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Live-attenuated virus and methods of production and use

一种减毒病毒、病毒的技术，应用在生物化学设备和方法、失活/减毒、病毒等方向，能够解决减毒病毒成本有效的方式产生、不能产生高病毒效价等问题

Active Publication Date: 2018-04-20
THE UNIVERSITY OF HONG KONG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods often convert the virus under study to a slow-replicating virus, and these attenuated viruses often fail to generate high viral titers in eggs
Because most vaccine manufacturers use fertilized eggs to produce influenza vaccines, these attenuated viruses cannot be produced in a cost-effective manner

Method used

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  • Live-attenuated virus and methods of production and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0277] Example 1. Production of Synthetic Viral Genes and Recombinant Viruses

[0278] Materials and methods

[0279] Influenza A / Brisbane / 59 / 2007 (H1N1) was used as the prototype virus in this study. Data sets of individual viral fragments from viruses with human or avian origin have been established in our previous studies (Wong et al. BMC Evolutionary Biology , 10:253 (2010)). The codon usage bias observed from each fragment-specific data set is compared to the corresponding counterpart (eg, human PB2 versus avian PB2). To generate recombinant A / Brisbane / 59 / 2007 virus with the largest open reading frame (ORF) in each viral segment with avian-like virus codon usage bias, segments of wild-type A / Brisbane / 59 / 2007 were compared Specific codon usage frequencies are comparable to those inferred from avian influenza sequences. This analysis allowed the determination of the number of mutations required to introduce the prototype virus to change its codon bias from human-li...

Embodiment 2

[0303] Example 2. 8-mut virus is attenuated in mammalian cells and in mice

[0304] Materials and methods

[0305] Viral replication kinetics of WT and 8-mut viruses were determined in mammalian cells (MDCK and A549 cells) and embryonated eggs. To evaluate whether the virus was attenuated in mice, 5 female BALB / C mice in each group were intranasally infected with 6.75x105 p.f.u. WT or 8-mut virus. To determine whether the 8-mut virus was still capable of inducing neutralizing antibodies in vivo, serum samples from infected mice were examined by a microneutralization assay 28 days after infection. Each group of female BALB / c mice (N=3) was intranasally infected with 6.75x10 5 p.f.u. WT or 8-mut virus. Sera were collected 28 days post-infection for microneutralization assays against WT and 8-mut virus. Equal volumes of serum from each mouse in the same group were pooled for analysis. Sera were heat inactivated and serially 2-fold diluted from 1:10 to 1:1280. Serum added...

Embodiment 3

[0316] Example 3. 8-mut virus protects mice from virus challenge.

[0317] Materials and methods

[0318] To determine whether 8-mut could induce immune protection in mice, infected mice were subsequently challenged either homologously or heterologously. Six BALB / c mice in each group were inoculated intranasally with 6.75 x10 5 8-mut virus of p.f.u., or mock-inoculated with PBS. On day 28 after inoculation, vaccinated or mock-vaccinated mice were treated with 4.3 x 10 5 Mouse-adapted A / Brisbane / 59 / 07 virus (MA-WT) virus challenge of p.f.u. Lung tissues from treated mice were harvested at days 3 and 7 post-challenge for virus titration and immunohistochemical staining.

[0319] result

[0320] Because WT virus can only cause mild weight loss in mice ( Figure 2A ), the more pathogenic mouse-adapted A / Brisbane / 59 / 07 virus (MA-WT) (Xu et al., PLoS ONE , 6(12):e28901 (2011)) for the attack. PBS-inoculated mice showed moderate curly hair and mild body protuberance, a...

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Abstract

The invention discloses a live-attenuated virus and methods of production and use. Presented herein are live-attenuated viruses and methods of generating thereof from a parental virus through a plurality of nucleotide substitutions in the viral genome. The nucleotide substitutions result in a change in codon usage bias within codons of one or more protein encoding sequences and no change in aminoacid sequences of the proteins. The live-attenuated viruses display unaltered replication in avian hosts for propagation, but attenuated replication in mammalian hosts, when compared to the replication of the parental virus. The live-attenuated viruses in a form of improved vaccines can be used to elicit protective immune responses.

Description

field of invention [0001] The present invention relates generally to the field of live attenuated viruses, and in particular to the field of making and using live attenuated influenza viruses. Background of the invention [0002] Influenza is one of the medically important viruses in various countries. Of all the well-established control measures for this virus, vaccination is one of the most effective ways to prevent influenza transmission. Currently commercially available vaccines can be mainly classified as inactivated vaccines or live attenuated vaccines. Both types of vaccines have their limitations. Inactivated vaccines, also known as killed vaccines, contain non-infectious viral protein products and, following vaccination, such vaccines induce antibody-mediated immunity, but not cell-mediated immunity. [0003] Therefore, inactivated vaccines cannot stimulate one of the important branches of the adaptive immune system. In contrast, live attenuated vaccines can ind...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/04A61K39/12A61P31/16
CPCA61K39/12C12N2760/16234C12N2800/22C12N7/00A61K2039/5254C12N2760/16134C12N2760/16162A61P31/16
Inventor 潘烈文范乐欣
Owner THE UNIVERSITY OF HONG KONG
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