Recombinant vaccinia virus induces neutralising antibodies in rabbits against Epstein-Barr virus membrane antigen gp340.

The Epstein-Barr virus membrane antigen gene gp340 was isolated, inserted into several strains of vaccinia virus and expressed under the control of a vaccinia virus promoter. The EBV-derived protein which was produced by the recombinant vaccinia viruses was heavily glycosylated, readily labelled with threonine, could be detected at the surface of infected cells and had a mol. wt. of approximately 340 kd, all of which are properties of the authentic gp340. Polyclonal rabbit antisera against gp340 and an EBV-neutralising anti-gp340 monoclonal antibody both recognised cells infected with the recombinant vaccinia viruses. Moreover, rabbits vaccinated with one of the recombinants produced antibodies that recognised EBV-containing lymphoblastoid cells and neutralised EBV.     

Antibody profiling by proteome microarray reveals the immunogenicity of the attenuated smallpox vaccine modified vaccinia virus ankara is comparable to that of Dryvax

Modified vaccinia virus Ankara (MVA) is a highly attenuated vaccinia virus that is under consideration as an alternative to the conventional smallpox vaccine Dryvax. MVA was attenuated by extensive passage of vaccinia virus Ankara in chicken embryo fibroblasts. Several immunomodulatory genes and genes that influence host range are deleted or mutated, and replication is aborted in the late stage of infection in most nonavian cells. The effect of these mutations on immunogenicity is not well understood. Since the structural genes appear to be intact in MVA, it is hypothesized that critical targets for antibody neutralization have been retained. To test this, we probed microarrays of the Western Reserve (WR) proteome with sera from humans and macaques after MVA and Dryvax vaccination. As most protein sequences of MVA are 97 to 99% identical to those of other vaccinia virus strains, extensive binding cross-reactivity is expected, except for those deleted or truncated. Despite different hosts and immunization regimens, the MVA and Dryvax antibody profiles were broadly similar, with antibodies against membrane and core proteins being the best conserved. The responses to nonstructural proteins were less well conserved, although these are not expected to influence virus neutralization. The broadest antibody response was obtained for hyperimmune rabbits with WR, which is pathogenic in rabbits. These data indicate that, despite the mutations and deletions in MVA, its overall immunogenicity is broadly comparable to that of Dryvax, particularly at the level of antibodies to membrane proteins. The work supports other information suggesting that MVA may be a useful alternative to Dryvax.     

Shope Fibroma Virus I. Biological and Molecular Properties of a Cytocidal and a Noncytocidal Strain

The biological and molecular properties of two strains of Shope fibroma virus (SFV) were compared. SFV-I was highly cytocidal to most of the cell lines tested and produced pocks in the chorioallantoic membrane of chick embryos. By contrast, SFV-W did not produce cytopathic effects in any of the cell lines or in the chorioallantoic membrane, but it induced characteristic foci 3 to 4 days after infection. Both strains produced tumors when inoculated into the skin of susceptible rabbits. Maximal infectivity in BSC-1 cells was reached by both strains between 24 to 48 h after inoculation. Viral DNA synthesis also took place at the same time, although cells infected with SFV-I incorporated three times more [(3)H]thymidine than cells infected with SFV-W. Sedimentation analysis and hydroxylapatite chromatography of the two viral DNAs indicated that their molecular weights were similar and that both were naturally cross-linked. Digestion with three restriction endonucleases, however, revealed that they had different restriction sites. When SFV-I and vaccinia DNA were compared, the restriction patterns were more alike. Analysis of the virion structural proteins by gel electrophoresis indicated that SFV-I, SFV-W, and vaccinia virus had many polypeptides in common, although there were distinctive differences among the three viruses. Finally, the results of plaque neutralization tests with different antisera showed that SFV-I and SFV-W shared common antigens and that vaccinia antiserum inhibited SFV-I but not SFV-W. We conclude that the SFV-I genome contains information for both cytolysis and tumorigenesis. This unusual virus may be a recombinant between an orthopoxvirus and a leporipoxvirus.     

Induction of protective immunity in animals vaccinated with recombinant vaccinia viruses that express PreM and E glycoproteins of Japanese encephalitis virus.

A cDNA clone representing the genome of structural proteins of Japanese encephalitis virus (JEV) was inserted into the thymidine kinase gene of vaccinia virus strains LC16mO and WR under the control of a strong early-late promoter for the vaccinia virus 7.5-kilodalton polypeptide. Indirect immunofluorescence and fluorescence-activated flow cytometric analysis revealed that the recombinant vaccinia viruses expressed JEV E protein on the membrane surface, as well as in the cytoplasm, of recombinant-infected cells. In addition, the E protein expressed from the JEV recombinants reacted to nine different characteristic monoclonal antibodies, some of which have hemagglutination-inhibiting and JEV-neutralizing activities. Radioimmunoprecipitation analysis demonstrated that two major proteins expressed in recombinant-infected cells were processed and glycosylated as the authentic PreM and E glycoproteins of JEV. Inoculation of rabbits with the infectious recombinant vaccinia virus resulted in rapid production of antiserum specific for the PreM and E glycoproteins of JEV. This antiserum had both hemagglutination-inhibiting and virus-neutralizing activities against JEV. Furthermore, mice vaccinated with the recombinant also produced JEV-neutralizing antibodies and were resistant to challenge with JEV.     

Demonstration of a tumor-associated surface antigen in Marek's disease.

Surface antigenic markers were detected on three classes of Marek's disease (MD) tumor cells, i.e., MD lymphoma cells, cultured cells of the MSB-1 lymphoblastoid cell line, and JMV lymphoblastic leukemia cells, by indirect membrane immunofluorescent staining with serum from chickens immunized with JMV cells or from rabbits immunized with MSB-1 cells. This surface antigen was not detected on normal chicken lymphocytes, RPL-16 tumor cells (tranedormed by an avian RNA virus, or MD virus-infected fibroblasts that were positive for viral membrane antigen (MA). Furthermore, the surface antigen appeared unrelated to embryonic or histocompatibility antigens. This antigen is provisionally designated as a Marek's disease tumor-associated surface antigen (MATSA). The MATSA's on JMV, MSB-1 and MD lymphoma cells were related but not identical as demonstrated by antiserum titration, absorption and blocking tests with homologous and heterologous systems.     

Assembly of vaccinia virus: role of the intermediate compartment between the endoplasmic reticulum and the Golgi stacks

Vaccinia virus, the prototype of the Poxviridae, is a large DNA virus which replicates in the cytoplasm of the host cell. The assembly pathway of vaccinia virus displays several unique features, such as the production of two structurally distinct, infectious forms. One of these, termed intracellular naked virus (INV), remains cells associated while the other, termed extracellular enveloped virus (EEV), is released from the cell. In addition, it has long been believed that INVs acquire their lipid envelopes by a unique example of de novo membrane biogenesis. To examine the structure and assembly of vaccinia virus we have used immunoelectron microscopy using antibodies to proteins of different subcellular compartments as well as a phospholipid analysis of purified INV and EEV. Our data are not consistent with the de novo model of viral membrane synthesis but rather argue that the vaccinia virus DNA becomes enwrapped by a membrane cisterna derived from the intermediate compartment between the ER and the Golgi stacks, thus acquiring two membranes in one step. Phospholipid analysis of purified INV supports its derivation from an early biosynthetic compartment. This unique assembly process is repeated once more when the INV becomes enwrapped by an additional membrane cisterna, in agreement with earlier reports. The available data suggest that after fusion between the outer envelope and the plasma membrane, mature EEV is released from the cell.     

Protein Kinase and Specific Phosphate Acceptor Proteins Associated with Vaccinia Virus Cores

Incubation of purified vaccinia virus with gamma-(32)P-adenosine triphosphate resulted in the incorporation of (32)P into hot trichloroacetic acid-insoluble material. Enzymatic activity was completely dependent on the addition of divalent cations and was stimulated by nonionic detergents and dithiothreitol. Chemical studies demonstrated that serine and threonine residues of 15,000 molecular weight viral polypeptides were phosphorylated. In contrast, the major structural proteins were not phosphorylated or were phosphorylated to a much lesser extent. Added histones and protamine, but not serum albumin, casein, or phosvitin were phosphorylated by the partially disrupted vaccinia virus preparations. The protein kinase was tightly associated with vaccinia virus particles since the specific enzymatic activity remained constant during the final steps of virus purification, the specific activities of many different preparations of virus were similar, and the enzymatic activity cosedimented with vaccinia virus during rate zonal sucrose gradient and potassium tartrate gradient equilibrium centrifugations. Controlled degradation of vaccinia virus, with nonionic detergents and dithiothreitol, indicated that both the protein kinase and the specific phosphate acceptor proteins were located in the virus core.     

Expression of a major bovine rotavirus neutralisation antigen (VP7c) in Escherichia coli

Sequences from genomic RNA segment 8 of the United Kingdom tissue-culture (t.c.)-adapted bovine rotavirus encoding a major viral neutralisation antigen VP7c have been expressed in Escherichia coli. Expression under the regulated control of the bacteriophage lambda pR promoter was as a C-terminal extension to E. coli beta-galactosidase (beta Gal). Following temperature induction, high levels of the fusion protein were synthesised and accumulated in induced cells, making up 5%-15% of total bacterial cell protein after 2 h of induction. Immunisation of sero-negative rabbits and mice with gel-purified fusion-protein raised antibodies, which gave specific immunofluorescence with virus-infected cells and were able to immunoprecipitate proteins of the VP7 complex from such cells. Hyperimmune sera also gave a virus-type-specific reaction in a solid-phase enzyme-linked immunoabsorbant assay and neutralised virus infectivity in standard plaque-reduction assays.     

Characterization of the major duck hepatitis B virus core particle protein.

The amino acid composition of the major duck hepatitis B virus (DHBV) core particle proteins was determined. The results of this analysis indicated that cores are composed of a single major protein that initiates translation from the second available AUG in the DHBV core gene. Proteins isolated from core particles purified from the cytoplasm of DHBV-infected duck hepatocytes exhibited heterogeneity in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, independent of the stage of viral DNA maturation. Incubation of native cores with alkaline phosphatase removed this heterogeneity, indicating that phosphorylation of external amino acids was responsible. Core protein isolated from mature DHBV purified from serum of infected animals did not display heterogeneity, suggesting a possible role for dephosphorylation in virus maturation.     

Neutralisation of herbicides. Effects on wall vegetation

The chemical control of wall vegetation growing on monuments has been stated as a suitable instrument of conservative intervention. However, often the use of herbicides has not been the most suitable, endangering either the environment or the monument in question. In particular, when dealing with limestone monuments, the chemical control has to be made very carefully because all the carbonate rocks are particularly sensitive to the weathering action of acids. Thus, to prevent this damage, two acid herbicides were neutralised with potassium hydroxide. This neutralisation did, in fact, decrease the damage caused by their acidity on the limestone. This paper deals with the effects of these neutralised herbicides on higher plants. Several experiments were made to determine whether the efficiency of the herbicides was affected. Also, the neutralisation did not affect the ability of the herbicides to eliminate the wall vegetation.     

Vaccinia virus leads to ATG12–ATG3 conjugation and deficiency in autophagosome formation

The interactions between viruses and cellular autophagy have been widely reported. On the one hand, autophagy is an important innate immune response against viral infection. On the other hand, some viruses exploit the autophagy pathway for their survival and proliferation in host cells. Vaccinia virus is a member of the family of Poxviridae which includes the smallpox virus. The biogenesis of vaccinia envelopes, including the core envelope of the immature virus (IV), is not fully understood. In this study we investigated the possible interaction between vaccinia virus and the autophagy membrane biogenesis machinery. Massive LC3 lipidation was observed in mouse fibroblast cells upon vaccinia virus infection. Surprisingly, the vaccinia virus induced LC3 lipidation was shown to be independent of ATG5 and ATG7, as the atg5 and atg7 null mouse embryonic fibroblasts (MEFs) exhibited the same high levels of LC3 lipidation as compared with the wild-type MEFs. Mass spectrometry and immunoblotting analyses revealed that the viral infection led to the direct conjugation of ATG3, which is the E2-like enzyme required for LC3-phosphoethanonamine conjugation, to ATG12, which is a component of the E3-like ATG12–ATG5-ATG16 complex for LC3 lipidation. Consistently, ATG3 was shown to be required for the vaccinia virus induced LC3 lipidation. Strikingly, despite the high levels of LC3 lipidation, subsequent electron microscopy showed that vaccinia virus-infected cells were devoid of autophagosomes, either in normal growth medium or upon serum and amino acid deprivation. In addition, no autophagy flux was observed in virus-infected cells. We further demonstrated that neither ATG3 nor LC3 lipidation is crucial for viral membrane biogenesis or viral proliferation and infection. Together, these results indicated that vaccinia virus does not exploit the cellular autophagic membrane biogenesis machinery for their viral membrane production. Moreover, this study demonstrated that vaccinia virus instead actively disrupts the cellular autophagy through a novel molecular mechanism that is associated with aberrant LC3 lipidation and a direct conjugation between ATG12 and ATG3.     

Proceedings: Protectants in the freeze-drying and the preservation of vaccinia virus

Purified and unpurified vaccinia virus suspension were prepared from calf dermal pulp or chorioallantoic membrane of developing hen eggs, infected with vaccinia virus strain Ikeda. For the preparation of more stable dried product, single and combined protectants composed of a mixture of sodium glutamate with soluble starch, polyvinylpyrrolidone (PVP) or sodium carboxymethyl cellulose (SCMC), were compared in the course of the freeze-drying and preservation process.Single protectants sodium glutamate or peptone were effective in the freeze-drying and the preservation of both purified and unpurificd vaccinia virus products. There was an optimal concentration of sodium glutamate to be added to the suspension for the preservation of the dried products, especially the purified products.Combined protectants were effective in the purified products when the concentration of sodium glutamate exceeded the optimum necessary for the preservation.     

Vaccinia virus leads to ATG12–ATG3 conjugation and deficiency in autophagosome formation

The interactions between viruses and cellular autophagy have been widely reported. On the one hand, autophagy is an important innate immune response against viral infection. On the other hand, some viruses exploit the autophagy pathway for their survival and proliferation in host cells. Vaccinia virus is a member of the family of Poxviridae which includes the smallpox virus. The biogenesis of vaccinia envelopes, including the core envelope of the immature virus (IV), is not fully understood. In this study we investigated the possible interaction between vaccinia virus and the autophagy membrane biogenesis machinery. Massive LC3 lipidation was observed in mouse fibroblast cells upon vaccinia virus infection. Surprisingly, the vaccinia virus induced LC3 lipidation was shown to be independent of ATG5 and ATG7, as the atg5 and atg7 null mouse embryonic fibroblasts (MEFs) exhibited the same high levels of LC3 lipidation as compared with the wild-type MEFs. Mass spectrometry and immunoblotting analyses revealed that the viral infection led to the direct conjugation of ATG3, which is the E2-like enzyme required for LC3-phosphoethanonamine conjugation, to ATG12, which is a component of the E3-like ATG12–ATG5-ATG16 complex for LC3 lipidation. Consistently, ATG3 was shown to be required for the vaccinia virus induced LC3 lipidation. Strikingly, despite the high levels of LC3 lipidation, subsequent electron microscopy showed that vaccinia virus-infected cells were devoid of autophagosomes, either in normal growth medium or upon serum and amino acid deprivation. In addition, no autophagy flux was observed in virus-infected cells. We further demonstrated that neither ATG3 nor LC3 lipidation is crucial for viral membrane biogenesis or viral proliferation and infection. Together, these results indicated that vaccinia virus does not exploit the cellular autophagic membrane biogenesis machinery for their viral membrane production. Moreover, this study demonstrated that vaccinia virus instead actively disrupts the cellular autophagy through a novel molecular mechanism that is associated with aberrant LC3 lipidation and a direct conjugation between ATG12 and ATG3.     

Mechanism of Vaccinia Virus Release and Its Specific Inhibition by N1-Isonicotinoyl-N2-3-Methyl-4- Chlorobenzoylhydrazine

The release of vaccinia virus from RK-13 cells and its specific inhibition by N(1)-isonicotinoyl-N(2)-3-methyl-4- chlorobenzoylhydrazine (IMCBH) was studied. Intracellular naked vaccinia virus (INV) was wrapped by intracytoplasmic membranes, forming an intracellular double-membraned virion. Wrapped virions migrated to the cell surface, where the outer virion membrane presumably fused with the plasma membrane, releasing virus surrounded by the inner membrane, referred to as extracellular enveloped vaccinia virus (EEV). At no time was there any evidence that vaccinia virus acquired an envelope by budding of naked virus from the cytoplasmic membrane. Naked virus and double-membraned virus each constituted about one-third of intracellular virus at 8 and 12 h postinfection (p.i.). Beginning at 16 h p.i., the proportion of intracellular virus occurring as double-membraned virus steadily decreased to 1% at 24 h while the proportion of naked virus rose to 87%. IMCBH inhibited the formation of the double-membraned virion and the appearance of EEV while not affecting the production of INV. IMCBH had no effect on INV infectivity or polypeptide composition, on vaccinia virus-specified membrane-associated proteins or glycoproteins, or on hemadsorption. The presence of IMCBH until 4 h p.i. did not decrease the amount of EEV at 48 h p.i., whereas less than 10% of the normal 48-h EEV yield was obtained if the drug was present during the first 16 h p.i. Cell cultures infected at very low multiplicities showed a rapid virus dissemination in the absence of the drug, whereas the presence of IMCBH very effectively inhibited this spread. We conclude that vaccinia virus is liberated via a double-membraned intermediate as an enveloped virion and that it is this extracellular enveloped virus that is responsible for dissemination of infection.     

Hepatitis B virus large surface protein is not secreted but is immunogenic when selectively expressed by recombinant vaccinia virus.

The envelope region of the hepatitis B virus (HBV) genome contains an open reading frame that begins upstream of the major surface protein gene. The two minor proteins that are initiated within this pre-s segment are immunogenic and may be involved in virus attachment to hepatocytes. We have constructed a recombinant vaccinia virus that contains the predicted coding segment for the large surface protein (LS) under control of a vaccinia virus that contains the predicted coding segment for the large surface protein (LS) under control of a vaccinia virus promoter. Cells infected with the recombinant virus synthesized HBV polypeptides of 39 and 42 kilodaltons, corresponding to the unglycosylated and glycosylated forms of LS, respectively. The presence of pre-s epitopes in the 39- and 42-kilodalton polypeptides was demonstrated by binding of antibody prepared against a synthetic peptide. Synthesis of the 42-kilodalton species was specifically inhibited by tunicamycin, suggesting that it is N-glycosylated. Despite apparent glycosylation, LS was not secreted into the medium of infected cells. Nevertheless, rabbits vaccinated with the purified recombinant virus made antibodies that recognized s and pre-s epitopes. Antibody to the NH2 terminus of LS appeared before or simultaneously with antibody that bound to the major surface protein. The additional immunogenicity provided by expression of LS may be advantageous for the development of an HBV vaccine.     

Antibodies directed against a synthetic peptide enable detection of a protein encoded by a vaccinia virus host range gene that is conserved within the Orthopoxvirus genus.

A vaccinia virus gene required for multiplication in some cell lines but not in others has been previously isolated and sequenced. A synthetic peptide predicted from the nucleotide sequence and corresponding to the carboxy-terminal 18 amino acids was used to raise antibodies in rabbits. The immune serum enabled detection of a 29-kilodalton (kDa) polypeptide by either immunoprecipitation or Western immunoblot assays. Synthesis of the 29-kDa polypeptide occurred immediately after infection and lasted for about 3 h. Shutoff of its synthesis was concomitant with the appearance of a delayed early polypeptide that may be antigenically related to the 29-kDa polypeptide. Analysis of cloned segments of the genomes of other orthopoxviruses by hybridization with the vaccinia virus host range gene demonstrates that it is well conserved within this genus.     

Transport to the cell surface of a peptide sequence attached to the truncated C terminus of an N-terminally anchored integral membrane protein.

Attempts to construct hybrid proteins that are transported to the plasma membrane are frequently unsuccessful because of perturbations in polypeptide folding. In seeking to minimize this problem, we have used the less common type of integral membrane protein, which has an uncleaved signal-anchor domain and an extracellular carboxyl portion, to transport a peptide sequence of interest to the cell surface. A set of plasmids was constructed that contained the gene encoding respiratory syncytial virus glycoprotein G (RSVG) interrupted immediately after one of several proline codons by a synthetic sequence containing unique restriction endonuclease sites and a stop codon. The shortened RSVG gene was flanked by vaccinia virus DNA to permit cloning and expression in a vaccinia virus vector. An open reading frame encoding four copies of the immunodominant repeating epitope of the circumsporozoite protein of Plasmodium falciparum was inserted into the tails of the truncated RSVG genes. Recombinant vaccinia viruses were isolated and shown to express hybrid proteins that reacted with a monoclonal antibody directed to the repeating circumsporozoite epitope. Moreover, immunofluorescence studies indicated that the peptide was on the external cell surface and available to react with antibodies. Expression of the hybrid protein also occurred in rabbits inoculated with the live recombinant vaccinia virus, as demonstrated by the generation of antibodies that bound to P. falciparum sporozoites in vitro.     

Postexposure immunoprophylaxis against B virus (Herpesvirus simiae) infection.

Local infiltration of antiserum into sites inoculated with B virus protected rabbits from an otherwise fatal encephalomyelitis. Treatment was effective when delayed for six hours but not after 24 hours. Homologous rabbit antisera were more effective than heterologous monkey antisera, and protection was unrelated to neutralisation titres. Protection apparently depended not on neutralisation of inoculated virus but on destruction of infected cells before they produced progeny virus. Normal human immunoglobulin able to neutralise B virus did not protect. Intravenously administered antibody was effective only if large doses were given. The findings suggest that persons bitten or scratched by monkeys latently infected with B virus may be treated successfully by immunoprophylaxis with specific antibody. Stocks of human or of more readily available simian antisera should be held in laboratories where such animals are used.