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Production of flu vaccine in myceliophthora thermophila

A technology for Myceliophthora thermophila and influenza, which is applied in the direction of antisense single-stranded RNA viruses, microorganisms, microorganisms, etc., and can solve problems such as expensive yields

Pending Publication Date: 2020-06-19
二进国际有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the methods described so far are relatively expensive and / or their yields are relatively low

Method used

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  • Production of flu vaccine in myceliophthora thermophila
  • Production of flu vaccine in myceliophthora thermophila
  • Production of flu vaccine in myceliophthora thermophila

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0122] Example 1 - Hemagglutinin (HA) and neuraminidase (NA) in Myceliophthora (Myceliophthora expression in thermophila) (C1)

[0123] Several series of expression constructs were designed for the expression of recombinant HA proteins of each of the different influenza strains in C1. The HA proteins tested in this study are detailed in Table 1 below. The expression constructs are detailed in Figures 1-4 (Figures 1-3 - Constructs with HA; Figure 4 - Constructs with NA. P = promoter, ss = signal sequence, T = terminator). The type of HA or NA included in each construct is marked in bold. A list of abbreviations used to describe the various elements in the construct is provided at Figure 5 middle. The generation of such constructs is described in detail below under "Materials and Methods".

[0124] Table 1. HA proteins / virus strains

[0125]

[0126]

[0127] Initially, HA was expressed under the cbh1 (cellobiohydrolase 1) or chi1 (chitinase 1) promoter and ...

Embodiment 2

[0227] Example 2 - Immunization of HA produced in Myceliophthora thermophila (C1 ) Originality

[0228] Animal experiments were performed to test the full-length recombinant hemagglutinin protein with a transmembrane domain (rHA-TMD) from the A / New Caledonia / 20 / 99 (H1N1) influenza strain produced in C1 immunogenicity. The biochemical evaluation of rHA-TMD is shown in Figure 9 , which shows analysis of SDS-PAGE, native PAGE, and oligomer status using TEM negative staining. The magnification in the TEM images is x29000. Scale bar = 200 nm. White arrows point to ~40 nm oligomers. White circles mark ~15 nm oligomers.

[0229] Previous immunogenicity studies were performed using a secreted form of rHA from the same strain produced in C1. The transmembrane domain of this rHA is truncated and it contains additional domains (8-Gly domain and T4 foldon domain) that are not present in native HA. The rHA-8Gly-T4 construct did not induce a functional antibody response. It was...

Embodiment 3

[0298] Example 3 - Stirred Tank Fermentation of HA

[0299] Experiments were carried out to measure the production levels of the aforementioned rHA-TMD on a larger scale in stirred tank fermenters using batch and fed-batch techniques. For this, use Minifors TM 3L bioreactor to grow C1 strain D389 expressing rHA-TMD.

[0300] Table 6 summarizes the fermentation conditions for an initial set of experiments performed for 50 hrs. "Batch" - the indicated sugar concentration at the beginning of the fermentation. "Fed-batch" - the concentration of sugar in the feed. Fermentations were performed at pH 7.5 using a starting volume of 1.5 liters.

[0301] Table 6. Fermentation conditions 50hrs

[0302] run number temperature(℃) in batches fed batch R465F2 25 4% xylose none R465F4 35 1% xylose 47% xylose. R465F5 35 1% glucose 50% glucose. R465F6 35 4.7% glucose none

[0303] After fermentation, the mycelium was collecte...

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Abstract

Recombinant expression of influenza virus surface proteins in the fungus Myceliophthora thermophila strain C1 is provided. The recombinant proteins are for use in influenza vaccine compositions.

Description

technical field [0001] The present invention relates to the production of recombinant influenza virus surface proteins in the fungus Myceliophthora thermophila. The recombinant protein is used in influenza vaccine composition. Background technique [0002] Influenza virus is a lipid-enveloped virus with a negative-sense single-stranded segmented RNA genome. The envelope of the virion contains two types of surface glycoproteins, hemagglutinin and neuraminidase, which play essential roles in viral infection. Hemagglutinin (HA) mediates virus association with its host cell and viral entry through membrane fusion. Neuraminidase (NA) is an enzyme that plays an important role in the release of new progeny and in preventing their aggregation. [0003] Influenza viruses are classified into types A, B, and C on the basis of differences in nucleoproteins and matrix proteins. Each type is further classified into subtypes according to the combination of HA and NA present on their su...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/145C07K14/005C12N1/14C12P21/02C12R1/645
CPCA61K39/12C07K14/005C12P21/02C12N2760/16134C12N2760/16234A61P31/16C12N15/80A61K2039/5256A61K39/145C12N7/00C12N2760/16251C12N2760/18134C12N1/145C12R2001/645
Inventor M·艾玛法布T·C·维沃尔德M·R·阿尔费尼托M·贝尔I·勒加斯特卢瓦M-P·卡泽克M-C·伯纳德J·杜拜勒R·肯辛格
Owner 二进国际有限公司
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