Biomedical subjects

Y Paterson

Publications and source records attributed to Y Paterson.

At least 37 records · Page 2Linked to original sources

PubMed1999

Th1 T cell responses to HIV-1 Gag protein delivered by a Listeria monocytogenes vaccine are similar to those induced by endogenous listerial antigens.

Listeria monocytogenes is a facultative intracellular bacterium that lives and grows in the cytoplasm of the host cell. The hallmark of a listerial infection is a cell-mediated immune response to its own secreted virulence factors. Thus, L. monocytogenes vaccines engineered to secrete HIV proteins may be ideal vectors for boosting cellular immune responses against HIV. Using strains of L. monocytogenes that stably express and secrete HIV Gag (Lm-Gag) to deliver this Ag to the immune system, we have previously shown strong MHC class I-restricted cytotoxic T cell responses to this protein. In this study, we examine MHC class II-restricted T cell responses to HIV-Gag delivered by Lm-Gag. We demonstrate the induction of CD4+ T cells that are HIV-Gag specific and identify three epitopes in two strains of mice, BALB/c (H-2d) and C57BL/6 (H-2b), two of which are both H-2d and H-2b restricted, but are not immunodominant for both haplotypes. In addition, we show that the CD4+ T cells induced are of the Th1 phenotype that produce IFN-gamma at levels similar to CD4+ T cells induced to endogenous listerial Ags. These studies suggest that chromosomally modified strains of L. monocytogenes may be useful as vaccine vectors for the induction of Th1 T cell responses against HIV.

Amino Acid Sequence↗

PubMed1999

Recognition of an MHC class I-restricted antigenic peptide can be modulated by para-substitution of its buried tyrosine residues in a TCR-specific manner.

Conformational dependence of TCR contact residues of the H-2Kb molecule on the two buried tyrosine side chains of the vesicular stomatitis virus (VSV)-8 peptide was investigated by systematic substitutions of the tyrosines with phenylalanine, p-fluorophenylalanine (pFF), or p-bromophenylalanine (pBrF). The results of peptide competition CTL assays revealed that all of the peptide variants, except for the pBrF analogues, had near-native binding to the H-2Kb molecule. Epitope-mapped anti-H-2Kb mAbs detected conformational differences among H-2Kb molecules stabilized with these VSV-8 variants on RMA-S cells. Selective recognition of the VSV-8 analogues was displayed by a panel of three H-2Kb-restricted, anti-VSV-8 TCRs. Thus, these substitutions result in an antigenically significant conformational change of the MHC molecular surface structure at both C and D pockets, and the effect of this change on cognate T cell recognition is dependent on the TCR structure. Our results confirm that the structure of buried peptide side chains can determine the surface conformation of the MHC molecule and demonstrate that even a very subtle structural nuance of the buried side chain can be incorporated into the surface conformation of the MHC molecule. The ability of buried residues to modulate this molecular surface augments the number of residues on the MHC-peptide complex that can be recognized as "foreign" by the CD8+ T cell repertoire and allows for a higher level of antigenic discrimination. This may be an important mechanism to expand the total number of TCR specificities that can respond to a single peptide determinant.

Animals↗

PubMed1999

The connection domain is implicated in metalloporphyrin binding and inhibition of HIV reverse transcriptase.

We have shown that heme and zinc protoporphyrin inhibit both human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) reverse transcriptases (RTs) and, in combination with other nucleoside and non-nucleoside inhibitors, exert an additive effect on HIV-1 RT inhibition. Screening of a phage peptide library against heme resulted in the isolation of a peptide with sequence similarity to sequence 398-407 from the connection subdomain of both HIV-1 and HIV-2 RTs, suggesting that this highly conserved region of HIV RTs corresponds to the binding site for metalloporphyrins and a new site for inhibition of enzyme activity. Inclusion of a synthetic peptide corresponding to the exact sequence 398-407 of HIV-1 RT in RT inhibition assays had a protective effect on metalloporphyrin inhibition, as it was able to reverse the inhibitory effect of both metalloporphyrins on HIV-1 RT activity. Furthermore, intrinsic fluorescence assays indicated that these metalloporphyrins bind to synthetic peptide 398-407 as well as to intact dimeric HIV-1 RT. The identification of this novel inhibition site will help to expand our understanding of the mode of action of metalloporphyrins in RT inhibition and will assist in the design and development of more potent metalloporphyrin RT inhibitors for the management of HIV infection.

Amino Acid Sequence↗

PubMed1998

The MHC class I-restricted immune response to HIV-gag in BALB/c mice selects a single epitope that does not have a predictable MHC-binding motif and binds to Kd through interactions between a glutamine at P3 and pocket D.

Using a strain of Listeria monocytogenes that stably expresses and secretes HIV gag to deliver this Ag to the MHC class I pathway of Ag processing, we have identified the immunodominant CTL epitope to gag in the BALB/c mouse and shown that it is Kd restricted. The specific motif for the peptides that bind the MHC class I molecule H-2 Kd is believed to be a nonamer with residues tyrosine or phenylalanine in the second amino acid position and leucine or isoleucine in the carboxyl-terminal or ninth amino acid position as dominant anchoring positions. Surprisingly, the identified gag peptide, AMQMLKETI, does not contain an anchoring aromatic residue in position two although competition assays with other Kd-restricted epitopes indicated that it binds to Kd with comparable affinity. Using a theoretical molecular dynamics approach to probe the stability of peptide binding to MHC class I molecules, we show that the absence of an appropriate anchor residue at P2 in AMQMLKETI is compensated by favorable interactions of the glutamine at P3 with pocket D of Kd. These findings were verified experimentally, demonstrating the predictive power of this theoretical approach in analyzing MHC class I/peptide interactions. These studies also indicate that CTL epitope prediction that relies on dominant peptide motifs may not always identify the correct epitope.

Amino Acid Sequence↗

PubMed1998

Structural basis for the binding of an anti-cytochrome c antibody to its antigen: crystal structures of FabE8-cytochrome c complex to 1.8 A resolution and FabE8 to 2.26 A resolution.

A complete understanding of antibody-antigen association and specificity requires the stereochemical description of both antigen and antibody before and upon complex formation. The structural mechanism involved in the binding of the IgG1 monoclonal antibody E8 to its highly charged protein antigen horse cytochrome c (cyt c) is revealed by crystallographic structures of the antigen-binding fragment (Fab) of E8 bound to cyt c (FabE8-cytc), determined to 1.8 A resolution, and of uncomplexed Fab E8 (FabE8), determined to 2.26 A resolution. E8 antibody binds to three major discontiguous segments (33 to 39; 56 to 66; 96 to 104), and two minor sites on cyt c opposite to the exposed haem edge. Crystallographic definition of the E8 epitope complements and extends biochemical mapping and two-dimensional nuclear magnetic resonance with hydrogen-deuterium exchange studies. These combined results demonstrate that antibody-induced stabilization of secondary structural elements within the antigen can propagate locally to adjacent residues outside the epitope. Pre-existing shape complementarity at the FabE8-cytc interface is enhanced by 48 bound water molecules, and by local movements of up to 4.2 A for E8 antibody and 8.9 A for cyt c. Glu62, Asn103 and the C-terminal Glu104 of cyt c adjust to fit the pre-formed VL "hill" and VH "valley" shape of the grooved E8 paratope. All six E8 complementarity determining regions (CDRs) contact the antigen, with CDR L1 forming 46% of the total atomic contacts, and CDRs L1 (29%) and H3 (20%) contributing the highest percentage of the total surface area of E8 buried by cyt c (550 A2). The E8 antibody covers 534 A2 of the cyt c surface. The formation of five ion pairs between E8 and flexible cyt c residues Lys60, Glu62 and Glu104 suggests the importance of mobile regions and electrostatic interactions in providing the exquisite specificity needed for recognition of this extremely conserved protein antigen. The highly homologous VL domains of E8 and anti-lysozyme antibody D1. 3 achieve their distinct antigen-binding specificities by expanding the impact of their limited sequence differences through the recruitment of different sets of conserved residues and distinctly different CDR L3 conformations.

Animals↗

PubMed1998

DR/CLIP (class II-associated invariant chain peptides) and DR/peptide complexes colocalize in prelysosomes in human B lymphoblastoid cells.

In APCs, MHC class II molecules (MHC class II) bind antigenic peptides after HLA-DM mediated removal of CLIP. To characterize intracellular sites of peptide loading in human B lymphoblastoid cell lines, we conducted immunoelectron microscopy studies with Abs recognizing MHC class II associated with CLIP or bound peptide, respectively, together with Abs to HLA-DM and endocytic markers. The distribution of these molecules indicates that peptide binding occurs in compartments with characteristics of normal late endosomes, and in compartments that show characteristics of late endosomes, but are not detectably accessed by endocytosed BSA-gold. The latter compartments may represent or give rise to recycling vesicles that deliver peptide-loaded class II molecules to the cell surface. In addition, we have compared cells in which HLA-DM and HLA-DR interaction is defective with cells in which this interaction is intact, and find that DM/DR interaction is not required for the proper localization of either molecule to peptide-loading compartments.

Antigens, Differentiation, B-Lymphocyte↗

PubMed1998

Rational approaches to immune regulation.

Our studies are mainly focused on developing strategies of immune regulation. In the case of infectious and neoplastic disease, our approach is to upregulate cell-mediated immunity to viral of tumor antigens using an intracellular bacterium as a vector for targeting these antigens to the major histocompatibility complex (MHC) class I and class II pathways of antigen processing, in addition to exploiting the adjuvant properties of the vector to stimulate innate immunity. In the area of autoimmunity, we are attempting to downregulate the immune response by specific immune intervention directed against autoreactive T cells. In these studies we use murine models for multiple sclerosis. Our approach is to use both rationally designed T cell receptor (TCR) peptide analogs and recombinant viral vectors that express TCR components to regulate the disease.

Animals↗

PubMed1997

Influenza-specific immunity induced by recombinant Listeria monocytogenes vaccines.

In this study, we evaluate two Listeria monocytogenes strains that express influenza nucleoprotein (NP) sequences for their ability to protect against challenge with influenza-virus. The construction of one strain, which expresses only the Kd restricted NP epitope (NP 147-155), is described in this study; the other strain, which expresses the full NP sequence in the form of a fusion protein, has been described previously. The ability of the two strains to present the Kd restricted NP epitope in vitro and induce NP-specific CTL in vivo is also described. Mice immunized by the intravenous route with either strain cleared a subsequent (3 weeks post-immunization) influenza virus infection more rapidly as indicated by reduced virus titers in the lungs 5 days after challenge. Efficacy of both recombinant L. monocytogenes strains as vaccines in this system was equivalent and equal to that of recombinant vaccinia expressing NP.

Animals↗

PubMed1997

Listeria monocytogenes: a potent vaccine vector for neoplastic and infectious disease.

Listeria monocytogenes (L. monocytogenes) is a promising candidate vaccine vector that naturally infects antigen-presenting cells, and targets antigen delivery to both the class I MHC pathway of endogenous antigen presentation and the class II pathway of exogenous antigen presentation. At the same time, L. monocytogenes stimulates the innate immune response to produce cytokines that enhance antigen-presenting function and induce a Th1-type cytokine profile associated with cell-mediated immune responses. Immune responses with these features are considered to be particularly important for clearance of viruses, tumors, and intracellular infections. In this review, we describe the development of methods to transform L. monocytogenes to express and secrete foreign antigens and the studies that have demonstrated that genetically engineered L. monocytogenes mutants are highly effective vectors for the induction of potent immune responses against viral antigens and tumor cells. In addition, we discuss the strengths and weaknesses of L. monocytogenes as a vaccine vector.

Animals↗

PubMed1997

CD4+ and CD8+ T cells are not major effectors of mouse hepatitis virus A59-induced demyelinating disease.

We examined murine hepatitis virus strain A59 (MHV-A59)-induced demyelinating disease in C57BL/6 mice which had previously been thymectomized at 25 days of age. Demyelination was observed in 51-96% of spinal cord quadrants examined 30 or 60 days post infection (dpi), indicating that neither an intact thymus nor thymic infection is a prerequisite to demyelination. Depletion of CD4+ or CD8+ T cells at 5, 7 or 10 dpi did not influence the extent of demyelination indicating that neither T cell subset is a major effector of demyelination. However, these findings do not exclude the possibility that T cells are involved in initiating demyelinating disease very early in infection.

Animals↗

PubMed1996

Exploitation of the Vbeta8.2 T cell receptor in protection against experimental autoimmune encephalomyelitis using a live vaccinia virus vector.

This study takes advantage of the predominant usage of Vbeta8.2 by the TCRs of encephalitogenic T cells specific for myelin basic protein. Vaccinia virus recombinants expressing Vbeta8.2 (VVbeta8.2) 8.2) and Vbeta3 (VVbeta 3) proteins were constructed, and their abilities to confer protection against experimental autoimmune encephalomyelitis (EAE) induction in H-2u mice were examined. Mice immunized with VVbeta8.2 developed very mild EAE by comparison with mice that were vaccinated with VVbeta3, which developed severe clinical symptoms. This reduction in EAE correlated with a diminished T cell proliferative response to myelin basic protein in the mice that received VVbeta8.2 compared with that in mice receiving VVbeta3.

Animals↗

PubMed1996

Nuclear Magnetic Resonance Spectroscopy for the Study of B-Cell Epitopes

High-resolution nuclear magnetic resonance (NMR) spectroscopy is a structural technique that is finding increasing use in the study of antibody-antigen interactions. In this review we describe how the dynamic structural parameters obtained from NMR spectroscopy can further our understanding of B-cell epitopes and their function. Specific applications of NMR spectroscopy to examine the residues on peptides and proteins that contact the antibody combining site are also described. These include "footprinting" techniques using H-D exchange-COSY NMR spectroscopy, which are particularly useful for epitope mapping of protein antigens. For smaller systems, such as Fab-or Fv-peptide complexes, nuclear magnetization transfer difference NMR spectroscopy, transferred nuclear Overhauser effect spectroscopy, double-quantum-filtered NOE spectroscopy, and isotope editing techniques have been applied. The interpretation and limitations of the data obtained from these procedures and anticipated improvements in these applications in the future are discussed.

Journal Article↗

PubMed1996

Recombinant Listeria monocytogenes cancer vaccines.

Listeria monocytogenes has recently been exploited as a vector for targeting antigens to the immune system. In the past year it has been shown to be particularly effective as a tumor antigen vector. Here we set its potency as a cancer vaccine in the context of the type of immunity induced by this facultative intracellular bacterium.

Animals↗

PubMed1995

Induction of cell-mediated immune responses to human immunodeficiency virus type 1 Gag protein by using Listeria monocytogenes as a live vaccine vector.

Cytolytic T cells, acting through cytokines or by direct lysis of infected target cells, have been shown to play a significant role in the control of viral infections and may be responsible for the prolonged asymptomatic phase following infection by HIV. Accordingly, methods that can generate strong cell-mediated immune responses may be useful in the development of prophylactic and therapeutic vaccines against HIV. Listeria monocytogenes is a Gram-positive intracellular microorganism that elicits strong cell-mediated immune responses against its own secreted proteins following infection. In this study we have modified the chromosome of L. monocytogenes so that it stably expresses and secretes the p55 HIV gag gene product and examined the cell-mediated immune response of BALB/c mice to infection with this recombinant organism. Infected animals were found to mount a specific, strong, long-lasting CD8+ cytolytic T cell response against a predominant epitope contained within the p24 fragment of the HIV Gag protein. This epitope previously has been shown to be recognized by CTLs obtained from some HIV-infected humans. Our results suggest that chromosomally modified strains of L. monocytogenes may provide valuable vaccine vectors for use against HIV.

AIDS Vaccines↗

PubMed1995

Regression of established tumors in mice mediated by the oral administration of a recombinant Listeria monocytogenes vaccine.

We have shown previously that Listeria monocytogenes, a gram-positive, facultative intracellular bacterium, is a potent vector for targeting tumor-specific antigens to the immune system. After parenteral administration, we observed protection against both renal and colorectal mouse tumors and regression of established renal tumors. In the present study, we have exploited the fact that the normal route of infection of this organism is through the gut. We show that an L. monocytogenes recombinant that expresses a model tumor antigen is an effective cancer immunotherapeutic agent when delivered orally in that it causes regression of established, macroscopic mouse renal and colorectal tumors expressing the same antigen.

Administration, Oral↗

PubMed1995

The self antigen heme evades immune recognition by sequestration in some hemoproteins.

Heme is a non-protein autoantigen which is ubiquitous in vivo, primarily complexed in various hemoproteins or bound to specialized carrier molecules. Nevertheless, heme is able to stimulate a high frequency of CD4+, class II-restricted T cells, freshly explanted from unprimed mice, to proliferate in vitro. In this study, we show that heme incorporated into various species of mammalian cytochrome c (cyt c), including murine cyt c, represents a facultative cryptic determinant, able to be recalled only at high doses of native cyt c. By contrast, avian cyt c is of comparable antigenicity to free heme. Artificially denatured carboxymethylated (CM) mammalian cyt c exhibited greatly increased antigenicity, comparable to that of heme and avian cyt c, indicating that the crypticity of heme in native mammalian cyt c is due to the resistance of the native conformation of this molecule to antigen processing within murine antigen-presenting cells. Thus, tolerance to the heme group of at least some hemoproteins, may be maintained by the crypticity of the heme, rather than by deletion of heme-reactive T cells. Given the high frequency of heme-reactive T cells in unprimed mice, these findings suggest that heme may become an important modulator during an inflammatory response.

Animals↗

PubMed1995

Rapid amide proton exchange rates in peptides and proteins measured by solvent quenching and two-dimensional NMR.

In an effort to develop a more versatile quenched hydrogen exchange method for studies of peptide conformation and protein-ligand interactions, the mechanism of amide proton exchange for model peptides in DMSO-D2O mixtures was investigated by NMR methods. As in water, H-D exchange rates in the presence of 90% or 95% DMSO exhibit characteristic acid- and base-catalyzed processes and negligible water catalysis. However, the base-catalyzed rate is suppressed by as much as four orders of magnitude in 95% DMSO. As a result, the pH at which the exchange rate goes through a minimum is shifted up by about two pH units and the minimum exchange rate is approximately 100-fold reduced relative to that in D2O. The solvent-dependent decrease in base-catalyzed exchange rates can be attributed primarily to a large increase in pKa values for the NH group, whereas solvent effects on pKW seem less important. Addition of toluene and cyclohexane resulted in improved proton NMR chemical shift dispersion. The dramatic reduction in exchange rates observed in the solvent mixture at optimal pH makes it possible to apply 2D NMR for NH exchange measurements on peptides under conditions where rates are too rapid for direct NMR analysis. To test this solvent-quenching method, melittin was exchanged in D2O (pH 3.2, 12 degrees C), aliquots were quenched by rapid freezing, lyophilized, and dissolved in quenching buffer (70% DMSO, 25% toluene, 4% D2O, 1% cyclohexane, 75 mM dichloroacetic acid) for NMR analysis. Exchange rates for 21 amide protons were measured by recording 2D NMR spectra on a series of samples quenched at different times. The results are consistent with a monomeric unfolded conformation of melittin at acidic pH. The ability to trap labile protons by solvent quenching makes it possible to extend amide protection studies to peptide ligands or labile protons on the surface of a protein involved in macromolecular interactions.

Amides↗

PubMed1995

Configurational effects in antibody-antigen interactions studied by microcalorimetry.

In this paper we study the binding of two monoclonal antibodies, E3 and E8, to cytochrome c using high-sensitivity isothermal titration calorimetry. We combine the calorimetric results with empirical calculations which relate changes in heat capacity to changes in entropy which arise from the hydrophobic effect. The change in heat capacity for binding E3 is -350 +/- 60 cal K-1 mol-1 while for E8 it is -165 +/- 40 cal K-1 mol-1. This result indicates that the hydrophobic effect makes a much larger contribution for E3 than for E8. Since the total entropy change at 25 degrees C is very similar for both antibodies, it follows that the configurational entropy cost for binding E3 is much larger than for binding E8 (-77 +/- 15 vs. -34 +/- 11 cal K-1 mol-1). These results illustrate a case of entropy compensation in which the cost of restricting conformational degrees of freedom is to a large extent compensated by solvent release. We also show that the thermodynamic data can be used to make estimates of the surface area changes that occur upon binding. The results of the present study are consistent with previous hydrogen-deuterium exchange data, detected using 2D NMR, on the two antibody-antigen interactions. The NMR study indicated that protection from exchange is limited to the binding epitope for E8, but extends beyond the epitope for E3.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗