Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Genes encoding virus-specific proteins with molecular masses of 36 kDa and 12 kDa were mapped in HindIII-P and HindIII-U DNA fragments of vaccinia strain LIVP and ectromelia strain K-1 viruses, respectively, by hybrid selection of RNA to cloned DNA fragments followed by in vitro translation. The 36K translation initiation codon was detected in the HindIII-J fragment. The nucleotide sequences of corresponding genes from vaccinia, ectromelia, cowpox and variola virus genomes were determined. The 12K protein has similarity to mammalian glutaredoxins. The derived amino acid sequence of the 36K polypeptide was compared with the protein bank PIR. No homology was found between the 36K protein and known structures of proteins. The 36K protein genes of vaccinia and ectromelia viruses were cloned in pUR290, which led to the production of E. coli chimeric proteins, consisting of the sequence of beta-galactosidase and the viral protein on their C-ends. The chimeric proteins were shown to possess viral antigenic specificity. To identify the protein product of the 36K gene monospecific antisera to chimeric proteins were obtained. The late 36K protein is associated with virosomes but is not incorporated into the virions of orthopoxviruses.
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PMID:Comparative analysis of the conserved region of the orthopoxvirus genome encoding the 36K and 12K proteins. 823 12

In anticipation of large-scale smallpox vaccination, clinical trials of new vaccine candidates with improved safety profiles, and new vaccinia immune globulin (VIG) products, there is an immediate need to develop new assays to measure vaccinia-specific immune responses. The classical assay to measure vaccinia neutralization, the plaque-reduction neutralization test (PRNT), is slow, labor intensive, and difficult to validate and transfer. Here we describe the development of a novel vaccinia-neutralization assay based on the expression of a reporter gene, beta-galactosidase (beta-Gal). Using a previously constructed vaccinia-beta-Gal recombinant virus, vSC56, we developed a neutralization assay that is rapid, sensitive, and reproducible. The readout is automated. We show that the neutralizing titers, ID(50), for several VIG products measured by our assay were similar to those obtained by PRNTs. A new Food and Drug Administration VIG standard was established for distribution to other laboratories. The new assay will serve as an important tool both for preclinical and clinical trials of new smallpox vaccines and for evaluation of therapeutic agents to treat vaccine-associated adverse reactions.
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PMID:Development of a novel vaccinia-neutralization assay based on reporter-gene expression. 1287 Jan 27

For the last 30 yr, interest in vaccinia virus immune monitoring has focused on the use of the vaccinia virus as a recombinant vaccine vector and the potential detrimental effect of antivector immunity on subsequent vaccination with a recombinant vaccinia virus. However, interest in this area has intensified after the publication of reports suggesting that smallpox may be a major pathogen selected for bioterrorist activities. Owing to the unacceptably high incidence of complications induced by previous effective smallpox vaccine strains, alternative safer strains (e.g., modified vaccinia Ankara [MVA]) are being assessed for their antigenicity in clinical trials. The exact immune effector mechanism responsible for vaccine-induced protection to smallpox infection has not been fully elucidated, although it is believed that neutralizing antibody plays a major role. This chapter describes a simple enzyme-linked immunosorbent assay (ELISA) to quantify vaccinia virus antibody titer. Additionally, to define serum-neutralizing activity, both a classical plaque reduction assay and a high-throughput 96-well plate method based on reduction of recombinant vaccinia virus expressed beta-galactosidase is described. Furthermore, details are given for a T-cell proliferation assay, primarily for monitoring T-helper CD4 activity and an enzyme-linked immunospot (ELISPOT) assay for CD8 analysis. The use of reliable immunological assays is vital in assessing the potential efficacy of new vaccines to protect against smallpox infection.
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PMID:Monitoring of human immunological responses to vaccinia virus. 1511 20

Viruses are one of four classes of biothreat agents, and bacteriophage MS2 has been used as a simulant for biothreat viruses, such as smallpox. A paramagnetic bead-based electrochemical immunoassay has been developed for detecting bacteriophage MS2. The immunoassay sandwich was made by attaching a biotinylated rabbit anti-MS2 IgG to a streptavidin-coated bead, capturing the virus, and then attaching a rabbit anti-MS2 IgG-beta-galactosidase conjugate to another site on the virus. beta-Galactosidase converts p-aminophenyl galactopyranoside (PAPG) to p-aminophenol (PAP). PAPG is electroinactive at the potential at which PAP is oxidized to p-quinone imine (PQI), so the current resulting from the oxidation of PAP to PQI is directly proportional to the concentration of antigen in the sample. The immunoassay was detected with rotating disk electrode (RDE) amperometry and an interdigitated array (IDA) electrode. With an applied potential of +290 mV vs Ag/AgCl and a rotation rate of 3000 rpm, the detection limit was 200 ng/mL MS2 or 3.2 x 10(10) viral particles/mL with RDE amperometry. A trench IDA electrode was incorporated into a poly(dimethyl siloxane) channel, within which beads were collected, incubated with PAPG, and PAP generation was detected. The two working electrodes were held at +290 and -300 mV vs Ag/AgCl, and electrochemical recycling of the PAP/PQI couple by the IDA electrode lowered the limit of detection to 90 ng/mL MS2, or 1.5 x 10(10) MS2 particles/mL.
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PMID:Bead-based electrochemical immunoassay for bacteriophage MS2. 1514 78

Development of a safe and effective vaccine for induction of mucosal immunity to the human immunodeficiency virus (HIV) envelope glycoprotein (Env, gp160) represents the best hope for containing the spread of an HIV epidemic worldwide. The highly attenuated modified vaccinia virus Ankara (MVA) is a laboratory virus well suited as a safe vaccine vector. However, the presence of pre-existing immunity to Vaccinia virus in the adult population represents a hindrance that limits the application of the MVA vector for inducing immunity to HIV antigens. Here, cationic liposomes were covalently attached to the surface of recombinant MVA expressing the HIV-1 strain IIIB Env glycoprotein and beta-galactosidase (MVA(IIIB/beta-gal)) using tresylmonomethoxypolyethylene glycol (TMPEG) grafted into a lipid membrane without compromising viral infectivity in vitro and in vivo. The orally administered MVA(IIIB/beta-gal)-TMPEG/liposome complexes were capable of delivering the transgenes to mucosal tissues in mice with pre-existing poxvirus immunity based on beta-galactosidase gene expression in intestinal tissues measured 18 h after infection. Importantly, the MVA(IIIB/beta-gal)-TMPEG/liposome complexes enhanced Env-specific cellular and humoral immune responses in the mucosal and systemic tissues after repeated oral immunization of BALB/c mice. This approach may prove useful for induction of protective immunity against infectious diseases and cancer in populations with pre-existing immunity to vaccinia from smallpox vaccination.
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PMID:Oral vaccination with modified vaccinia virus Ankara attached covalently to TMPEG-modified cationic liposomes overcomes pre-existing poxvirus immunity from recombinant vaccinia immunization. 1717 Apr 37

Concerns about the possible use of Variola virus, the causative agent of smallpox, as a weapon for bioterrorism have led to renewed efforts to identify new antivirals against orthopoxviruses. We identified a peptide, EB, which inhibited infection by Vaccinia virus with an EC(50) of 15 microM. A control peptide, EBX, identical in composition to EB but differing in sequence, was inactive (EC50>200 microM), indicating sequence specificity. The inhibition was reversed upon removal of the peptide, and EB treatment had no effect on the physical integrity of virus particles as determined by electron microscopy. Viral adsorption was unaffected by the presence of EB, and the addition of EB post-entry had no effect on viral titers or on early gene expression. The addition of EB post-adsorption resulted in the inhibition of beta-galactosidase expression from an early viral promoter with an EC(50) of 45 microM. A significant reduction in virus entry was detected in the presence of the peptide when the number of viral cores released into the cytoplasm was quantified. Electron microscopy indicated that 88% of the virions remained on the surface of cells in the presence of EB, compared to 37% in the control (p<0.001). EB also blocked fusion-from-within, suggesting that virus infection is inhibited at the fusion step. Analysis of EB derivatives suggested that peptide length may be important for the activity of EB. The EB peptide is, to our knowledge, the first known small molecule inhibitor of Vaccinia virus entry.
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PMID:Inhibition of Vaccinia virus entry by a broad spectrum antiviral peptide. 1939 56

Because of the bioterrorism threat posed by agents such as variola virus, considerable time, resources, and effort have been devoted to biodefense preparation. One avenue of this research has been the development of rapid, sensitive, high-throughput assays to validate immune responses to poxviruses. Here we describe the adaptation of a beta-galactosidase reporter-based vaccinia virus neutralization assay to large-scale use in a study that included over 1,000 subjects. We also describe the statistical methods involved in analyzing the large quantity of data generated. The assay and its associated methods should prove useful tools in monitoring immune responses to next-generation smallpox vaccines, studying poxvirus immunity, and evaluating therapeutic agents such as vaccinia virus immune globulin.
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PMID:Statistical approach to estimate vaccinia-specific neutralizing antibody titers using a high-throughput assay. 1953 40