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Method for production of ph stable enveloped viruses

A technology for enveloped viruses and viruses, applied in biochemical equipment and methods, viruses, antiviral agents, etc., can solve the problems of reducing virus infectivity, reducing immunogenicity, reducing

Active Publication Date: 2011-10-19
NANOTHERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Destabilizing mutations commonly found in the interface HA1-HA2 or HA2-HA2 region or in the N-terminus of HA2 can in turn lead to reduced binding to cell surface receptors (Korte et al., 2007, Rachakonda et al., 2007, Faseb J 21,995-1002, Shental-Bechor et al., 2002, Biochim Biophys Acta 1565.81-9), which lead to reduced viral infectivity and subsequently reduced immunogenicity of live virus preparations

Method used

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  • Method for production of ph stable enveloped viruses
  • Method for production of ph stable enveloped viruses
  • Method for production of ph stable enveloped viruses

Examples

Experimental program
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Effect test

Embodiment 1

[0073] Two influenza strains were constructed by reverse genetics carrying surface glycoproteins from the circulating strain A / Wisconsin / 67 / 05 (H3N2) and from the WHO vaccine strain IVR-116 (A / New Caledonian / 20 / 99 and A / Puerto Rico / 8 / 34 reassortant) all other genes, and the NS gene (ΔNS1) lacking the NS1 open reading frame. Due to the different passage conditions of Vero cells, the obtained virus differed by one amino acid substitution in the sequence of the HA molecule. The first virus, named Wisc.ΔNS1, was always passaged on Vero cells, and the virus inoculum was first treated with a low pH buffer, namely:

[0074] MES infection buffer (0.1M MES, 150mM NaCl, 0.9mM CaCl) supplemented with 0.25μg / ml amphotericin B 2 , 0.5 mM MgCl 2 ; pH=5.6) to dilute the virus to the appropriate moi. Vero cells were washed with infection buffer, and viral inoculum was applied to the cells and incubated for 30 minutes. The inoculum was then removed and cells were incubated in serum-free O...

Embodiment 2

[0083] Two H1N1 influenza strains were constructed by reverse genetics, carrying the surface glycoprotein from the circulating strain A / Brisbane / 59 / 07 (H1N1) and all other genes from the WHO strain IVR-116, and lacking NS1 NS gene ([Delta]NS1) combination of open reading frames. The obtained virus differed by one amino acid substitution in the sequence of the HA molecule, which appeared to be due to the different passage conditions of the Vero cells. The first virus, designated Brisbane ΔNS1, was passaged in the presence of amphotericin B under low pH conditions. This procedure resulted in the maintenance of the HA sequence, which appeared to be similar to that of the virus (passage 1 ) isolated in MDCK cells from clinical samples.

[0084] A second virus, designated Brisbane ΔNS1_HA2_N16I, was passaged by standard methods and acquired a substitution in the HA2 subunit, N16I (Table 2). Table 2 shows a sequence comparison of the HA molecules, compared to the original isolates...

Embodiment 3

[0091] Cultivation of influenza B strains on Vero cells under standard conditions also resulted in the appearance of destabilizing mutations at the interface of the HA1-HA2 or HA2-HA2 regions in the HA molecule, which involved a decrease in stability and subsequent reduction in the mutant virus in animal models. Immunogenicity in (data not shown).

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Abstract

The present invention provides a method for producing pH-stable enveloped viruses wherein said viruses are used for infection of host cells under low pH conditions and for incubation with cell culture cells under conditions of low pH, as well as influenza viruses obtainable by this method which exhibit a high growth rate in cell culture, increased pH and temperature stability and which have human receptor specificity.

Description

field of invention [0001] The present invention provides a method for producing a pH-stable enveloped virus, wherein the virus is incubated with cell culture cells under low pH conditions. Such viruses include novel influenza viruses that display high growth rates in cell culture, elevated pH and temperature stability, and have human receptor specificity. Background of the invention [0002] Vaccination is the most important public health measure to prevent diseases caused by annual viral infection epidemics. Efficient supply of vaccines depends on the ability to rapidly produce large quantities of vaccine material, such as viruses. The rapid development of vaccines and their abundant availability are critical in the fight against many human and animal diseases. Delays and insufficient quantities of vaccine production can cause problems when addressing disease outbreaks. [0003] Growth of viruses, especially influenza viruses, in embryonated eggs has been shown to result...

Claims

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Application Information

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IPC IPC(8): A61K39/145
CPCA61K2039/5254C12N2760/16134A61K39/12A61K39/145A61P31/12A61P31/16A61P37/04C12N7/00C12N2760/16021C12N2760/16034C12N2760/16051
Inventor 朱莉亚.罗马诺瓦安德雷杰.埃戈罗夫布里吉特.克伦马库斯.沃尔谢克萨拜因.纳科维奇
Owner NANOTHERAPEUTICS INC
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