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Evidence for the modification of vaccinia virus core proteins by ADP-ribosylationChild, Stephanie J. 07 January 1992 (has links)
Vaccinia virus (VV), the prototype member of the orthopoxvirus family, is
a large virus of complex morphology which contains a 191 Kbp double-stranded
DNA genome whose expression is tightly regulated in a temporal
fashion during viral replication. The regulation of gene expression can be
exerted at various of levels, including transcriptional, translational, and post-translational
points of control. In addition to transcriptional regulatory
mechanisms, the occurrence of a variety of post-translational modifications
in VV has been demonstrated. In an effort to better understand the role
played by post-translational modifications during the viral replication cycle,
we chose to focus on one specific modification event, ADP-ribosylation.
Experiments were designed to determine whether any VV proteins might
be subject to ADP-ribosylation. The ability to metabolically label a subset of
viral proteins by growth of the virus in the presence of [3H]adenosine, in
addition to the effects of the ADP-ribosylation inhibitor nicotinamide on viral
core protein precursor processing and replication, provided evidence that this
or some similar modification is an obligatory event during VV replication.
Immunological reagents were used to identify several of the modified
proteins. Biochemical evidence obtained via labeling with various precursor
compounds, boronate affinity chromatography, and reverse phase HPLC
analysis confirmed that the proteins were modified by ADP-ribose or a closely
related compound.
Additional ADP-ribosylation inhibitor studies provided further support for
the initial finding that the viral proteins are subject to ADP-ribosylation or
some related modification, and the evidence obtained from these
experiments supports a model where this modification event might serve a
function in either the proteolytic processing of the core protein precursors, or
in localization of the mature core proteins to sites of VV replication within
infected cells. / Graduation date: 1992
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Development and characterization of murine monoclonal antibodies capable of neutralizing vaccinia virusChen, Ran 24 October 2007 (has links)
INTRODUCTION: Since the eradication of smallpox in 1977, mass vaccination efforts against it have been discontinued. Thus, the majority of the younger population is susceptible to both smallpox virus and vaccinia virus (VV). The re-emergence or intentional release of smallpox will present a serious threat to global health. There are limited supplies of smallpox vaccine, which is associated with significant complications, and pooled anti-VV human immune globulin (VIG) that can be used as prophylaxis or to treat smallpox-exposed individuals. We are developing murine monoclonal antibodies (MAbs) able to neutralize VV. The developed MAbs may be useful in establishing a rapid diagnostic test for the detection of VV infection or providing the genetic materials needed for developing recombinant antibodies suitable for human use.
METHODS: VV Western Reserve (WR) strain was propagated in HeLa or Chicken Embryo Fibroblast (CEF) cell lines, purified through a 36% sucrose cushion and inactivated by binary ethyleneimine (BEI). Female BABL/c mice were immunized with inactivated VV. Hybridoma cell lines (HCLs) were developed from spleen cells of the mice with high neutralizing antibody titers. Tissue culture supernatants from the developed HCLs were screened by Enzyme-Linked Immunosorbent Assay (ELISA) and Plaque Reduction Assay (PRA) for their abilities to produce neutralizing antibodies against VV. HCLs producing neutralizing antibodies were sub-cloned by limiting dilution method. Highly neutralizing MAbs were isotyped and purified. The effect of using increasing microgram amounts of each MAb or mixtures of two MAbs on VV neutralization has been determined. Specific target proteins recognized by MAbs were detected by western blot assay (WB). The abilities of the developed MAbs to neutralize other three VV strains, Large-variant (L-variant), IHD-W and New York City Board of Health (NYCBH), were measured.
RESULTS: We have developed 261 HCLs producing anti-VV antibodies; 65 of them neutralized VV. Twelve HCLs were sub-cloned. We developed 79 sub-clones producing neutralizing MAbs. The majority of them were immunoglobulin IgG1/κ isotype. Four highly neutralizing MAbs were concentrated and purified. They were able to neutralize 50% of VV infection at 0.01-0.1 µg in PRAs. Synergistic effects on VV neutralization were observed when mixing two MAbs from clones, 1-E9-1-E4 and 2-B7-9-E6, at the amounts giving about 20% and 40% VV neutralization. Based on the WB results, the developed MAbs are recognizing 75 kilodalton (kDa), 45 kDa, 35 kDa or 8 kDa WR VV proteins. The abilities of the developed MAbs to neutralize other strains of VV varied.
CONCLUSIONS: Several HCLs producing antibodies against VV were developed. Highly neutralizing MAbs against WR VV have been produced and purified. Virus neutralization is dose dependent and some of MAbs have synergistic neutralization effects on each other. Most of the MAbs were targeting the same three virus envelope proteins indicating that these proteins contain important epitope(s) responsible for the neutralizing effects by the developed MAbs. Variable neutralization abilities were observed on three other VV strains indicating their immunobiologic differences with WR VV strain. The developed MAbs may be used as a research tool to study VV pathogenesis or for the development of chimeric antibodies for clinical applications. / October 2006
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Development and characterization of murine monoclonal antibodies capable of neutralizing vaccinia virusChen, Ran 24 October 2007 (has links)
INTRODUCTION: Since the eradication of smallpox in 1977, mass vaccination efforts against it have been discontinued. Thus, the majority of the younger population is susceptible to both smallpox virus and vaccinia virus (VV). The re-emergence or intentional release of smallpox will present a serious threat to global health. There are limited supplies of smallpox vaccine, which is associated with significant complications, and pooled anti-VV human immune globulin (VIG) that can be used as prophylaxis or to treat smallpox-exposed individuals. We are developing murine monoclonal antibodies (MAbs) able to neutralize VV. The developed MAbs may be useful in establishing a rapid diagnostic test for the detection of VV infection or providing the genetic materials needed for developing recombinant antibodies suitable for human use.
METHODS: VV Western Reserve (WR) strain was propagated in HeLa or Chicken Embryo Fibroblast (CEF) cell lines, purified through a 36% sucrose cushion and inactivated by binary ethyleneimine (BEI). Female BABL/c mice were immunized with inactivated VV. Hybridoma cell lines (HCLs) were developed from spleen cells of the mice with high neutralizing antibody titers. Tissue culture supernatants from the developed HCLs were screened by Enzyme-Linked Immunosorbent Assay (ELISA) and Plaque Reduction Assay (PRA) for their abilities to produce neutralizing antibodies against VV. HCLs producing neutralizing antibodies were sub-cloned by limiting dilution method. Highly neutralizing MAbs were isotyped and purified. The effect of using increasing microgram amounts of each MAb or mixtures of two MAbs on VV neutralization has been determined. Specific target proteins recognized by MAbs were detected by western blot assay (WB). The abilities of the developed MAbs to neutralize other three VV strains, Large-variant (L-variant), IHD-W and New York City Board of Health (NYCBH), were measured.
RESULTS: We have developed 261 HCLs producing anti-VV antibodies; 65 of them neutralized VV. Twelve HCLs were sub-cloned. We developed 79 sub-clones producing neutralizing MAbs. The majority of them were immunoglobulin IgG1/κ isotype. Four highly neutralizing MAbs were concentrated and purified. They were able to neutralize 50% of VV infection at 0.01-0.1 µg in PRAs. Synergistic effects on VV neutralization were observed when mixing two MAbs from clones, 1-E9-1-E4 and 2-B7-9-E6, at the amounts giving about 20% and 40% VV neutralization. Based on the WB results, the developed MAbs are recognizing 75 kilodalton (kDa), 45 kDa, 35 kDa or 8 kDa WR VV proteins. The abilities of the developed MAbs to neutralize other strains of VV varied.
CONCLUSIONS: Several HCLs producing antibodies against VV were developed. Highly neutralizing MAbs against WR VV have been produced and purified. Virus neutralization is dose dependent and some of MAbs have synergistic neutralization effects on each other. Most of the MAbs were targeting the same three virus envelope proteins indicating that these proteins contain important epitope(s) responsible for the neutralizing effects by the developed MAbs. Variable neutralization abilities were observed on three other VV strains indicating their immunobiologic differences with WR VV strain. The developed MAbs may be used as a research tool to study VV pathogenesis or for the development of chimeric antibodies for clinical applications.
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Development and characterization of murine monoclonal antibodies capable of neutralizing vaccinia virusChen, Ran 24 October 2007 (has links)
INTRODUCTION: Since the eradication of smallpox in 1977, mass vaccination efforts against it have been discontinued. Thus, the majority of the younger population is susceptible to both smallpox virus and vaccinia virus (VV). The re-emergence or intentional release of smallpox will present a serious threat to global health. There are limited supplies of smallpox vaccine, which is associated with significant complications, and pooled anti-VV human immune globulin (VIG) that can be used as prophylaxis or to treat smallpox-exposed individuals. We are developing murine monoclonal antibodies (MAbs) able to neutralize VV. The developed MAbs may be useful in establishing a rapid diagnostic test for the detection of VV infection or providing the genetic materials needed for developing recombinant antibodies suitable for human use.
METHODS: VV Western Reserve (WR) strain was propagated in HeLa or Chicken Embryo Fibroblast (CEF) cell lines, purified through a 36% sucrose cushion and inactivated by binary ethyleneimine (BEI). Female BABL/c mice were immunized with inactivated VV. Hybridoma cell lines (HCLs) were developed from spleen cells of the mice with high neutralizing antibody titers. Tissue culture supernatants from the developed HCLs were screened by Enzyme-Linked Immunosorbent Assay (ELISA) and Plaque Reduction Assay (PRA) for their abilities to produce neutralizing antibodies against VV. HCLs producing neutralizing antibodies were sub-cloned by limiting dilution method. Highly neutralizing MAbs were isotyped and purified. The effect of using increasing microgram amounts of each MAb or mixtures of two MAbs on VV neutralization has been determined. Specific target proteins recognized by MAbs were detected by western blot assay (WB). The abilities of the developed MAbs to neutralize other three VV strains, Large-variant (L-variant), IHD-W and New York City Board of Health (NYCBH), were measured.
RESULTS: We have developed 261 HCLs producing anti-VV antibodies; 65 of them neutralized VV. Twelve HCLs were sub-cloned. We developed 79 sub-clones producing neutralizing MAbs. The majority of them were immunoglobulin IgG1/κ isotype. Four highly neutralizing MAbs were concentrated and purified. They were able to neutralize 50% of VV infection at 0.01-0.1 µg in PRAs. Synergistic effects on VV neutralization were observed when mixing two MAbs from clones, 1-E9-1-E4 and 2-B7-9-E6, at the amounts giving about 20% and 40% VV neutralization. Based on the WB results, the developed MAbs are recognizing 75 kilodalton (kDa), 45 kDa, 35 kDa or 8 kDa WR VV proteins. The abilities of the developed MAbs to neutralize other strains of VV varied.
CONCLUSIONS: Several HCLs producing antibodies against VV were developed. Highly neutralizing MAbs against WR VV have been produced and purified. Virus neutralization is dose dependent and some of MAbs have synergistic neutralization effects on each other. Most of the MAbs were targeting the same three virus envelope proteins indicating that these proteins contain important epitope(s) responsible for the neutralizing effects by the developed MAbs. Variable neutralization abilities were observed on three other VV strains indicating their immunobiologic differences with WR VV strain. The developed MAbs may be used as a research tool to study VV pathogenesis or for the development of chimeric antibodies for clinical applications.
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Über Immunisierungsversuche mit Schutzpockenlympfe an KaninchenAmelung, Heinrich. January 1919 (has links)
Inaug.-Diss.-Munich.
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An analysis of vaccinia virus DNA ligaseOdell, Mark January 1996 (has links)
No description available.
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Functional Genomic Studies of Vaccinia Virus Provide Fundamental Insights into Virus-Host InteractionsKeller, Brian Andrew January 2017 (has links)
The oncolytic virus field is in the midst of strong and sustained growth. The clinical utility of this class of therapeutics has been bolstered in recent years by the rise of immune checkpoint inhibition, which has the potential to work synergistically with oncolytic viruses to increase the scope of patients who respond favourably to therapy. This growth has been further driven by clear industry support with several pharmaceutical companies acquiring or developing oncolytic virus products following the 2015 FDA approval of Talimogene laherparepvec and the generally-accepted potential of immunotherapeutic approaches to cancer treatment. Vaccinia virus is a double-stranded DNA virus with an extensive history of vaccine use in humans and a desirable safety profile. It is a large virus with a complex lifecycle, and its history of use as a vaccine has resulted in the generation of dozens of unique strains. Although it has been studied extensively, much remains unknown about many vaccinia virus gene function(s) and the virus’ interactions with cellular hosts. Vaccinia virus-based oncolytic viruses have been developed, however clinical outcomes thus far have been unsatisfactory. A more complete understanding of vaccinia virus gene functions must therefore precede the effective design of a next-generation vaccinia virus-based oncolytic candidate. With this downstream goal, we sought to (1) understand the unique oncolytic virus-relevant phenotypic properties of five clinical candidate vaccinia virus strains, and (2) generate and characterize a library of single-gene mutants of the Copenhagen strain of vaccinia virus. These studies resulted in the selection of vaccinia virus-Copenhagen as the wild-type strain of choice that will be utilized for future oncolytic virus development. Furthermore, the generation and initial characterization of an 89-member clonal library of vaccinia-Copenhagen single-gene mutants will be an important tool as we seek to generate a next-generation oncolytic virus candidate. Completed characterization studies challenge the role that viral thymidine kinase should play in oncolytic virus design, demonstrate novel functions of the vaccinia virus gene A47L, and provide an understanding of the role of the vaccinia virus gene F15L. These studies also raise the concept of the personalized selection of oncolytic virotherapeutics. This virus library has the potential to increase the fundamental understanding of vaccinia virus biology in this field as well as in the study of vaccine development and pathogen-host interactions.
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Changes in the level of free nucleotides of vaccinia infected chorioallantoic membrane of the chick embryo in vivoWylie, Vivian January 1964 (has links)
The ribonucleotides in the chorioallantoic membrane of 12-day-old chick embryos have been isolated by ion-exchange chromatography and characterized by their spectrophotometric and paper chromatographic properties. The following nucleotides were identified: adenosine-5' phosphate (AMP), uridine-5' phosphate (UMP), cytidine-5' phosphate (CMP), uridine-5' diphosphate galactose (UDPGal), uridine-5' diphosphate N-acetyl hexosamine (UDPNAHexosamine), guanosine-5' phosphate (GMP), cytidine-5' diphosphate (CDP), uridine-5' diphosphate (UDP), adenosine-51 diphosphate (ADP), guanosine-5' diphosphate (GDP), cytidine-5' triphosphate (CTP), uridine-5' triphosphate (DTP), adenosine-5' triphosphate (ATP), and guanosine-5' triphosphate (GTP).
Quantitative determinations of these nucleotides were made on the basis of their ultraviolet absorption at 260 mμ. Similarly, concentrations of these nucleotides were estimated in 12-day-old chorioallantoic membranes after infection with vaccinia virus.
Larger amounts of ribonucleoside-5' phosphates were present in the infected tissue at 4 and 12 hours after infection. The amounts of ribonucleoside-5' triphosphates were decreased. In tissues where, it is believed, synchronous infection occurred, the amounts of ribonucleoside-5' diphosphates and triphosphates were markedly lower than in controls after 12 hours of infection.
Infection in the presence of tritium₌labelled thymidine showed that the amount of labelled thymidine-5' mono-, di-, and triphosphates had increased after 4 hours and that the amounts of these nucleotides subsequently decreased. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate
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Vaccina Virus Binding and Infection of Primary Human B CellsShepherd, Nicole Elizabeth 12 1900 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / Vaccinia virus (VACV), the prototypical poxvirus, was used to eradicate
smallpox worldwide and, in recent years, has received considerable attention as
a vector for the development of vaccines against infectious diseases and
oncolytic virus therapy. Studies have demonstrated that VACV exhibits an
extremely strong bias for binding to and infection of primary human antigenpresenting
cells (APCs) including monocytes, macrophages, and dendritic cells.
However, very few studies have evaluated VACV binding to and infection of
primary human B cells, a main type of professional APC. In this study, we
evaluated the susceptibility of primary human peripheral B cells at different
developmental stages to VACV binding, infection, and replication. We found that
VACV exhibited strong binding but little entry into ex vivo B cells. Phenotypic
analysis of B cells revealed that plasmablasts were the only subset resistant to
VACV binding. Infection studies showed that plasma and mature-naïve B cells
were resistant to VACV infection, while memory B cells were preferentially
infected. Additionally, VACV infection was increased in larger and proliferative B
cells suggesting a bias of VACV infection towards specific stages of
differentiation and proliferative ability. VACV infection in B cells was abortive, and
cessation of VACV infection was determined to occur at the stage of late viral
gene expression. Interestingly, B cell function, measured by cytokine production,
was not affected within 24 hours post-infection. In contrast to ex vivo B cells, stimulated B cells were permissive to productive VACV infection. These results
demonstrate the value of B cells as a tool to aid in deciphering the intricacies of
poxvirus infection in humans. Understanding VACV infection in primary human B
cells at various stages of differentiation and maturation is important for the
development of a safer smallpox vaccine and better vectors for vaccines against
cancers and other infectious diseases.
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Bio-Engineering Vaccinia Viruses for Increased Oncolytic PotentialPelin, Adrian 02 December 2019 (has links)
Vaccinia virus has a large and still incompletely understood genome although several strains of this virus are already in clinical development. For the most part, clinical candidates have been attenuated from their wild type vaccine strains through deletion of metabolic genes like the viral thymidine kinase gene.In the present work, we thoroughly examined the genetic elements of vaccinia which could be modulated to tailor the virus as a cancer therapeutic. Using a variety of cancer cell lines and primary tumor explants, we performed a fitness assay that directly compares multiple wild-type Vaccinia strains to identify the genetic elements that together create an optimal “oncolytic engine”. Using a transposon insertion strategy and deep sequencing of viral populations we systematically examined Vaccinia genes that do or do not play a role in the therapeutic activity of the virus. Our studies allowed us to identify a variety of genes in the vaccinia genome that when deleted, augment the oncolytic activity of a newly engineered Vaccinia virus. In the context of this thesis, I define enhanced oncolytic activity as superior therapeutic activity, increased immunogenicity and an improved safety profile, all aspects which we used to compare this novel virus to Vaccinia viruses currently in the clinic.
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