Association of antigens to ISCOMATRIX adjuvant using metal chelation leads to improved CTL responses.

The association of antigen with ISCOMATRIX trade mark adjuvant has been shown to be important for the optimal induction of cytotoxic T lymphocyte (CTL) responses. Here, we describe a simple broadly applicable method for associating recombinant proteins with hexa-histidine tags to ISCOMATRIX trade mark adjuvant utilising metal-affinity chelating interactions. The metal chelation binding step can be performed in a wide range of buffers, including commonly used denaturants such as urea, which makes it an ideal strategy for formulating proteins which are otherwise insoluble.

Immunogenicity of recombinant HBsAg/HCV particles in mice pre-immunised with hepatitis B virus-specific vaccine.

Due to their spatial structure virus-like particles (VLPs) generally induce effective immune responses. VLPs derived from the small envelope protein (HBsAg-S) of hepatitis B virus (HBV) comprise the HBV vaccine. Modified HBsAs-S VLPs, carrying the immunodominant hypervariable region (HVR1) of the hepatitis C virus (HCV) envelope protein E2 within the exposed 'a'-determinant region (HBsAg/HVR1-VLPs), elicited HVR1-specific antibodies in mice.

Antibody responses to HPV6b E polyproteins and production of monoclonal antibodies.

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.

Exogenous peptides presented by transporter associated with antigen processing (TAP)-deficient and TAP-competent cells: intracellular loading and kinetics of presentation.

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.

Studies on experimental adjuvanted influenza vaccines: comparison of immune stimulating complexes (Iscoms) and oil-in-water vaccines.

Detergent-disrupted influenza virus vaccines, formulated as Iscoms, or oil-in-water (o/w) emulsions, were administered parenterally to mice and evaluated for immunogenicity and protective efficacy. Both formulations enhanced both primary and secondary serum antibody responses. The magnitude of these responses with o/w emulsions was further enhanced by the addition of the non-ionic block copolymer L121 in the emulsion.

Stimulation of cytotoxic T-lymphocyte responses by rabies virus glycoprotein and identification of an immunodominant domain.

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.

Differences in cell-to-cell spread of pathogenic and apathogenic rabies virus in vivo and in vitro.

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.

T cell responses to cleaved rabies virus glycoprotein and to synthetic peptides.

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.

Protection from rabies by a vaccinia virus recombinant containing the rabies virus glycoprotein gene.

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).

Macrophages bind directly to Semliki Forest virus-infected cells and mediate antibody-dependent cell-mediated cytotoxicity.

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.

Antigenic analysis of rabies and Mokola virus from Zimbabwe using monoclonal antibodies.

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.

Antigenic sites on the CVS rabies virus glycoprotein: analysis with monoclonal antibodies.

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.

Antigenic structure of rabies virus glycoprotein: ordering and immunological characterization of the large CNBr cleavage fragments.

After rabies virus glycoprotein was treated with CNBr, the peptide mixture was fractionated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. CNBr-cleaved peptide fragments were resolved into seven peptide bands under reducing conditions and six peptide bands under nonreducing conditions. The isolated nonreduced polypeptides were further analyzed by electrophoresis under reducing conditions.

Further characterization of natural killer cells induced by Kunjin virus.

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.

Comparison of natural killer cells induced by Kunjin virus and Corynebacterium parvum with those occurring naturally in nude mice.

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.

Two cytotoxic cells in peritoneal cavity of virus-infected mice: antibody-dependent macrophages and nonspecific killer cells.

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.