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1.
J Virol ; 75(7): 3469-73, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11238873

ABSTRACT

As early as 5 days after DNA copies of the hepatitis delta virus (HDV) genome or even in vitro-transcribed HDV RNA sequences were injected into the mouse tail vein using the hydrodynamics-based transfection procedure of F. Liu et al. (Gene Ther. 6:1258-1266, 1999), it was possible to detect in the liver by Northern analyses of RNA, immunoblots of protein, and immunostaining of liver sections what were considered typical features of HDV genome replication. This transfection strategy should have valuable applications for in vivo studies of HDV replication and pathogenesis and may also be useful for studies of other hepatotropic viruses.


Subject(s)
DNA Replication , Genome, Viral , Hepatitis Delta Virus/genetics , Hepatocytes/virology , RNA, Viral/biosynthesis , Virus Replication , Animals , Humans , Immunoblotting , Injections, Intravenous , Mice , Transfection
2.
J Exp Med ; 192(8): 1143-50, 2000 Oct 16.
Article in English | MEDLINE | ID: mdl-11034604

ABSTRACT

Bone marrow (BM)-derived professional antigen-presenting cells (pAPCs) are required for the generation of cytotoxic T lymphocyte (CTL) responses to vaccinia virus and poliovirus. Furthermore, these BM-derived pAPCs require a functional transporter associated with antigen presentation (TAP). In this report we analyze the requirements for BM-derived pAPCs and TAP in the initiation of CTL responses to lymphocytic choriomeningitis virus (LCMV) and influenza virus (Flu). Our results indicate a requirement for BM-derived pAPCs for the CTL responses to these viruses. However, we found that the generation of CTLs to one LCMV epitope (LCMV nucleoprotein 396-404) was dependent on BM-derived pAPCs but, surprisingly, TAP independent. The study of the CTL response to Flu confirmed the existence of this BM-derived pAPC-dependent/TAP-independent CTL response and indicated that the TAP-independent pathway is approximately 10-300-fold less efficient than the TAP-dependent pathway.


Subject(s)
Antigen-Presenting Cells/immunology , Bone Marrow Cells/immunology , Bone Transplantation/immunology , Lymphocytic choriomeningitis virus/immunology , Poliovirus/immunology , T-Lymphocytes, Cytotoxic/immunology , Transplantation Chimera , Vaccinia virus/immunology , Animals , Antigen-Presenting Cells/cytology , Bone Marrow Cells/cytology , Crosses, Genetic , Cytotoxicity, Immunologic , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , T-Lymphocytes, Cytotoxic/virology , Tumor Cells, Cultured
3.
Nature ; 398(6722): 77-80, 1999 Mar 04.
Article in English | MEDLINE | ID: mdl-10078533

ABSTRACT

Cytotoxic T lymphocytes (CTLs) are thought to detect viral infections by monitoring the surface of all cells for the presence of viral peptides bound to major histocompatibility complex (MHC) class I molecules. In most cells, peptides presented by MHC class I molecules are derived exclusively from proteins synthesized by the antigen-bearing cells. Macrophages and dendritic cells also have an alternative MHC class I pathway that can present peptides derived from extracellular antigens; however, the physiological role of this process is unclear. Here we show that virally infected non-haematopoietic cells are unable to stimulate primary CTL-mediated immunity directly. Instead, bone-marrow-derived cells are required as antigen-presenting cells (APCs) to initiate anti-viral CTL responses. In these APCs, the alternative (exogenous) MHC class I pathway is the obligatory mechanism for the initiation of CTL responses to viruses that infect only non-haematopoietic cells.


Subject(s)
Antigen Presentation , Antigen-Presenting Cells/immunology , Antigens, Viral/immunology , Histocompatibility Antigens Class I/immunology , Membrane Proteins , T-Lymphocytes, Cytotoxic/immunology , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/immunology , Bone Marrow Cells/virology , Chimera , Hematopoietic Stem Cells/immunology , Hematopoietic Stem Cells/virology , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Poliovirus , Receptors, Virus/genetics , Receptors, Virus/metabolism , Vaccinia virus/immunology
4.
J Immunol ; 161(6): 2740-5, 1998 Sep 15.
Article in English | MEDLINE | ID: mdl-9743331

ABSTRACT

The role of B7-1 and B7-2 costimulatory molecules in the generation of Ag-specific CD8+ CTLs is not well understood. In this paper, we analyze the role of both B7-1 and B7-2 in the generation of CTLs to nonliving, exogenous Ag and to live virus. To analyze the role of B7 costimulation in the induction of CTLs, we blocked B7-1 and/or B7-2 in vivo by injecting C57BL/6 mice with anti-B7-1 and/or anti-B7-2 mAbs; the mice were subsequently immunized with either chicken OVA that had been cross-linked to beads as a model of exogenous Ags or with wild-type and recombinant vaccinia virus expressing different forms of chicken OVA as models of viral Ags. Our results indicate that B7 costimulation is necessary in the generation of CTLs for all of these Ags. Since the B7 molecules could be costimulating CD8+ and/or CD4+ T cells in wild-type animals, we also examined the role of costimulation in the generation of CTLs to exogenous and viral Ag in MHC class II-deficient mice lacking most CD4+ T cells. In these animals, a combination of both mAbs also blocked all CTL responses, indicating that the Th cell-independent activation of CTLs is dependent upon the B7-costimulatory signals supplied to the CD8+ cell. These findings contribute to the understanding of the role of costimulation for the generation of CTLs. We also discuss the implications of these findings on the role of professional APCs in the initiation of CTL responses.


Subject(s)
Antigens, CD/physiology , B7-1 Antigen/physiology , Cytotoxicity, Immunologic/immunology , Lymphocyte Activation/immunology , Membrane Glycoproteins/physiology , T-Lymphocytes, Cytotoxic/immunology , Animals , Antibodies, Blocking/pharmacology , Antigen-Presenting Cells/immunology , Antigens, CD/immunology , Antigens, Viral/immunology , B7-1 Antigen/immunology , B7-2 Antigen , Cell Line , Female , Immune Sera/pharmacology , Injections, Subcutaneous , Membrane Glycoproteins/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Microspheres , Ovalbumin/administration & dosage , Ovalbumin/genetics , Ovalbumin/immunology , Stem Cells/immunology , Vaccinia virus/genetics , Vaccinia virus/immunology
5.
Proc Natl Acad Sci U S A ; 95(14): 8216-21, 1998 Jul 07.
Article in English | MEDLINE | ID: mdl-9653167

ABSTRACT

Recombinant polioviruses expressing foreign antigens may provide a convenient vaccine vector system to induce protective immunity against diverse pathogens. Replication-competent chimeric viruses can be constructed by inserting foreign antigenic sequences within the poliovirus polyprotein. When inserted sequences are flanked by poliovirus protease recognition sites the recombinant polyprotein is processed to mature and functional viral proteins plus the exogenous antigen. It previously has been shown that poliovirus recombinants can induce antibody responses against the inserted sequences but it is not known whether poliovirus or vaccine vectors derived from it can elicit effective cytotoxic T lymphocyte (CTL) responses. To examine the ability of the recombinant poliovirus to induce CTL responses, a segment of the chicken ovalbumin gene, which includes the H2-Kb-restricted CTL epitope SIINFEKL, was cloned at the junction of the P1 and P2 regions. This recombinant virus replicated with near wild-type efficiency in culture and stably expressed high levels of the ovalbumin antigen. Murine and primate cells infected with the recombinant virus appropriately processed the SIINFEKL epitope and presented it within major histocompatibility complex class I molecules. Inoculation of mice with recombinant poliovirus that expresses ovalbumin elicits an effective specific CTL response. Furthermore, vaccination with these recombinant poliovirus induced protective immunity against challenge with lethal doses of a malignant melanoma cell line expressing ovalbumin.


Subject(s)
Epitopes/immunology , Genetic Vectors , Melanoma, Experimental/immunology , Poliovirus Vaccine, Inactivated , T-Lymphocytes, Cytotoxic/immunology , Animals , Antigen Presentation , Cytotoxicity, Immunologic , Epitopes/genetics , HeLa Cells , Humans , Melanoma, Experimental/genetics , Mice , Ovalbumin/genetics , Ovalbumin/immunology
6.
Mol Immunol ; 33(17-18): 1323-33, 1996 Dec.
Article in English | MEDLINE | ID: mdl-9171892

ABSTRACT

To understand better, the role of non-anchor residues of class I restricted T cell epitopes in class I binding and TCR stimulation, a panel of peptides was synthesized in which each of the non-anchor positions of the Db-restricted influenza peptide, ASNENMETM, was changed to each of the 20 natural amino acids (AAs). The relative affinity of all the peptides for Db was determined and their ability to stimulate anti-ASNENMETM cytotoxic T cell hybridomas was also assessed. The results illustrated that for Db binding, the AAs with the most solvent exposure had the smallest effect on binding. Changes at other positions affected binding to different degrees. Results for the recognition by the T cell hybridomas indicated that a peptide-MHC complex represents a multitude of epitopes, as each hybridoma recognized a different subset of peptides. Most changes in the highly solvent-exposed residues negatively affected recognition by all hybridomas while changes in other positions affected each hybridoma differently, independent of the direction of the side chain of the AA at that position. Furthermore, the use of saturating concentrations of low and high binding peptides showed that, as long as the class I-peptide complex is formed, the T-cell receptor does not differentiate between high and low binding peptides. This indicates that, although the stability of the class I-peptide complex is highly dependent on peptide affinity, the class I MHC conformation induced by low affinity peptides does not necessarily differ significantly from that induced by high affinity peptides. The results of peptide-class I recognition by one ASNENMETM-specific hybridoma was used to construct a peptide that differed from ASNENMETM at four of the nine residues, yet stimulated the hybridoma to a level comparable to ASNENMETM. In addition, peptides bearing the canonical Db-binding motif but unable to bind to the class I molecule with high affinity could be made to bind Db, by changing unfavorable AAs to favourable ones at appropriate positions. The extended motif determined was used to identify more accurately the peptides derived from Coxsakie b3 virus that would bind Db. It was also shown that some of the canonical characteristics of the peptide motif could be obviated and still obtain high affinity binding, provided optimal AAs, were present at secondary anchor positions.


Subject(s)
H-2 Antigens/genetics , Nucleoproteins/immunology , Peptides/immunology , Peptides/physiology , RNA-Binding Proteins , Receptors, Antigen, T-Cell/metabolism , Viral Core Proteins/immunology , Amino Acid Sequence , Animals , H-2 Antigens/chemistry , H-2 Antigens/metabolism , Histocompatibility Antigen H-2D , Hybridomas/immunology , Hybridomas/metabolism , Influenza A virus/immunology , Mice , Nucleocapsid Proteins , Nucleoproteins/metabolism , Nucleoproteins/physiology , Peptides/chemical synthesis , Protein Binding/immunology , Protein Conformation , Receptors, Antigen, T-Cell/immunology , Structure-Activity Relationship , Viral Core Proteins/metabolism , Viral Core Proteins/physiology
7.
Mol Immunol ; 32(9): 623-32, 1995 Jun.
Article in English | MEDLINE | ID: mdl-7643854

ABSTRACT

Relative affinities were determined for the interaction of H-2Db with all the peptides from the A/PR/8/34 strain of influenza virus that contained the Db-binding motif. The results indicated that, even though 23 peptides with the appropriate motif were identified and analysed, binding of only five of them could be detected at peptide concentrations lower than 10(-7) M. Of these five, only one, TGICNQNII, bound with better affinity than the nucleoprotein-derived natural epitope, ASNENMETM. The origin of the higher binding peptide was the influenza neuraminidase, a protein for which little cytosolic processing would be expected since it is a surface glycoprotein. To establish why many of the influenza-derived peptides did not bind, the role of non-anchor residues on Db-peptide interactions was analysed, using a scheme where QDIENEEKI, a non-binding peptide from the influenza virus polymerase 1, was sequentially converted to ASNENMETI, which binds to Db with an affinity similar to that of ASNENMETM. Although all positions examined influenced peptide binding, peptide residue no. 2 (P2) was of particular importance. Therefore, each of the 20 naturally occurring amino acids were inserted at this position to investigate their effects on peptide-MHC interaction. The results indicated that amino acids having side chains with charged or ring structures were deleterious, while non-polar and polar residues were either neutral or facilitated binding to different degrees. Our data also indicated that every residue of the peptide contributes to the stability of the MHC-peptide complex, and the final affinity is dependent on the nature of the amino acids at each position, not just on those at a small number of anchor positions. The results also suggested that increased stability, as indicated by the half-life of the peptide-MHC class I complex, might play an important role in selecting the immunodominant epitope.


Subject(s)
Antigens, Viral/immunology , H-2 Antigens/immunology , Influenza A virus/immunology , Amino Acid Sequence , Animals , Antigens, Viral/metabolism , Epitope Mapping , H-2 Antigens/metabolism , Mice , Molecular Sequence Data , Peptides/immunology , Protein Binding , Structure-Activity Relationship
8.
J Immunol Methods ; 177(1-2): 261-8, 1994 Dec 28.
Article in English | MEDLINE | ID: mdl-7822833

ABSTRACT

A lactate dehydrogenase (LDH)-based immunoassay, referred to as CPEIA (cell panning enzyme immunoassay), has been developed for the detection of cell-surface antigens. CPEIA is similar to a panning assay, in that it is based on the capture of cells bearing an antigen of interest by means of an antibody immobilized to a 96-well microtiter plate. Attachment of the cells is then measured by addition of a substrate for the intracellular enzyme lactate dehydrogenase. The substrate solution also contains the nonionic detergent Triton X-100 to lyse the cells and release LDH, which converts the substrate p-iodonitrotetrazolium violet (INT) from yellow to red. The intensity of the color resulting from the LDH-catalyzed reaction is proportional to the number of cells bound to the plate. The procedure does not require fixation of the cells, centrifugation, and blocking steps, resulting in a more convenient assay. CPEIA has been used for the detection of MHC class I antigens and other molecules on the surfaces of mouse cell lines and concanavalin A (ConA)-stimulated T lymphocytes. In addition, the assay has been used to detect peptide binding to Db and Kb MHC class I molecules on the surface of the mutant cell line RMA-S. The half-maximal responses for peptide-MHC class I interactions at different peptide concentrations can be determined with the assay, allowing the apparent dissociation constants to be calculated.


Subject(s)
Antigens, Surface/analysis , Antigens, Viral/immunology , H-2 Antigens/analysis , Immunoenzyme Techniques , Peptides/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Antigens, Viral/chemistry , Antigens, Viral/metabolism , H-2 Antigens/metabolism , In Vitro Techniques , L-Lactate Dehydrogenase/metabolism , Lymphocyte Activation , Mice , Molecular Sequence Data , Peptides/metabolism , T-Lymphocytes/immunology
9.
Biotechniques ; 17(4): 776-80, 1994 Oct.
Article in English | MEDLINE | ID: mdl-7833041

ABSTRACT

A method for detection of cell-surface antigens, referred to as cell-bead immunoassay (CBIA), has been developed by cross-linking monoclonal antibodies specific for cell-surface antigens to protein G-agarose beads. In this case, the antibodies were specific for different murine class I major histocompatibility complex (MHC) antigens and murine beta 2 microglobulin. The antibody-conjugated beads were incubated with cells expressing the relevant MHC molecule and observed microscopically for rosette formation. The number of cells bound per bead correlated with the amount of class I MHC expressed per cell, as measured by fluorescence activated cell sorting (FACS) analysis. In addition, changes in the amount of surface antigen expressed after induction could be followed by CBIA. The advantages of CBIA over other commonly used techniques, such as FACS and immunofluorescence, are that it requires only a few minutes incubation after beads are prepared, and no further manipulations are needed after the cells and beads are mixed together. Although CBIA is primarily a qualitative technique, it can also be used semiquantitatively by determining the number of cells bound per bead.


Subject(s)
Histocompatibility Antigens Class I/analysis , Immunoassay/methods , Animals , Antibodies, Monoclonal/immunology , Cell Line , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Rosette Formation
10.
Vet Immunol Immunopathol ; 30(4): 431-8, 1992 Jan 31.
Article in English | MEDLINE | ID: mdl-1347666

ABSTRACT

The percentages and absolute numbers of mononuclear peripheral blood cells (MNC) were studied in vaccinated (Vac) and non-vaccinated (control) cattle, challenged with foot-and-mouth disease virus (FMDV). All Vac cattle but none of the controls resisted challenge. Cell populations were studied immediately before and one week after challenge, by direct and indirect immunofluorescence, using polyclonal and monoclonal antibodies against different bovine markers. Total B-lymphocytes, as assessed with polyclonal anti-IgG (H + L) antisera, as well as total mononuclear cells, were normal before and after infection, and did not change in Vac or control groups. Before challenge Vac cattle had higher numbers of ILA-29+ (a putative marker for null cells or, alternatively, for gamma delta T-cells) than control cattle. After challenge, in control cattle, the number of total T-cells, BoT4-bearing (helper) T-cells and BoT8-bearing (cytotoxic/suppressor) T-cells were decreased, while IgM-bearing B-lymphocytes, as well as monocyte/macrophage cells were increased. The number of IL-A29-bearing T-cells did not change after infection in either group. After challenge, Vac cattle also showed increased numbers of IgM-bearing B-lymphocytes and monocyte/macrophage cells, whereas T-subpopulations did not change significantly.


Subject(s)
Foot-and-Mouth Disease/prevention & control , Leukocytes, Mononuclear/immunology , Vaccination , Animals , Antibodies, Monoclonal , Aphthovirus/immunology , B-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/immunology , Cattle , Fluorescent Antibody Technique , Foot-and-Mouth Disease/immunology , Macrophages/immunology , Monocytes/immunology , Receptors, Antigen, T-Cell, gamma-delta/immunology , T-Lymphocytes, Helper-Inducer/immunology , T-Lymphocytes, Regulatory/immunology , Viral Vaccines
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