RESUMO
A range of fusion constructs (expressed in Escherichia coli) were produced that contained two or more HPV6b E proteins, producing a single continuous amino acid sequence corresponding to the sequences of the individual E proteins. The constructs also included a C-terminal hexahistidine tag fused in-frame to aid purification. The fusion proteins (polyproteins) were semipurified by Ni(++) metal affinity chromatography under denaturing conditions. Immunization of BALB/c mice with these polyproteins resulted in the production of specific E protein antibodies. The draining lymph nodes from these mice were used to produce monoclonal antibodies (MAbs). The specificity of the polyclonal and MAbs was confirmed by immunoblotting and by screening for reaction with a series of synthetic peptides of E proteins. HPV E polyproteins were found to be immunogenic and immunization with the polyproteins resulted in specific antibody responses to the component E proteins.
Assuntos
Proteínas Oncogênicas Virais/química , Proteínas Oncogênicas Virais/imunologia , Aminoácidos/química , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Western Blotting , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Hibridomas , Immunoblotting , Técnicas Imunoenzimáticas , Linfonodos , Camundongos , Camundongos Endogâmicos BALB C , Níquel/farmacologia , Papillomaviridae/química , Poliproteínas/farmacologia , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/químicaRESUMO
This study investigates the differential capacity of TAP-deficient T2 cells, TAP-competent EBV cells, and immature and mature dendritic cells to present peptides to preformed CTL lines. It demonstrates that presentation of exogenous peptides involves peptide uptake and loading onto newly synthesized MHC class I molecules. This mechanism was best demonstrated for low affinity peptides in the presence of irrelevant peptides competing for HLA binding sites. Under these circumstances, inhibition of protein synthesis with cycloheximide or vesicular trafficking with brefeldin A significantly reduced the presentation of low affinity peptides. This was not restored by adding exogenous beta(2)-microglobulin to stabilize the MHC complex on the cell surface. In contrast, presentation of high affinity peptides was not sensitive to cycloheximide or brefeldin A, which suggests that different mechanisms may operate for presentation of high and low affinity peptides by TAP-competent cells. High affinity peptides can apparently compete with peptides in preloaded MHC class I molecules at the cell surface, whereas low affinity peptides require empty MHC molecules within cells. Accordingly, very high concentrations of exogenous low affinity peptides in conjunction with active MHC class I metabolism were required to allow successful presentation against a background of competing intracellular high affinity peptides in TAP-competent cells. These findings have implications for the design of peptide and protein-based vaccines.
Assuntos
Transportadores de Cassetes de Ligação de ATP/imunologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Apresentação de Antígeno/fisiologia , Peptídeos/imunologia , Peptídeos/metabolismo , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Sequência de Aminoácidos , Antígenos de Neoplasias , Ligação Competitiva , Diferenciação Celular , Linhagem Celular , Membrana Celular/imunologia , Membrana Celular/metabolismo , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Antígeno HLA-A2/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Interferon gama/biossíntese , Líquido Intracelular/imunologia , Líquido Intracelular/metabolismo , Cinética , Antígeno MART-1 , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/metabolismo , Linfócitos T Citotóxicos/imunologiaAssuntos
Antígenos Virais , Glicoproteínas/imunologia , Raiva/história , Vacinas Sintéticas/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Glicoproteínas/genética , História do Século XX , Imunoterapia Ativa , Camundongos , Raiva/imunologia , Raiva/prevenção & controle , Vacinas Sintéticas/genética , Vaccinia virus/genética , Proteínas do Envelope Viral/genéticaRESUMO
The cytotoxic T-lymphocyte (CTL) response to rabies virus glycoprotein has been studied. A primary in vivo CTL response was obtained following inoculation of A/J mice with 10 micrograms of glycoprotein, but only when in the form of reconstituted glycoprotein-lipid vesicles. These glycoprotein-lipid vesicles were prepared with lipids from BHK-21 cells, and did not incorporate mouse major histocompatibility complex (MHC) antigens. Secondary in vitro stimulation of rabies virus-specific CTL was obtained with inactivated virus and with larger quantities of glycoprotein. This response, but not that induced by rabies virus-infected stimulator cells, was dependent on the presence of radiation-resistant accessory cells which could not be replaced with T-cell growth factors. An analysis of the molecular requirements for stimulation of CTL by glycoprotein revealed that cleavage by cyanogen bromide (CNBr) or limited tryptic digestion actually enhanced stimulation of CTL. In contrast, reduction and alkylation destroyed activity. Following separation of CNBr or tryptic fragments by gel electrophoresis or high-pressure liquid chromatography (HPLC) (under nonreducing conditions), a nominal determinant of glycoprotein was identified. One CNBr peptide (residues 103-178) and one peak of tryptic peptides were found to stimulate rabies virus-specific CTL. The tryptic peak was further analyzed by Edman degradation-sequencing, and found to consist of three peptides with amino terminals at residues 130, 251 and 279. This evidence suggests that a nominal determinant of glycoprotein responsible for stimulating rabies virus-specific CTL is located between residues 130-178 of the glycoprotein, and incorporates a single disulfide loop (159-169) which is necessary for biologic activity.
Assuntos
Antígenos Virais/imunologia , Glicoproteínas/imunologia , Vírus da Raiva/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Brometo de Cianogênio/farmacologia , Citotoxicidade Imunológica , Feminino , Camundongos , Camundongos Endogâmicos , Baço/imunologia , Tripsina/farmacologiaRESUMO
Pathogenic parental rabies virus and apathogenic variant virus were shown to differ in their ability to infect neurons in vivo and neuroblastoma cells in vitro. After intracerebral inoculation, the distribution of infected neurons in the brain was similar for both viruses, but the rate of spread throughout the brain, the number of infected neurons, and the degree of cellular necrosis were much lower in the case of apathogenic virus. After adsorption to mouse neuroblastoma cells, apathogenic virus was less rapidly internalized than pathogenic virus, and cell-to-cell spread of apathogenic variant virus was completely prevented by the addition of rabies virus-neutralizing antibody, whereas the spread of pathogenic virus was not affected.
Assuntos
Neurônios/microbiologia , Vírus da Raiva/patogenicidade , Animais , Células Cultivadas , Cerebelo/microbiologia , Cricetinae , Técnicas Imunoenzimáticas , Camundongos , Vírus da Raiva/metabolismo , Receptores Virais/metabolismo , Proteínas Virais/biossíntese , Replicação ViralRESUMO
Inoculation of rabbits and mice with a vaccinia-rabies glycoprotein recombinant (V-RG) virus resulted in rapid induction of high concentrations of rabies virus-neutralizing antibodies and protection from severe intracerebral challenge with several strains of rabies virus. Protection from virus challenge also was achieved against the rabies-related Duvenhage virus but not against the Mokola virus. Effective immunization by V-RG depended on the expression of a rabies glycoprotein that registered proline rather than leucine as the eighth amino acid from its NH2 terminus (V-RGpro8). A minimum dose required for effective immunization of mice was 10(4) plaque-forming units of V-RGpro8 virus. beta-propiolactone-inactivated preparations of V-RGpro8 virus also induced high levels of rabies virus-neutralizing antibody and protected mice against intracerebral challenge with street rabies virus. V-RGpro8 virus was highly effective in priming mice to generate a secondary rabies virus-specific cytotoxic T-lymphocyte response following culture of lymphocytes with either ERA or PM strains of rabies virus.
Assuntos
Glicoproteínas/imunologia , Vacina Antirrábica/imunologia , Vírus da Raiva/imunologia , Animais , DNA Recombinante , Feminino , Genes Virais , Vetores Genéticos , Glicoproteínas/genética , Imunidade Celular , Memória Imunológica , Camundongos , Vírus da Raiva/genética , Linfócitos T Citotóxicos/imunologia , Vaccinia virus/genética , Proteínas Virais/genética , Proteínas Virais/imunologiaRESUMO
The antigenic structure of the rabies virus glycoprotein has been studied. A limited number of fragments were obtained by cyanogen bromide (CNBr) cleavage of viral glycoprotein, and eight large peptides were isolated by using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. These were tested for their capacity to stimulate the proliferation of nylon wool-purified T cells obtained from spleens of rabies-immune A/J mice. Three peptides (Cr1, Cr2 plus Cr2A, and Cr3) stimulated antigen-specific proliferation, indicating that at least three T cell determinants of the native molecule are sequential or continuous in nature. Stimulation was also obtained with 27-residue and 13-residue synthetic peptides (designated R21 and R20, respectively) that included sequences towards the carboxy terminal end of Cr1, but not with synthetic peptides that included sequences of Cr2 and Cr3 (which are both glycosylated in virus-derived material). The intact viral glycoprotein and synthetic peptide R21 stimulated T lymphocytes with surface characteristics of helper cells, and induced the production of interleukin 2 by these lymphocytes. Synthetic peptides R20 and R21 also stimulated a minor population of Lyt-2-positive cells, which were not yet identified as either suppressor or cytotoxic T lymphocytes.
Assuntos
Peptídeos/imunologia , Vírus da Raiva/imunologia , Linfócitos T/imunologia , Proteínas Virais/imunologia , Animais , Anticorpos Monoclonais , Anticorpos Antivirais/biossíntese , Antígenos Virais/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos A , Fragmentos de Peptídeos/imunologia , Peptídeos/síntese química , Fenótipo , Linfócitos T/classificaçãoRESUMO
Macrophages were found to bind directly to Semliki Forest virus (SFV)-infected, but not uninfected, P815 cells. In the presence of anti-H-2d or anti-BALB/c antibody, macrophages lysed SFV-infected, but not uninfected, P815 cells. It is proposed that macrophage-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) proceeds via two functionally distinguishable initial steps: (1) adhesion of effector to target cell, which may be mediated through antibody or through viral protein, as in the case of SFV-infected target cells suboptimally sensitized with antibody; (2) antibody-dependent initiation of cytolysis.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Macrófagos/imunologia , Infecções por Togaviridae/imunologia , Animais , Adesão Celular , Antígenos H-2/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Vírus da Floresta de SemlikiRESUMO
Eighteen strains of virus were recovered by tissue culture techniques from 20 samples of mouse brain received from Harare, Zimbabwe, and typed with monoclonal antibodies at The Wistar Institute. On the basis of reactivity with these monoclonal antibodies specific for rabies and rabies-related viruses, seven strains were identified as Mokola viruses, and the remaining 11, as rabies viruses. Seventeen of 36 monoclonal antibodies against the nucleocapsid antigen reacted with the Mokola strains, but none of 42 monoclonal antibodies against the glycoprotein that neutralized rabies virus was active against Mokola strains. Mokola virus was, however, neutralized by two monoclonal antibodies produced from mice immunized with Mokola, by a specific anti-Mokola serum prepared in rabbits, and to a lesser extent, by three polyclonal high titer antirabies sera of human or rabbit origin. Immunization of mice with a rabies vaccine (antigenic value, 10 international units) at a concentration 30-fold high than that necessary for complete protection against homologous challenge with rabies virus was not protective against Mokola infection. No cross-reactivity between Mokola and rabies viruses was seen with cytotoxic T lymphocytes.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Vírus da Raiva/imunologia , Rhabdoviridae/imunologia , Animais , Gatos , Cães , Camundongos , Testes de Neutralização , Raiva/prevenção & controle , Raiva/veterinária , Vírus da Raiva/isolamento & purificação , Rhabdoviridae/isolamento & purificação , Especificidade da Espécie , ZimbábueRESUMO
Antigenic variation in the glycoprotein of rabies (CVS-11) virus was studied. Neutralization-resistant variant viruses were isolated in vitro at high frequency (10(-4) to 10(-5)) in the presence of anti-glycoprotein monoclonal antibody. Analysis of these variants identified at least three functionally independent antigenic sites, based on the grouping of variants that were no longer neutralized by one or more of a panel of 24 monoclonal antibodies. Competition radioimmunoassay suggested that one of these three antigenic sites was topologically distinct, with the other two in close proximity. In addition, it was shown that most (but not all) neutralization-resistant variants failed to bind the relevant monoclonal antibody. Viruses with altered antigenicity were shown to accumulate in virus stocks following several passages in vitro in the absence of antibody. In addition, variants were isolated in vivo following treatment of mice with monoclonal antibody.
Assuntos
Antígenos Virais/análise , Glicoproteínas/análise , Vírus da Raiva/imunologia , Proteínas Virais/análise , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Variação Genética , RadioimunoensaioRESUMO
The natural killer (NK) cell induced one to two days after Kunjin virus infection of BALB/c mice is cytotoxic for a wide range of syngeneic, allogeneic and xenogeneic cell lines. It is also weakly cytotoxic for some non-malignant cells including mouse fibroblasts, macrophages and thymocytes, but not lymph node cells. Levels of lysis of non-tumour target cells are dependent on their genotype. Furthermore, malignant cell lines may become resistant following transplantation in vivo then susceptible again after culture in vitro. The virus-induced NK cell is elicited as readily in athymic (nude) as in normal mice. X-irradiation inhibits its development if administered prior to infection. It is labile on culture at 37 degrees. The cell carries Fc receptors but its NK activity is not antibody-dependent.
Assuntos
Células Matadoras Naturais/imunologia , Togaviridae/imunologia , Animais , Anticorpos/imunologia , Testes Imunológicos de Citotoxicidade , Interferons/fisiologia , Células Matadoras Naturais/efeitos da radiação , Camundongos , Neoplasias Experimentais/imunologia , Receptores Fc/análiseRESUMO
Natural killer (NK) cells are rapidly elicited in the spleen and peritoneal cavity of mice inoculated intravenously or intraperitoneally with live Kunjin virus, and more slowly in the peritoneal cavity of mice inoculated intraperitoneally with Formalin-inactivated Corynebacterium parvum. NK cells induced by either agent display cytotoxicity for a similar spectrum of syngeneic, allogeneic, and xenogeneic cultured cell lines. By contrast, the cells occurring naturally in the spleen of congenitally athymic (nude) mice show substantially lower NK activity and are cytotoxic for a more restricted range of target cell lines. The distinction suggests that there may be more than one type of NK cell or that activation enhances the cytotoxicity and perhaps broadens the range of target specificity of endogenous NK cells.
Assuntos
Arbovírus/imunologia , Citotoxicidade Imunológica , Células Matadoras Naturais/imunologia , Propionibacterium acnes/imunologia , Animais , Líquido Ascítico/citologia , Adesão Celular , Linhagem Celular , Células Matadoras Naturais/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias/imunologia , Ratos , Organismos Livres de Patógenos Específicos , Baço/citologiaRESUMO
Two new types of cell-mediated immune cytolysis of togavirus-infected cells are compared. The peritoneal cavity of mice 2 days after infection contains a nonadherent, non-phagocytic, non-O-bearing, trypsin-resistant, EDTA-sensitive cell displaying broadly specific cytotoxicity for uninfected or virus-infected syngeneic or xenogeneic cell lines. Peritoneal macrophages from normal mice are cytotoxic to infected target cells sensitized with minute amounts of homologous antiviral IgG.