Technologies

Pharmaceuticals & Vitamin D : Vaccines

Pharmaceuticals & Vitamin D Portfolios

Technologies

Improved Influenza B Virus Replication for Vaccine Development

UW–Madison researchers led by Yoshihiro Kawaoka and Gabriele Neumann have identified growth enhancing mutations that increase the yield of influenza B viruses, potentially enabling more rapid and cost-effective vaccine production.

Virus libraries were generated for each lineage (B/Victoria and B/Yamagata) and passaged in cultured cells to identify several mutations in the ‘internal’ genes of influenza B viruses that confer high-yield in cultured cells and/or embryonated chicken embryos. The use of one or more of these mutations in vaccine virus master strains results in higher viral titers (e.g., 108 PFU/mL or more) in cultured cells and/or embryonated chicken eggs.
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Mutations That Improve Genetic Stability of Influenza Virus for Vaccination, Gene Therapy & More

UW–Madison researchers have identified mutations in influenza virus gene segments that increase the stability and/or replication of the virus, particularly virus that contains a heterologous gene sequence. The mutations are found in the PA, PB1, PB2, NS and HA segments.

Recombinant influenza virus with one or more stabilizing mutations may be used as a vector for vaccines or gene delivery. In addition, the mutations allow influenza virus to stably maintain fluorescent reporter genes, making it possible to visualize the in vivo.
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Vaccine for Fungal Infections

UW–Madison researchers have developed a vaccine that could prevent infection by many strains of pathogenic fungi.

The vaccine contains calnexin, a common folding protein found in fungi and other eukaryotes. Administered in an effective amount, the vaccine helps a patient’s immune system recognize and destroy fungus it may encounter.
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Potential for Vaccine Against Johne’s Disease

UW–Madison researchers have developed MAP strains with mutated global gene regulators (GGRs) that may be utilized in a vaccine against Johne’s disease.

GGRs are proteins needed for initiating RNA synthesis, for example, sigma factors and transcriptional regulators. By deleting, inactivating or reducing some key GGR sequences in MAP bacteria, non-virulent strains could be produced and administered to animals to confer immunity.
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Safer Influenza Vaccine from Replication-Knock Out Virus

UW–Madison researchers have developed methods for generating novel influenza vaccines that can elicit robust immune response without the risk of symptoms or genetic reversion.

The recombinant influenza A virus is made to lack the genetic sequence necessary for replication in normal host cells. Specifically, the coding region of PB2 viral RNA can be deleted, disrupted or replaced with a harmless reporter gene useful for tracing during cell culture preparation. With the mutant gene segment, the virus is ‘biologically contained’—capable of replicating only in specially developed PB2-expressing cells.
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Improved Production of Influenza Virus, Including H1N1, for Vaccine Manufacture

UW-Madison researchers have identified a single point mutation in the haemagglutinin (HA) gene of the H1N1 virus that enhances viral titer.  Introducing the mutation into vaccine seed viruses could lead to higher titer production of the viruses, improving vaccine manufacture.
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Attenuated Influenza Viruses for Development of Live Influenza Vaccine

UW-Madison researchers now have developed new mutated influenza viruses that grow normally in cell culture but whose growth is attenuated in mice.  These viruses are useful for vaccines and vaccine production.

The mutations are in the influenza A virus nuclear export protein, known as NEP or NS2.  Because the NS genes are highly conserved, they likely mutate less frequently than other influenza genes, making viruses with NS2 mutations ideal for the development of live attenuated vaccines. 
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Novel Candidates for an Improved Tuberculosis Vaccine

UW-Madison researchers have developed four candidates for a live attenuated TB vaccine.  They disrupted regions of the M. tuberculosis genome that are associated with pathogenicity and identified viable but attenuated mutants with disruptions in the ctpV, rv0990c, rv0971c or rv0348 genes.  These mutants may be useful for eliciting an immune response against tuberculosis.
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Novel Peptide Adjuvant Improves Response to Influenza Vaccination

UW-Madison researchers have developed an effective influenza vaccine adjuvant known as EB. EB is a 20 amino acid oligopeptide that specifically binds to the viral haemagglutinin protein. It induces influenza virus particles to aggregate, leading to increased viral uptake by antigen presenting cells.

This peptide can be added to influenza vaccines to increase immune response to the vaccine. The inventors found that mice vaccinated with inactivated H5N1 virus pretreated with EB adjuvant had no detectable infectious virus in their lungs at three and six days post infection, in contrast to mice vaccinated with H5N1 alone or with alum as an adjuvant, which had viral levels of up to 104 infectious viral particles in their lungs. 
 
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Efficient Generation of Influenza Virus with Adenoviral Vectors

UW-Madison researchers now have modified the reverse genetics approach to make it possible to introduce viral genetic information into more cell types. Instead of multiple plasmids, adenoviral vectors are used to introduce the viral genes and other sequences needed for replication and transcription into the cells. Because these vectors are highly efficient at transferring genes, they allow vaccine viruses to be generated in a greater variety of cell lines than the plasmids used in the original system.
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Neuraminidase-Deficient Live Influenza Vaccine

UW–Madison researchers have developed a modified influenza virus that is infectious but completely avirulent. The virus can be used to prepare live attenuated virus stock for vaccines.

The virus was created by reverse genetics and lacks an NA gene segment necessary for functional sialidase activity. For this reason the seven-segment virus is innocuous but can still infect animals to induce the appropriate humoral and cellular immune responses.
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Nanoparticles That Target Dendritic Cells

UW-Madison researchers have developed a system for delivering vaccines and other biomolecules to dendritic cells. This system includes carbon nanoparticles that are preferentially taken up by dendritic cells, rather than macrophages.

Antigens, dendritic cell-targeting antibodies and dendritic cell-activating substances may be attached to the nanoparticles. The antigens are capable of inducing a specific T-cell response and can be associated with infectious disease or a tumor. When delivered to dendritic cells, these nanoparticles enhance immune response.

Other biomolecules, including targeting compounds, therapeutic agents and detectable labels, can be attached to the nanoparticles as well. Targeting compounds may be attached to enhance the uptake of the nanoparticles by dendritic cells. To treat autoimmune diseases, a cytotoxic agent could be attached to the nanoparticles to selectively target and kill aberrant dendritic cells. Fluorescent or radioactive labels could be added to make it easier to isolate dendritic cells. 
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Global Regulator of Morphogenesis and Pathogenicity in Dimorphic Fungi

UW-Madison researchers have identified strains of dimorphic fungi that are useful in vaccine development because they do not become virulent, along with a method of identifying compounds that prevent dimorphic fungi from becoming virulent. The researchers discovered that the fungal histidine kinase is responsible for the transformation of these organisms into virulent yeast. Knocking out or otherwise inactivating the histidine kinase gene results in a fungal strain that does not become virulent.

To determine if a test compound may be useful as an anti-fungal therapeutic, it is exposed to the fungal histidine kinase. Because histidine kinases play a key role in the ability of many fungi to sense and respond to environmental changes, compounds that reduce the activity of the kinase may be used to prevent or treat infection with pathogenic fungi, including dimorphic fungi.
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High Titer Recombinant Influenza Viruses for Vaccines

UW-Madison researchers have developed an improved reassortant virus for use in producing high levels of the H5N1 avian influenza strain, as well as seasonal influenza strains. They discovered that using the NA segment from a “harmless” strain that grows well in eggs resulted in significantly greater amounts of infectious virus in eggs or cell lines. The inventors found that using a different isolate of this strain as a source for the other six viral segments also improves yield.
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Improved Reverse Genetics Method to Produce Influenza Virus

UW-Madison researchers have developed a set of one to four plasmids that contain all sequences necessary for influenza virus generation. For the efficient production of vaccine, one plasmid might contain cDNAs for the HA and NA genes, while a second plasmid is used for the remaining viral segments. The viral proteins required for influenza virus production are then provided from two additional plasmids.

With this approach, only one plasmid (the one encoding HA and NA) has to be updated annually. Using fewer plasmids increases the efficiency of virus production in cell lines that cannot be transfected efficiently. Vaccine viruses can be generated more quickly, which would be especially important in responding to a pandemic.
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High Titer Recombinant Influenza Viruses for Vaccines and Gene Therapy

A UW-Madison researcher has developed an efficient technique and system for producing high titer influenza A virus in vertebrate cells in the absence of helper virus. The technology takes advantage of a reverse genetics system created by Dr. Kawaoka that allows efficient production of influenza virus for vaccines and gene therapy applications (see WARF reference number P03252US). In the technology described here, the inventor developed a new set of plasmids for use with the reverse genetics system. The plasmids contain cDNAs from a high titer influenza virus isolate; the promoter for RNA polymerase I or RNA polymerase II; and the terminator sequence for RNA polymerase I. When these constructs are transfected into host cells, the cells consistently generate high yields of infectious influenza particles.
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Recombinant Influenza Vectors with a Pol II Promoter and Ribozymes for Vaccines and Gene Therapy

UW-Madison resarchers have developed an improved reverse genetics system for producing influenza virus in vertebrate cells in the absence of helper virus. The system starts with a set of plasmids containing viral genome cDNAs flanked by ribozymes. Each plasmid carries a cDNA for one of the eight influenza A viral RNA segments. On each plasmid, the cDNA sits between a polymerase II promoter and a poly-A addition signal at the 3-prime end. When the plasmids are transfected into a vertebrate cell, the host cell’s RNA polymerase II transcribes each construct into a capped viral RNA with a proper poly-A tail. The flanking ribozyme RNAs then undergo site-specific, self-catalyzed cleavage to precisely trim each end of the viral RNA. Next, viral polymerase, which is provided by a protein expression plasmid, acts upon the viral RNAs, resulting in replication and mRNA synthesis. This system does not require a helper virus and allows the creation of transfectants with mutations in any gene segment.
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Methods for Engineering Influenza Viruses to Carry Defined Mutations

A UW-Madison reseasrcher has developed a method of preparing viruses with defined mutations. The method uses a reverse genetics system created by Dr. Kawaoka (see WARF reference number P03252US), which consists of plasmids containing the promoter for RNA polymerase I or RNA polymerase II; a cDNA for each of the influenza virus RNA segments; and the terminator for RNA polymerase I. These plasmids are transfected into cells along with protein expression plasmids to generate live virus.

The technology featured here allows mutations to be introduced into any of the cDNAs to generate viruses with defined mutations. For example, viruses lacking the NB protein -- an integral membrane glycoprotein that promotes efficient replication in vivo -- were created with this method. The NB knockout viruses replicated as efficiently as wild type virus in cell culture, but were attenuated in mice.
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Methods and Compositions for Treating Prostate Cancer with DNA Vaccines

A UW-Madison researcher has developed a DNA vaccine for treating prostate cancer. The vaccine consists of a plasmid vector that contains a DNA sequence encoding the enzyme prostatic acid phosphatase (PAP) and a transcription regulatory element. PAP is expressed almost exclusively in prostate tissue. Serum levels of PAP are low in healthy individuals, but elevated in individuals with prostate cancer. When the vaccine is administered to a patient, it induces a cytotoxic immune reaction against cells expressing PAP. This leads to destructive prostatitis (inflammation of the prostate gland), killing the prostate cells.
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Reverse Genetics Approach for Generating Ebola Virus and Other Filoviruses from Cloned DNA

Kawaoka and his colleagues have now developed a reverse genetics approach for generating Ebola virus entirely from cloned cDNA. They prepared the full Ebola genome through reverse transcription of viral RNA, followed by PCR amplification and cloning of Ebola cDNA. They then successfully produced infectious viral particles by transfecting host cells with plasmids carrying Ebola cDNA, along with plasmids expressing Ebola proteins L, NP, VP30 and VP35 (needed for transcription and replication of negative strand RNA viruses), and one encoding the T7 RNA polymerase.

The researchers also used the system to make mutant virus particles containing an altered furin recognition motif. Furin cleaves Ebola virus glycoprotein at a highly conserved sequence motif, an event hypothesized to be critical to viral pathogenicity. However, viral particles carrying the altered motif still showed pathogenicity and ability to replicate in culture. This result illustrates the system’s utility for hastening our understanding of the Ebola virus life cycle and the development of anti-viral agents.
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Replicating Influenza Mutants with Reduced Sialidase Activity

A UW-Madison researcher has developed mutant cells useful for propagating influenza A virus mutants with reduced sialidase activity. The mutant cells have decreased levels of sialic acid and sialic acid-containing cell receptors as compared to wild type cells.

To generate the mutant cells, the researcher started with cells that supported the growth of influenza virus. These cells were incubated with a growth-inhibiting agent. Surviving cell colonies were cloned and infected with influenza virus variants with known sialic acid receptor-linkage specificity. A small percentage of cells, designated as MaKS cells, continued to grow without any evidence of virus production, and showed lowered levels of sialic acid-containing receptors. Two influenza virus variants that were able to grow well in these cells after several rounds of selection showed large deletions in the gene for NA as compared to the parent viruses.
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Attenuated Viruses with Mutant Ion Channel Protein

A UW-Madison researcher has developed—for the first time—a mutant virus that does not contain M2 ion channel activity. The recombinant influenza A virus was prepared using the inventor’s reverse genetics system (see WARF reference number P99264US). Although it replicates well in tissue culture, it is attenuated in mice. It does not cause disease symptoms, but is able to generate an antibody-mediated immune response, making it an excellent candidate for a live influenza vaccine or a vaccine vector for another pathogen.
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Recombinant, Attenuated Vaccine for Blastomyces Dermatitidis to Prevent Fungal Infection

UW-Madison researchers have successfully engineered an attenuated, replication-competent strain of B. dermatitidis that is incapable of expressing the cell wall protein WI-1. This recombinant, attenuated strain serves as a highly effective vaccine against the systemic, lethal infectious disease caused by B. dermatitidis and possibly against related fungi. In vitro, the number of lung colony forming units, as a sign of lung infection, revealed a substantial decrease in wild type infection.

The present invention describes use of the genetically engineered fungus as a vaccine given subcutaneously in multiple doses. Dosages can be administered at high levels to provide infection resistance yet low enough so that the fungus is cleared from the body. The researchers are currently conducting in vivo studies with dogs.
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Cell Line for Evaluating Influenza Virus Sensitivity to NA Inhibitors

A UW-Madison researcher has developed a cell line that is capable of universally monitoring the sensitivity of human influenza virus isolates to NA inhibitors. This Madin-Darby canine kidney (MDCK) cell line has been modified so it overexpresses the human β-galactosidase α2,6-sialyltransferase I (ST6Gal I) gene.

Several influenza virus isolates were tested in this cell line. The sensitivity of the viruses to an NA inhibitor correlated with the sensitivity of viral sialidase to the compound, demonstrating the potential utility of this cell line for detecting viruses that are resistant to NA inhibitors.
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Plasmids Encoding Avian Influenza Genes

A UW-Madison researcher has developed plasmids encoding either the H3 N1 or the H5 N2 genes of avian influenza. These genes were cloned directly from viral isolates and are under the control of the pol II promoter.
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Safer Ebola Virus and Vaccines

UW–Madison researchers have developed recombinant, biologically-contained Ebola ‘ΔVP30’ virus for use as a vaccine and research tool. The infectious virus cannot spread because it is made to lack a gene essential for transcription.

Specifically, sequences encoding the protein VP30 are deleted from the virus genome, making it unable to replicate. Prepared and administered pharmaceutically, the modified virus evokes an antibody response against Ebola virus glycoprotein, GP. Selectable markers, reporters and other heterologous sequences can be inserted into the genome.
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Influenza Variant Grows Well in CHO Cells

UW–Madison researchers have developed a modified version of the PR8 virus to grow in high yields in CHO cells. The modified strain could be used as the donor ‘backbone’ in the annual influenza vaccine. The backbone combines six gene segments encoding ‘internal’ viral products with gene variants for the two cell surface proteins (NA and HA) that characterize a given year’s influenza threat.
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Wis.L Master Cell Bank for Vaccine Development

UW–Madison researchers have developed a master cell bank of Wis.L cells (primary human fetal pulmonary fibroblasts) for commercial use as a substrate for the production of viral vaccines. The cells were first isolated by the State Laboratory of Hygiene (SLH) in 1968 and have been used extensively by SLH in viral diagnostic laboratories to isolate viruses from clinical specimens. The cells also have been used to grow rhinoviruses for inoculation studies. They have been subjected to testing as recommended by the FDA for use in vaccine production or experimental inoculation studies, and are free from extraneous contaminants.
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