Prospects of the use of bacteriophage-based virus-like particles in the creation of anthrax vaccines

The profitability of vaccine production is less than that of other pharmaceutical goods worldwide. Thus, the cost of the vaccine substance determines the range of vaccines available for use. This is of particular importance for veterinary vaccines. In this review, we have surveyed the published data on exploited vaccines and concluded that the immunogenicity of antigen substances based on whole virions is higher than that of soluble antigens. The physiological basis of this phenomenon remains unknown; however, it may explain why most of the described recombinant vaccines have not yet been put into practice. All practically implemented antiviral vaccines (except that for hepatitis B) are based on viral substances produced by conventional cultural technologies. In light of this observation, an approach to the development of a universal platform for recombinant vaccines produced in the form of virus-like particles is suggested. To this end, a technique of designing fused bifunctional derivatives of bacteriophage proteins containing antigens of interest should be involved. The approach is depicted with the use of the protective anthrax antigen, a conventional vaccine antigen.     

Viral immunoblotting: A sensitive method for detecting viral-specific oliogoclonal bands in unconcentrated cerebrospinal fluid

A new method for detecting viral antibodies in cerebrospinal fluid is described. The technique has many advantages over previously published methods in that it is highly sensitive eliminating the need to concentrate the CSF, takes 5 h to complete, avoids the use of radionucleides, and most importantly circumvents problems associated with prozone effects which occur in immunoprecipitation reaction since the viral antigen is immobilized on nitrocellulose membranes.     

Synthesis of Highly Immunogenic Multiple Antigenic Peptides for Epitopes of Viral Antigen to Use in ELISA

Synthesis of multiple antigenic peptides (MAPs) for predicted antigenic determinants of a viral antigen is described. The method includes prediction of linear epitopes using predictive computer algorithms, synthesis of peptides for the predicted regions, testing of peptides to find the most reactive sites, synthesis of MAPs and their testing. The procedure involves manual synthesis of MAPs by solid phase peptide synthesis with Wang resin as solid support. The MAPs were prepared in eight copies and used for immunization of rabbits to generate anti-peptide antibodies. Further, the reactivity of MAPs in detecting the native cognate antigen in the whole virus was confirmed by ELISA. The MAP and anti-peptide antibodies could serve as diagnostic tools for viral diseases. MAPs have efficiently been used to confirm the presence of linear antigenic and immunogenic epitopes on viral proteins, for possible use in diagnostic and vaccine. It was suggested that this method could help in epitope mapping of dreadful human or animal pathogens as it involves production of safe, chemically defined and non-infectious materials for use as antigen as well as immunogen.     

Growth experiment of Hantaan virus in A549 cells: an attempt to improve the immunofluorescent antibody technique for hemorrhagic fever with renal syndrome

An assay method for the infectivity of Hantaan virus, a causative agent of HFRS (hemorrhagic fever with renal syndrome), was developed by the use of IFA (immunofluorescent antibody technique). With the aid of this method, the growth characteristics of Hantaan virus, 76-118 strain, were followed in A549 cells. At a maximal MOI (multiplicity of infection) of 1.6 VAIU (viral antigen-inducing units) per cell, the conventionally available value, plateau level potencies of the viral antigen and virus infectivity were attained at eight and ten days postinfection, respectively, and most of the infective virus produced accumulated in the culture fluids of infected cells. When infections were defined with MOI values in terms of VAIU per cell, development of the viral antigen was highly consistent and followed a given pattern of kinetics. Based on these findings, a protocol for preparation of the viral antigen in IFA was presented, wherein spot culture and FBS treatment were emphasized as effective procedures to minimize non-specific staining.     

Hepatitis delta virus ribonucleoproteins shuttle between the nucleus and the cytoplasm

Hepatitis delta virus (HDV) infection of individuals infected with hepatitis B virus (HBV) is associated with more severe liver damage and an increased risk of fulminant disease. HDV is a single-stranded RNA virus that encodes a single protein, the delta antigen, which is expressed in two forms, small (S-HDAg) and large (L-HDAg). Here we show that although HDV ribonucleoproteins are mainly detected in the nucleus, they are also present in the cytoplasm of cells infected with HDV or transfected with HDV cDNA. Making use of an heterokaryon assay, we demonstrate that HDV ribonucleoproteins shuttle continuously between the nucleus and the cytoplasm. In the absence of HDV RNA, both forms of the delta antigen are retained in the nucleus, whereas in the absence of the delta antigen, HDV RNA is predominantly detected in the cytoplasm. Coexpression of HDV RNA and S-HDAg (which binds to the viral RNA and contains a nuclear localization signal) results in nuclear accumulation of the viral RNA. This suggests that HDV RNA mediates export of viral particles to the cytoplasm whereas the delta antigen triggers their reimport into the nucleus.     

Phenotypic complementation of the SV40 tsA mutant defect in viral DNA synthesis following microinjection of SV40 T antigen.

African green monkey cells (CV-1P) were microinjected with highly purified SV40 T antigen using protein-loaded red cell ghosts and polyethylene glycol as fusagen. The microinjected cells were infected with a temperature-sensitive mutant of SV40 (tsA209) which is defective in the initiation of viral DNA synthesis. Using in situ hybridization as an assay method, we found that PEG-microinjection of both partially and highly purified T antigen resulted in an increase in the amount of viral DNA sequences in the monolayer. Moreover, 3H-thymidine-labeled and unlabeled Hirt supernatant from microinjected, tsA209-injected cells contained significantly more SV40 DNA than comparable extracts from sham-injected, tsA209-infected or uninfected cells, which were tested in parallel. Thus the introduction of highly purified, "large" SV40 T antigen led to phenotypic complementation of the tsA defect in viral DNA synthesis.     

An immunosorbent method for the quantitative determination of antiliver autoantibodies in patients with viral hepatitis

Autoimmune processes in patients with mild, moderate and severe forms of viral hepatitis B have been studied with the use of a new immunosorbent method for the quantitative determination of autoantibodies (auto-Ab), developed by the authors. The immunosorbent has been obtained on the basis of granulated aerosil, an active protein-adsorbent agent used as a solid antigen carrier. The level of auto-Ab in patients with all forms of viral hepatitis B has been found to exceed the content of such antibodies in practically healthy persons. Besides, in patients with severe and prolonged forms of the disease the content of antihepatic auto-Ab has been found to remain high even after clinical convalescence, which is indicative of the continuation of the autoimmune process.     

Mechanisms of infection with Epstein-Barr virus. I. Viral DNA replication and formation of noninfectious virus particles in superinfected Raji cells.

Human lymphoblastoid Raji cells, which do not produce virus, supported replication of Epstein-Barr virus (EBV) upon superinfection. Early antigen, viral capsid antigen, and virions were produced in Raji cells superinfected with EBV. Viral DNA replicated under complete inhibition of host cell DNA synthesis to the extent that a few micrograms of EBV DNA were recovered from 107 superinfected Raji cells, corresponding to 5,000 viral genomes/cell. Homology of the synthesized viral DNA to parental EBV DNA was more than 90%. Virions produced by the Raji cells contained a 55S DNA but failed to induce early antigen, viral capsid antigen, and viral DNA synthesis after a second superinfection of Raji cells.     

Studies of novel bioactive substances in the spent media of cell lines using protein-free culture system]

Many human cell lines have been maintained in fetal bovine serum (FBS)-supplemented medium. These produce and secrete many substances such as transferrin, alpha 1-antitrypsin, alpha 2-macroglobulin, alkaline phosphatase, gamma-glutamyltranspeptidase, creatine kinase, carcino-embryonic antigen, alpha-fetoprotein, and cytokines including colony stimulating factors and transforming growth factors and further they may produce small amounts of unknown substances. Usually, small amounts of substances have to be concentrated as highly as possible for detection, but FBS interferes with procedures. A protein-free culture system in an ideal method for detecting small quantities of substances which originate from cell lines without interference by FBS. Our protein-free culture system can be available in every laboratory since this is not only an economical method, but also an effective method for the saving of purification procedures. Moreover, this is a most suitable method for surveying unknown substances derived from cell lines.     

Association of simian virus 40 T antigen with the nuclear matrix of infected and transformed monkey cells.

The subnuclear distribution of simian virus 40 large T antigen within nuclei of transformed Cos and C6 monkey cells was examined. Cos cells express wild-type T antigen but lack viral sequences required for DNA replication, whereas C6 cells contain a functional viral origin but express a replication-defective mutant T antigen which is unable to bind specifically to viral DNA. Discrete subpopulations of T antigen were isolated from the soluble nucleoplasm, chromatin, and nuclear matrix of both cell lines. Although only a small quantity (2 to 12%) of the total nuclear T antigen from Cos cells was associated with the nuclear matrix, a high proportion (25 to 50%) of C6 T antigen was bound to this structure. Results obtained from lytically infected monkey cells showed that early in infection, before viral replication was initiated, a higher proportion (22%) of T antigen was found associated with the nuclear matrix compared with amounts found associated with this structure later in infection (5 to 8%). These results suggest that an increased association of T antigen with this structure is not correlated with viral replication. T antigen isolated from the C6 nuclear matrix was more highly phosphorylated than was soluble C6 T antigen and was capable of binding to the host p53 protein. C6 DNA contains three mutations: two corresponding to N-terminal changes at amino acid positions 30 and 51 and a third located internally at amino acid position 153. By analysis of the subnuclear distribution of T antigen from rat cells transformed by C6 submutant T antigens, it was determined that one or both of the mutations at the NH2 terminus are responsible for the increased quantity of C6 T antigen associated with the nuclear matrix. These results suggest that neither a functional viral DNA replication origin nor the origin binding property of T antigen is required for association of this protein with the nuclear matrix.     

Description of a murine B lymphoma tumor-specific antigen

By using monoclonal antibodies, a tumor-specific antigen (TSA 41.5) was detected on the cell surface of a B lymphoma CH-1 tumor variant, CH-1.1. This antigen is not expressed by normal lymphocytes (spleen cells, lymph node cells, thymocytes, bone marrow cells, or blast cells) of B10.A mice, the host strain of CH-1.1, or by the CH-1 lymphoma. Immunoprecipitation and biochemical characterization of TSA 41.5 with the use of two-dimensional gel electrophoresis showed this antigen to be a surface protein of CH-1.1 cells with an Mr of 80k and pI of 4.6. TSA 41.5 is not related to the murine transferrin receptor, and not to gp70, a viral envelope protein expressed by CH-1.1 cells, shown by comparative peptide map analysis of these three proteins. TSA 41.5 is a surface antigen unique to the CH-1.1 tumor, which is not expressed by the 19 different murine tumor lines that were tested nor by spleen cells of 15 independent mouse strains. In addition, treatment of spleen cells with bacterial lipopolysaccharide did not induce the expression of TSA 41.5. These characteristics of TSA 41.5 make it unlikely to be a product of viruses. Additional evidence against TSA 41.5 being a viral protein was obtained by the observation that antisera against viral proteins could not block the binding of the anti-TSA monoclonal antibody to its antigen. In vitro treatment of CH-1.1 cells with anti-TSA monoclonal antibody specifically inhibited the in vitro growth of the tumor cells in a dose-dependent fashion. The CH-1.1 tumor and monoclonal antibodies could be a useful murine model system for the exploration of the use of monoclonal antibodies for the in vivo treatment of cancer.     

Epstein-Barr Viral Antigen in Single Cell Clones of Two Human Leukocytic Lines

A method was devised for obtaining single cell clones of human leukocytic cell lines in the presence of a human placental cell feeder layer. Clones of two lines, LS-B and EB₃, which contain Epstein-Barr viral (EBV) antigen in approximately 1% of cells were tested for EBV antigen. Since all EB₃ clones and LS-B clones contained EBV antigen, it is concluded that in vitro EBV genome is associated with all of the cells.     

Induction of Simian Virus 40 Antigen in BSC1 Transformed Cells

Heating to 45 C induced in virus-free clones of simian virus 40 (SV40) transformed BSC₁ cells the synthesis of SV40 viral antigen, as evidenced by immunofluorescence. Up to 3.8% of the cells exhibited viral antigen 72 hr after heating to 45 C for 30 min. Depletion of arginine from the medium of the heated cells enhanced and increased the percentage of cells synthesizing viral antigen to 11%. Cytosine arabinoside completely inhibited the induction of the viral antigen. No infectious virus was recovered from the cells in which synthesis of viral antigen was induced. However, small amounts of infectious SV40 virus were rescued from the BSC₁ transformed cells by fusion with rabbit kidney cells or by treatment with mitomycin C.     

Specific mutation of a gammaherpesvirus-expressed antigen in response to CD8 T cell selection in vivo

Herpesviruses are thought to be highly genetically stable, and their use as vaccine vectors has been proposed. However, studies of the human gammaherpesvirus, Epstein-Barr virus, have found viral isolates containing mutations in HLA class I-restricted epitopes. Using murine gammaherpesvirus 68 expressing ovalbumin (OVA), we examined the stability of a gammaherpesvirus antigenic locus under strong CD8 T cell selection in vivo. OVA-specific CD8 T cells selected viral isolates containing mutations in the OVA locus but minimal alterations in other genomic regions. Thus, a CD8 T cell response to a gammaherpesvirus-expressed antigen that is not essential for replication or pathogenesis can result in selective mutation of that antigen in vivo. This finding may have relevance for the use of herpesvirus vectors for chronic antigen expression in vivo.