibodies. CONCLUSIONS: The results indicate that induced anti-PCNA antibodies recognize well defined linear epitopes, in contrast to PCNA autoepitopes which are strongly dependent on the protein conformation.

Amino Acid Sequence↗

PubMed1992

Immunization with soluble protein-pulsed spleen cells induces class I-restricted cytotoxic T lymphocytes that recognize immunodominant epitopic peptides from Plasmodium falciparum and HIV-1.

CTL lines specific for two different proteins derived from the human pathogens, Plasmodium falciparum (malaria) circumsporozoite protein and HIV-1 reverse transcriptase, were obtained by immunizing mice with protein-pulsed syngeneic spleen cells. The lysis of the target cells was dependent on a class I MHC molecule and the accessory molecule CD8. Immunodominant epitopic peptides were identified previously in the two proteins using murine CTL derived after immunization with recombinant virus or sporozoites, or using CTL from HIV-1-infected patients. These peptides were also recognized by the CTL lines obtained after protein-pulsed spleen-cell immunization. A new CTL antigenic determinant was localized in HIV-1 reverse transcriptase to residues 514 to 528, a sequence that, if folded as an alpha-helix, would be strongly amphipathic. The determinant was tentatively narrowed, using overlapping peptides, to a core of at least nine residues, 515 to 523. This site was also recognized by CTL obtained by the two different methods of immunization. Therefore, extracellular proteins incubated with spleen cells can be processed and presented in vivo in the same way as intracellular proteins, resulting in recognition of the same epitopes in association with the same class I MHC molecules. The potential implications for vaccine development and for the understanding of class I-restricted Ag presentation are discussed.

Amino Acid Sequence↗

PubMed1992

Induction of cytotoxic T cells to a cross-reactive epitope in the hepatitis C virus nonstructural RNA polymerase-like protein.

Cytotoxic T lymphocytes (CTL) have been found to mediate protection in vivo against certain virus infections. CTL also may play an important role in control of infection by hepatitis C virus (HCV), but no CTL epitopes have yet been defined in any HCV protein. The nonstructural protein with homology to RNA polymerase should be a relatively conserved target protein for CTL. To investigate the epitope specificity of CTL specific for this protein, we used 28 peptides from this sequence to study murine CTL. Mice were immunized with a recombinant vaccinia virus expressing the HCV nonstructural region corresponding to the flavivirus NS5 gene (RNA polymerase), and the primed spleen cells were restimulated in vitro with peptides. CTL from H-2d mice responded to a single 16-residue synthetic peptide (HCV 2422 to 2437). This relatively conserved epitope was presented by H-2d class I major histocompatibility complex (MHC) molecules to conventional CD4- CD8+ CTL but was not recognized by CTL restricted by H-2b. Moreover, exon shuffle experiments using several transfectants expressing recombinant Dd/Ld and Kd demonstrated that this peptide is seen in association with alpha 1 and alpha 2 domains of the Dd class I MHC molecule. This peptide differs from the homologous segments of this nonstructural region from three other HCV isolates by one residue each. Variant peptides with single amino acid substitutions were made to test the effect of each residue on the ability to sensitize targets. Neither substitution affected recognition. Therefore, these conservative mutations affected peptide interaction neither with the Dd class I MHC molecule nor with the T-cell receptor. Because these CTL cross-react with all four sequenced isolates of HCV in the United States and Japan, if human CTL display similar cross-reactivity, this peptide may be valuable for studies of HCV diagnosis and vaccine development. Our study provides the first evidence that CD8+ CTL can recognize an epitope from the HCV sequence in association with a class I MHC molecule.

Amino Acid Sequence↗

PubMed1991

Autoantibody production in hepatitis B e antigen transgenic mice elicited with a self T-cell peptide and inhibited with nonself peptides.

Studies in hepatitis B e antigen (HBeAg)-expressing transgenic mice indicate that self tolerance to two T-cell determinants on the same transgenic self molecule can differ markedly. The dominant T-cell site on HBeAg is tolerogenic, whereas a proportion of T cells recognizing a second T-cell site evade tolerance induction, persist in the periphery, and can be activated in vivo by a single injection of a 12-residue T-cell self peptide. The self-reactive T cells mediate in vivo autoantibody production sufficient to neutralize detection of the autoantigen in serum. Furthermore, autoantibody production can be inhibited by nonself peptides that compete with the self peptide for binding to major histocompatibility complex molecules. This model illustrates that T cells specific for an immunogenic T-cell site on a nonsequestered autoantigen can escape tolerance induction and, more importantly, can mediate autoreactivity in vivo. Furthermore, these results suggest that synthetic T-cell sites may be useful as immunotherapeutic agents for the purpose of circumventing nonresponse to HBeAg during persistent hepatitis B virus infection.

Amino Acid Sequence↗

PubMed1991

Development of cross-reactive antibodies to plasminogen during the immune response to dengue virus infection.

The four serotypes of dengue virus (a mosquito-borne flavivirus) cause an acute febrile illness (dengue fever) or a more prolonged illness with plasma leakage resulting in hypovolemia (dengue hemorrhagic fever). Hemorrhage may accompany either. Epidemiologic data suggest a role for dengue antibodies in pathogenesis. Computer analysis revealed a 20-residue region of similarity in amino acid sequence between the dengue type 4 envelope glycoprotein (E) and a family of clotting factors, including plasminogen, the prime mediator of fibrinolysis. By use of synthetic peptides in ELISA, E antibodies that potentially bind plasminogen were detected in 75% of 40 Thai patients acutely infected with dengue virus type 1, 2, 3, or 4. Plasminogen cross-reactivity of dengue antibodies was shown to be specific for the related sites in E and plasminogen. The dengue E sequence with similarity to plasminogen is largely conserved within the currently known flavivirus E sequences. However, 15 Thai patients hospitalized for illness caused by Japanese encephalitis virus (a flavivirus not associated with hemorrhage) did not develop plasminogen-cross-reactive antibodies, and this finding correlated with failure of Japanese encephalitis virus antibodies to bind to the plasminogen-cross-reactive site in E.

Adolescent↗