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In this study, we have compared the effector functions and fate of a number of human CTL clones in vitro or ex vivo following contact with variant peptides presented either on the cell surface or in a soluble multimeric format. In the presence of CD8 coreceptor binding, there is a good correlation between TCR signaling, killing of the targets, and FasL-mediated CTL apoptosis. Blocking CD8 binding using alpha3 domain mutants of MHC class I results in much reduced signaling and reduced killing of the targets. Surprisingly, however, FasL expression is induced to a similar degree on these CTLs, and apoptosis of CTL is unaffected. The ability to divorce these events may allow the deletion of antigen-specific and pathological CTL populations without the deleterious effects induced by full CTL activation.

We produced a B cell hybridoma (TW2.3) from vaccinia virus-infected mice that secreted a monoclonal antibody (MAb) reactive with a 25-kDA early viral protein that was localized by laser scanning confocal microscopy to the nucleus and cytoplasmic viral factory regions of infected cells. By cell-free translation of mRNA selected by hybridization to a complete library of vaccinia virus DNA fragments, the immunoreactive polypeptide was mapped to open reading frame E3L. The RNA start site of an early promoter was located 26 nucleotides upstream of the first methionine codon of E3L. Evidence was obtained that translation initiation occurs in vivo and in vitro at both the first and second methionine codons to produce major and minor polypeptides of 25 and 19 kDa, respectively. Both polypeptides bound double-stranded RNA, confirming the recent report of H.-W. Chang, J. C. Watson, and B. L. Jacobs (Proc. Natl. Acad. Sci. USA 89, 4825-4829, 1992). Other vaccinia virus proteins were not required for the nuclear localization of the E3L protein, since MAb TW2.3 bound to the nuclei of uninfected cells that were transfected with the E3L gene under the control of the SV40 early promoter. We also demonstrated that the E3L protein can bind to nuclei of aldehyde fixed and detergent permeabilized uninfected cells. This binding was abrogated by treatment of the cells with RNase but not DNase. The nuclear and cytoplasmic locations of the double-stranded RNA binding protein are consistent with multiple functions in the vaccinia virus infectious cycle.

Of the three major proteins associated with the rabies virus membrane, only the glycoprotein was found to be located on the external surface of the virus membrane. Glycoprotein prepared by treatment of rabies virus with Triton X-100 and purified by isoelectric focusing was found to be homogeneous with respect to size and isoelectric point. This material, which is free of phospholipids, is able to protect in vaccination experiments against a lethal challenge infection with rabies virus. The apparent mol. wt. of this component isolated under non-denaturing conditions is approx. 400000. The same material analysed by SDS polyacrylamide gel electrophoresis (PAGE) was found to consist solely of polypeptide chains of the G protein (mol. wt. 80000). A minor glycoprotein (gp 50), detected by PAGE of the Triton X-100 released material, appeared to be a breakdown product of the G-protein. Therefore the detergent released material represents homopolymers of the G-protein. Whether the antigenic determinants reside on the monomeric subunit or are a property of the polymeric form of the G-protein is discussed.

Purified rabies virus glycoprotein (G) was shown by complement fixation and immunodiffusion tests to be a second distinct antigen of the virus. It it the only structural protein of the virus that induces the formation of virus-neutralizing antibodies and which confers immunity to animals. When the G protein is taken as antigen, the complement fixation test can be used for the assay of virus-neutralizing antibodies. The total protective activity of the virus was recovered in the purified G protein preparation. The protective activity of G protein increased with purification: 9 ng of G protein was required to protect 50% of the mice as compared to 1.63 micrograms of the virus. Selective immunofluorescent membrane staining and immunocytolysis of rabies virus-infected cells were shown to be G protein specific. Due to its purity and potency, the G protein preparation can be considered the ideal human antirabies vaccine.

The three major proteins of vesicular stomatitis virus-Indiana, glycoprotein (G), nucleoprotein (N), and membrane protein (M), were isolated and characterized by means of specific monocomponent antisera. G, N, and M proteins are distinct, nonrelated antigens with specific serological properties. The G protein is the only antigen inducing the formation of virus-neutralizing antibodies and was shown to confer immunity to mice. Specific complement-fixing and precipitating activity was demonstrated for each of the three antisera. The future use of isolated rhabdovirus components and of monospecific antisera is considered for therapeutic and diagnostic purposes as well as for virus strain differentiation and classification work.

The immunogenicity of synthetic peptide dimers based on epitope sequences derived from the mycobacterial 65-kDa antigen and the foot and mouth disease virus (FMDV) VP1 protein was examined in inbred mice. The analysis was directed towards the potential helper role of a T cell stimulatory mycobacterial epitope (65-85) with respect to poorly immunogenic sites either from the same molecule (422-436) or from VP1 (141-160). The 65-85 repeat homodimer induced an antibody response in CBA/ca but not in C57BL/6 mice, both nonresponders to the 65-85 monomer, and amplified the antibody response in BALB/c, monomer-responder mice. Analysis of the immunogenicity of hybrid dimers in BALB/c mice showed that the orientation of peptides within the dimer is critical for the extent of the produced antibody response. Only the 422-436/65-85 but not the 65-85/422-436 induced antibodies binding to the 422-436 sequence which was nonimmunogenic when injected either as a monomer or dimer. Despite the striking difference in immunogenicity, both tested hybrid dimers reacted equally in the solid-phase immunoassay with antisera raised to 65-85-dimer or 422-436/65-85 peptides or with a monoclonal antibody to the 422-436 epitope. The described differences in antibody responsiveness also cannot be attributed merely to the extent of T cell stimulation since the proliferative responses were uniformly expressed for all relevant combinations of peptides. Antisera to 65-85 dimer and 422-436/65-85 hybrid also reacted with the native 65-kDa protein. Furthermore, the production of FMDV-neutralizing antibodies in response to the 141-160 (VP1-derived)/65-85 hybrid peptide in 141-160 nonresponder B10.D2 mice also confirmed the helper activity of the 65-85 epitope. Thus, combining heterologous peptides with the N-terminal of the mycobacterial 65-85 sequence may be generally applicable for the potentiation of peptide vaccines.

The five structural proteins of rabies virus, L, G, N, M1 and M2 have been re-evaluated in light of recent results. It has been found that the M1 protein is associated with the N protein which together with the L protein and the RNA forms the nucleocapsid complex. Only the G and M2 proteins could be visualized on the surface of infected cells. Therefore, it is proposed that the M1 protein be renamed NS analogous to vesicular stomatitis virus and that the M2 protein, being the only membrane protein, now be called simply the M protein.

Of the three major proteins associated with the rabies virus membrane, only the glycoprotein (G-protein) was found to be located on the external surface of the viral membrane. A minor glycoprotein (gp 50) detected by SDS-polyacrylamide gel electrophoresis (PAGE) of rabies virus appeared to be a breakdown product of the G-protein. Glycoprotein prepared by treatment of rabies virus with Triton X100 and purified by isoelectric focusing was found to be homogeneous with respect to size and isoelectric point. The apparent molecular weight of this component isolated under nondenaturing conditions is approximately 400,000 daltons. The same material analyzed by PAGE was found to be comprised solely of polypeptide chains of the G-protein (MW 80,000 daltons). Therefore, the Triton X100-released material represents homopolymers of the G-protein. The purified rabies virus glycoprotein (G) is only structural protein of the virus that induces the formation of virus-neutralizing antibodies and which confers immunity to animals. The total protective activity of the virus was recovered in the purified G protein preparation. The protective activity of G protein increased with purification : 9 ng of G protein was required to protect 50% of the mice as compared to 1.63 microgram of the virus. The number of oligosaccharide side chains on rabies virus glycoprotein was investigated. Analysis of glycopeptides obtained by protease digestion of desialated glycoprotein revealed three discrete glycopeptides. Comparison of the protease digestion products from desialated and from untreated glycoprotein indicated a heterogeneity among the glycopeptides in the sialic acid content. Two major tryptic glycopeptides were isolated from desialated rabies virus glycoprotein and were analyzed after protease digestion; one contained two oligosaccharide side chains and the other contained a single oligosaccharide side chain.