Method of inactivating and preserving respiratory syncytial virus (RSV)

A kind of technology of syncytial virus, respiratory tract, be used in preserving RSV virus and stabilizing the pre-F protein in described RSV, prevent or treat RSV infection or the disease associated with RSV infection, respiratory syncytial virus and stabilizing in described RSV The pre-F protein domain

Pending Publication Date: 2020-01-14
XIAMEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the pre-F protein is in a metastable state and is easily transformed into a stable post-F protein, there are still great difficulties and challenges in developing the pre-F protein into an effective vaccine

Method used

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  • Method of inactivating and preserving respiratory syncytial virus (RSV)
  • Method of inactivating and preserving respiratory syncytial virus (RSV)
  • Method of inactivating and preserving respiratory syncytial virus (RSV)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0121] The inactivation of embodiment 1.RSV virus

[0122] 1. Materials and instruments:

[0123] Hep-2 cells ( CCL-23 TM ), Vero cells (ATCC): obtained from ATCC.

[0124] hRSV (pSynkRSV A2D46F): Standard strain of human respiratory syncytial virus, obtained from NIH, National Institutes of Health, USA.

[0125] 5C4 antibody: self-made in the laboratory. The 5C4 antibody specifically recognizes and binds to the pre-F protein, but does not recognize or bind to the post-F protein. The 5C4 antibody recognizes the Siteφ epitope on the pre-F protein, and it is a strong neutralizing antibody with significantly higher neutralizing activity than Palivizumab. For detailed information about the 5C4 antibody, please refer to Chinese patent application 201480013927.7 and PCT international application PCT / CN2014 / 073505.

[0126] 8C2 antibody: self-made in the laboratory. 8C2 antibody can specifically bind to both pre-F protein and post-F protein. The 8C2 antibody recognizes the ...

Embodiment 2

[0179] The preservation of embodiment 2.RSV virus

[0180] In this example, we further studied the effect of dialyzed saline solution (i.e. storage solution) on the pre-F protein on the surface of the virus after immobilization. Generally, we remove fixative from post-fixed samples (ie, solutions containing inactivated virus and fixative) by dialysis, filtration, or centrifugation. In Example 1, we adopted the method of placing the fixed samples in different concentrations of dialysis saline solution to replace the fixative, so as to preserve the inactivated virus in the corresponding storage solution. In this embodiment, the fixed sample was further dialyzed in different salt solutions to monitor the stability of the inactivated / fixed viral protein over time in different salt solutions. Thereby selecting a more suitable storage salt solution environment, so that the pre-F protein on the surface of the virus is more stable.

[0181] Use sodium chloride (Nacl, Sodium chloride...

Embodiment 3

[0187] Example 3. Detection of immune protection

[0188] In this example, we studied the immunoprotection of RSV virus treated with formaldehyde fixative. Briefly, formaldehyde was prepared in 2×PBS to a concentration of 0.01% and 0.0527%, respectively, and allowed to stand at 25° C. for 30 min. Subsequently, at 25° C., the prepared formaldehyde solutions and viruses were mixed slowly and evenly at a volume ratio of 1:1 for 12 hours. Subsequently, as described above, the fixative was removed by dialysis (dialysis at 25°C for 18 h), the dialysate was 550 mM saline solution, and then the dialyzed sample was centrifuged to remove a part of soluble impurities, and a certain volume of serum-free medium was used The precipitate was resuspended and mixed with AL adjuvant at a volume ratio of 1:1, and used for muscle immunization of SPF Balb / C mice (n=6-10). The group inactivated with 0.0527% formaldehyde had three immune doses, which were 2.52×10 5 (L), 2×10 7 (M) and 1×10 8 (H...

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Abstract

The invention relates to the field of virology and immunology, in particular to an inactivated and isolated respiratory syncytial virus (RSV) and a method of stabilizing pre-F proteins in the RSV, further relates to a method of preserving the RSV and stabilizing the pre-F proteins in the RSV and further relates to a vaccine containing the inactivated RSV, the inactivated RSV prepared and / or preserved by the method of the present invention and a use of the vaccine for the preventing or treating RSV infection or diseases associated with RSV infection.

Description

technical field [0001] This application relates to the fields of virology and immunology. Specifically, the present application relates to a method for inactivating an isolated respiratory syncytial virus (Respiratory Syncytial Virus, RSV) and stabilizing the pre-F protein in the RSV. In addition, the present application also relates to a method for preserving RSV virus and stabilizing the pre-F protein in said RSV. The present application also relates to a vaccine containing an inactivated RSV virus prepared and / or preserved by the method of the present invention, and the vaccine is used for preventing or treating RSV infection or a disease associated with RSV infection the use of. Background technique [0002] Since its discovery in the 1950s, human respiratory syncytial virus (RSV) has been the leading cause of lower respiratory tract infection in infants and young children. RSV is the leading cause of hospitalization in infants under 1 year of age in the United States...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/06C12N1/04A61K39/155A61P31/14
CPCC12N7/00C12N1/04A61K39/12A61P31/14C12N2760/18563A61K2039/5252C12N2760/18534A61P11/00C12N2760/18561A61K2039/575A61K39/155C07K14/005C12N2760/18571
Inventor 郑子峥张伟张璐婧孙永鹏陈莉夏宁邵
Owner XIAMEN UNIV
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