双抗体夹心ELISA法检测狂犬病疫苗抗原活性组分

采用多克隆双抗体夹心ＥＬＩＳＡ法快速检测狂犬病毒抗原含量。结果表明,灵敏度达到１５μｇ／ｍｌ,同时特异性及变异系数均合乎要求。本方法用于精制狂苗制备过程中有效抗原活性组分的检测准确、快速、重复性好,且抗原的ＥＬＩＳＡ效价与ＮＩＨ动物法测定效价具有平行趋势。     

Antigenic site II of the rabies virus glycoprotein: structure and role in viral virulence.

Twelve monoclonal antibodies neutralizing the CVS strain of rabies virus were used to characterize antigenic site II of the viral glycoprotein. Nineteen antigenic mutants resistant to neutralization by some of these antibodies were selected; some continued to normally or partially bind the antibody, whereas others did not. Mutations conferring resistance to neutralization by site II-specific monoclonal antibodies were localized into two clusters, the first between amino acids 34 and 42 (seven groups of mutants) and the second at amino acids 198 and 200 (three groups of mutants). Two intermediate mutations were identified at positions 147 and 184. Four mutations resulted in reduced pathogenicity after intramuscular inoculation of the virus in adult mice. One of the mutants, M23, was 300 times and the others were 10 to 30 times less pathogenic than CVS. In three cases the attenuated phenotype was related to an important modification of antigenic site II, whereas the other known antigenic sites were unchanged.     

Antigenic properties of the human immunodeficiency virus envelope during cell-cell fusion

Human immunodeficiency virus (HIV) fusion and entry involves sequential interactions between the viral envelope protein, gp120, cell surface CD4, and a G-protein-coupled coreceptor. Each interaction creates an intermediate gp120 structure predicted to display distinct antigenic features, including key functional domains for viral entry. In this study, we examined the disposition of these features during the fusion of HeLa cells expressing either HIV(HXB2) envelope (Env cells) or CXCR4 and CD4 (target cells). Cell-cell fusion, indicated by cytoplasmic dye transfer, was allowed to progress for various times and then arrested. The cells were then examined for reactivity with antibodies directed against receptor-induced epitopes on gp120. Analyses of cells arrested by cooling to 4( degrees )C revealed that antibodies against the CD4-induced coreceptor-binding domain, i.e., 17b, 48d, and CG10, faintly react with Env cells even in the absence of target cell or soluble CD4 (sCD4) interactions. Such reactivity increased after exposure to sCD4 but remained unchanged during fusion with target cells and was not intensified at the Env-target cell interface. Notably, the antibodies did not react with Env cells when treated with a covalent cross-linker either alone or during fusion with target cells. Immunoreactivity could not be promoted or otherwise altered on either temperature arrested or cross-linked cells by preventing coreceptor interactions or by using a 17b Fab. In comparison, two other gp120-CD4 complex-dependent antibodies against epitopes outside the coreceptor domain, 8F101 and A32, exhibited a different pattern of reactivity. These antibodies reacted with the Env-target cell interface only after 30 min of cocultivation, concurrent with the first visible transfer of cytoplasmic dye from Env to target cells. At later times, the staining surrounded entire syncytia. Such binding was entirely dependent on the formation of gp120-CD4-CXCR4 tricomplexes since staining was absent with SDF-treated or coreceptor-negative target cells. Overall, these studies show that access to the CD4-induced coreceptor-binding domain on gp120 is largely blocked at the fusing cell interface and is unlikely to represent a target for neutralizing antibodies. However, new epitopes are presented on intermediate gp120 structures formed as a result of coreceptor interactions. Such findings have important implications for HIV vaccine approaches based on conformational alterations in envelope structures.     

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. The N-terminal amino acid sequences were determined for the peptides in each of the isolated bands. The sequence data identified eight CNBr peptides and allowed the peptide fragments to be ordered within the deduced amino acid sequence of the glycoprotein. Analysis of the nonreduced CNBr peptides revealed two conformations of the glycoprotein. Two CNBr peptide fragments were specifically immunoprecipitated with a hyperimmune anti-rabies glycoprotein serum. These two and one other CNBr peptide induced the production of rabies virus-neutralizing antibodies, indicating the existence of at least three distinct antigenic sites on the rabies virus glycoprotein.     

Antigenic properties of the human immunodeficiency virus transmembrane glycoprotein during cell-cell fusion

Human immunodeficiency virus (HIV) entry is triggered by interactions between a pair of heptad repeats in the gp41 ectodomain, which convert a prehairpin gp41 trimer into a fusogenic three-hairpin bundle. Here we examined the disposition and antigenic nature of these structures during the HIV-mediated fusion of HeLa cells expressing either HIV(HXB2) envelope (Env cells) or CXCR4 and CD4 (target cells). Cell-cell fusion, indicated by cytoplasmic dye transfer, was allowed to progress for various lengths of time and then arrested. Fusion intermediates were then examined for reactivity with various monoclonal antibodies (MAbs) against immunogenic cluster I and cluster II epitopes in the gp41 ectodomain. All of these MAbs produced similar staining patterns indicative of reactivity with prehairpin gp41 intermediates or related structures. MAb staining was seen on Env cells only upon exposure to soluble CD4, CD4-positive, coreceptor-negative cells, or stromal cell-derived factor-treated target cells. In the fusion system, the MAbs reacted with the interfaces of attached Env and target cells within 10 min of coculture. MAb reactivity colocalized with the formation of gp120-CD4-coreceptor tricomplexes after longer periods of coculture, although reactivity was absent on cells exhibiting cytoplasmic dye transfer. Notably, the MAbs were unable to inhibit fusion even when allowed to react with soluble-CD4-triggered or temperature-arrested antigens prior to initiation of the fusion process. In comparison, a broadly neutralizing antibody, 2F5, which recognizes gp41 antigens in the HIV envelope spike, was immunoreactive with free Env cells and Env-target cell clusters but not with fused cells. Notably, exposure of the 2F5 epitope required temperature-dependent elements of the HIV envelope structure, as MAb binding occurred only above 19 degrees C. Overall, these results demonstrate that immunogenic epitopes, both neutralizing and nonneutralizing, are accessible on gp41 antigens prior to membrane fusion. The 2F5 epitope appears to depend on temperature-dependent elements on prefusion antigens, whereas cluster I and cluster II epitopes are displayed by transient gp41 structures. Such findings have important implications for HIV vaccine approaches based on gp41 intermediates.     

Induction and biological properties of defective interfering particles of rabies virus.

A method for obtaining large quantities of defective interfering (DI) rabies virus particles that fulfill all the criteria delineated by Huang and Baltimore (1970) is described. The purified rabies DI virion was found to be much shorter (60 to 80 nm) than the complete virion (180 nm) and to have a viral genome of about half the size of normal rabies RNA but with all of the structural proteins of standard virions. Rabies DI virions were noninfectious for both cells in culture and for animals. As determined by in vitro and in vivo techniques, interference with the replication of standard virus was specific to rabies virus. The possible role of rabies DI virion in the pathogenicity of rabies virus infection and in the establishment of attenuated strains for use as live rabies vaccines is discussed.     

Antigenic properties of synthetic fragments of the heavy chain of influenza virus A hemagglutinin

The antigenic properties of the synthetic peptides corresponding to the regions 122-133, 136-147, 154-164 of the virus A/Aichi/2/68 hemagglutinin heavy chain have been studied. The 122-133 and 136-147 peptides comprise together almost whole antigenic determinant A, while the 154-164 peptide is a part of determinant B. Rabbits immunized by the peptides conjugated with carrier-protein BSA gave the high immune response to hapten-peptides. Each antiserum reacted only with homologous conjugate. All the antipeptide serums reacted with the virus A/Aichi/2/68 fixed on the base. Conjugate of the 136-147 peptide reacted with the rabbit antiserum against the virus A/Aichi/2/68 rendering the direct evidence to location of at least one hemagglutinin antigenic determinant in the region 136-147.     

Apoptosis and rabies virus neuroinvasion

Rabies virus (RV) causes a non-lytic infection of neurons leading to a fatal myeloencephalitis in mammals including humans. By comparing the infection of the nervous system of mice by a highly pathogenic neuroinvasive strain of RV (CVS) and by a strain of attenuated pathogenicity (PV) with restricted brain invasion, we showed that RV neuroinvasiveness results of three factors: not only neurotropic RV avoids induced neuron cell death but also "protective" T cells that migrate into the infected nervous system are killed by apoptosis and finally inflammation of the infected nervous system is limited. Our data suggest that the preservation of the neuronal network, the limitation of the inflammation and the destruction of T cells that invade the CNS in response to the infection are crucial events for RV neuroinvasion and for transmission of RV to another animal.