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Influenza b virus mutants and uses therefor

An influenza virus and mutant technology, applied in the direction of virus/phage, virus, virus peptide, etc., can solve the problem of unclear mutation of attenuated virus

Pending Publication Date: 2019-12-13
FLUGEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, mutations in attenuated viruses are often ambiguous, and reversion is a problem

Method used

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  • Influenza b virus mutants and uses therefor
  • Influenza b virus mutants and uses therefor
  • Influenza b virus mutants and uses therefor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0199] Example 1: Generation of BM2 virus mutants

[0200] Based on section 7 of B / Florida / 4 / 2006 ( figure 2 ) designed a series of five novel BM2 null mutant constructs (BM2SR-1, BM2SR-2, BM2SR-3, BM2SR-4 and BM2SR-5), which were then synthesized using standard techniques known in the art into suitable Double-stranded DNA fragments from standard in vitro gene-assembly procedures. Constructs BM2SR-1 (SEQ ID NO:1), BM2SR-2 (SEQ ID NO:2), BM2SR-3 (SEQ ID NO:3), BM2SR-4 (SEQ ID NO:4) and BM2SR-5 ( The single-stranded mRNA or sense DNA nucleotide sequence of SEQ ID NO:5) is provided in Table 1.

Embodiment 2

[0201] Example 2: Production and cultivation of BM2 mutant virus

[0202] This example demonstrates that BM2SR-1 (SEQ ID NO:1), BM2SR-2 (SEQ ID NO:2), BM2SR-3 (SEQ ID NO:3), BM2SR-4 (SEQ ID NO:4) or BM2SR-5 (SEQ ID NO:5) mutated B / Florida / 04 / 2006 segment 7 and B / Lee / 1940 control, BM2-deleted segment 7 to grow BM2SR mutants. Mutant viruses, such as those carrying mutant BM2 nucleic acids, can be generated by plasmid-based reverse genetics as described by Neumann et al., Proc. Natl. Acad. Sci. USA 96:9345-9350 (1999). Briefly, 293T host cells were transfected with one or more plasmids encoding eight viral RNAs (corresponding to each of the eight influenza B segments). Each viral RNA sequence is flanked by an RNA polymerase I promoter and an RNA polymerase I terminator. Notably, viral RNAs encoding BM2 proteins include mutant BM2 nucleic acid sequences. The host cells are additionally transfected with one or more expression plasmids encoding viral proteins such as polymerase...

Embodiment 3

[0206] Embodiment 3: BM2SR viral protein expression

[0207] This example demonstrates the proteins expressed by the 12 BM2-deficient influenza B BM2SR viruses described in Table 4.

[0208] Vero cells (200,000) were cultured in TC-12 plate wells in MEM medium containing 10% fetal calf serum (FCS) for 24 hours. After washing twice with D-PBS, Vero cells were infected with 12 constructed B / Florida / 4 / 2006BM2SR-1 to BM2SR-5 and B / Lee / 40BM2SR mutant viruses together with two wild-type at a high MOI of 1.0 Viral controls B / Lee / 1940 and B / Florida / 4 / 2006 were infected. at 35°C, 5% CO 2 After 6 hours of incubation in atmosphere to allow a single round of replication, samples were harvested for protein analysis.

[0209]Infected cells were gently lysed in 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 1% Triton X-100 containing protease inhibitors. Nuclei and insoluble debris were pelleted by centrifugation at 14,000 xG. The clarified supernatant was subjected to sodium dodecyl sulfate pol...

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Abstract

Disclosed herein are compositions and methods related to mutant viruses, and in particular, mutant influenza viruses. The mutant viruses disclosed herein include a mutant BM2 sequence, and are usefulin immunogenic compositions, e.g., as vaccines. Also disclosed herein are methods, compositions and cells for propagating the viral mutants, and methods, devices and compositions related to vaccination.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of and priority to US Application Serial No. 62 / 463,994, filed February 27, 2017, the contents of which are incorporated herein by reference in their entirety. Background technique [0003] Influenza is the leading cause of death among American adults. About 36,000 people die from the flu each year, and more than 200,000 are hospitalized. Influenza is a highly contagious disease spread by coughing, sneezing and direct physical contact with virus-laden objects such as doorknobs and phones. Symptoms of the flu include fever, extreme fatigue, headache, chills, and body aches; about 50 percent of infected people are asymptomatic but remain contagious. Immunization is 50% to 60% effective in preventing influenza in healthy people under the age of 65, as long as the antigenicity of the circulating virus strain matches that of the vaccine. [0004] Vaccination is the main method of preven...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N15/44A61K39/12A61K39/145
CPCA61K39/12C07K14/005C12N7/00C12N2760/16121C12N2760/16122C12N2760/16134C12N2760/16162C12N2760/16221C12N2760/16222C12N2760/16234C12N2760/16262A61K2039/70A61P31/16C07K7/00A61K39/145A61K39/39C12N2760/16161C12N2760/16261
Inventor 迈克尔·J·莫泽八田靖子帕穆克·比尔赛尔
Owner FLUGEN
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