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Ectodomains of influenza matrix 2 protein, expression system, and uses thereof

A technology of influenza virus and ectodomain, applied in the direction of viruses, viral peptides, antiviral agents, etc., can solve problems such as insufficient efficacy

Inactive Publication Date: 2013-05-22
EMERGENT PROD DEV GAITHERSBURG INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, DNA vaccines are easy to produce and safe to administer, but are not effective enough, especially in clinical trials, where large (milligram) doses are required

Method used

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  • Ectodomains of influenza matrix 2 protein, expression system, and uses thereof
  • Ectodomains of influenza matrix 2 protein, expression system, and uses thereof
  • Ectodomains of influenza matrix 2 protein, expression system, and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0282] Embodiment 1 recombinant vector construction

[0283] Type A influenza virus gene Pr8HA sequence (SEQ ID NO:51-52), NP consensus sequence (SEQ ID NO:19-20), Pr8M2 (SEQ ID NO:13-14), Pr8M2e_TML (SEQ ID NO:53-54) , METR_S (SEQ ID NO:17-18 and 56) and METR_C (SEQ ID NO:15-16 and 55) were cloned into the vEM11 recombinant vector ( figure 1 ). The resulting recombinant vectors vEM47 (encoding NP consensus sequence), vEM58 (encoding METR_S peptide), vEM57 (encoding METR_C peptide), vEM61 (encoding Pr8M2), vEM62 (encoding Pr8M2e-TML) and vEM65 (encoding Pr8HA) are shown in Figure 2A-F .

Embodiment 2

[0284] Example 2 Homologous recombination and recombinant virus isolation

[0285] For the modified MVA virus vector MVAtor TM (EBS company (Emergent Biosolutions)) into the influenza virus gene, with MVAtor to infect CEF cells and then with Figure 2A-F Transfection with recombinant vectors indicated in . 2-3 sets of parallel settings were carried out in each carrier. First, 5X10 5 CEF cells were incubated at 37°C and 5% CO 2 Incubate for 24 hours. The next day, the cell density was assumed to be 10 6 cell. The MVAtor standard was diluted in Opti-Pro SFM containing 4 mM L-glutamine and 2.5 μg gentamicin per ml so that 500 μl contained 5X10 4 TCID 50 , that is, the working concentration is 1X10 5 TCID 50 / ml and yielded a moi of 0.05. For infection, growth medium was removed from cells, 500 μl of diluted MVAtor standard was added to each well and incubated for one hour at room temperature with shaking. Virus inoculum was removed and cells were washed with Opti-Pro ...

Embodiment 3

[0292] Embodiment 3 is used for getting rid of the PCR of empty vector contamination

[0293] To confirm that residual non-recombinant MVAtor was completely excluded from the filled vials of the purified virus stock, samples from purified and filled MVAtor-NP for mEM10, MVAtor-METR_C for mEM18, MVAtor-METR_S for mEM19, MVAtor-METR_S for mEM19, DNA for MVAtor-Pr8M2 for mEM22, or MVAtor-Pr8M2e_TML for mEM23 was isolated and used for PCR. The recombinant vector vEM47 was used as a positive control for the recombinant virus (vEM47). DNA isolated from MVAtor was used as a positive control for empty vector (MVA). Use H 2 O and CEF cells were used as negative controls.

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Abstract

The present invention provides a polynucleotide, polypeptide, recombinant modified vaccinia virus Ankara (rMVA) and related vaccine compositions and methods useful in the prevention and treatment of an influenza viral infection. Provided is an isolated polynucleotide encoding multiple copies of M2 infuenza ectodomain peptides or rMVA comprising the polynucleotide. Also provided are methods for inducing an immune response to a subject against an influenza virus or for treating a disease or symptom caused by or resulting from infection with an influenza virus.

Description

technical background [0001] Influenza viruses are negative-sense RNA members of the Orthomyxoviridae family that cause disease in humans and animals. Influenza virus infections are common and can be either epidemic or seasonal. Although influenza virus infection rarely causes death in infected individuals, the morbidity is severe. Therefore, influenza virus epidemics can cause significant economic losses. Also, influenza virus infection may be more dangerous for certain groups of individuals, such as those with heart disease, C.A.R.A. patients, or the elderly. [0002] Influenza viruses cause disease in a recurrent manner due to a number of complicating factors, including: 1) the presence of established reservoirs of different subtypes of influenza A viruses in waterfowl and waterfowl; 2) the interaction of avian influenza viruses with influenza viruses from other animals such as swine the ability to recombine, a process called 'antigenic shift'; 3) mutations that accumulat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K35/17A61K35/30A61K35/44A61K35/48A61K35/545A61K35/57A61K35/76A61K35/761A61K38/16A61K39/00
CPCA61K39/145C12N2760/16122C12N2750/14143C07K14/005C12N2760/16134A61K2039/5256A61K2039/543C12N2710/24143A61K39/12A61P31/16A61P43/00
Inventor T·吉南M·莱西M·斯基多普鲁斯N·迈托
Owner EMERGENT PROD DEV GAITHERSBURG INC
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