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Hendra and nipah virus g glycoprotein immunogenic compositions

An immunogenicity, Nipah virus technology, applied in the field of immunogenicity and vaccine composition, can solve the problems of reducing cell lysis effect, reducing the ability to extract cholesterol, reducing reactivity, etc.

Inactive Publication Date: 2014-12-24
ZOETIS SERVICE LLC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This is mainly due to the reduced reactivity of Quil A when incorporated into immunostimulatory complexes, as its binding to cholesterol in the complex reduces its ability to extract cholesterol from the cell membrane and therefore reduces its cell lysis effect

Method used

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  • Hendra and nipah virus g glycoprotein immunogenic compositions
  • Hendra and nipah virus g glycoprotein immunogenic compositions
  • Hendra and nipah virus g glycoprotein immunogenic compositions

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1: Vector constructs

[0078] Vectors for the expression of HeV G or NiV G deleted for the transmembrane / cytoplasmic tail were constructed. The cloned cDNA of the full-length HeV or NiV G protein was amplified by PCR to generate a ~2600 nucleotide fragment encoding the transmembrane domain / cytoplasmic tail deleted HeV or NiV G protein.

[0079] The following oligonucleotide primers for HeV G amplification were synthesized.

[0080] sHGS: 5'-GTCGACCACCATGCAAAATTACACCAGAACGACTGATAAT-3' (SEQ ID NO: 5).

[0081] sHGAS: 5'-GTTTAAACGTCGACCAATCAACTCTCTGAACATTGGGCAGGTATC-3'. (SEQ ID NO: 6).

[0082] The following oligonucleotide primers for NiV G amplification were synthesized.

[0083] sNGS: 5'-CTCGAGCACCATGCAAAATTACACAAGATCAACAGACAA-3' (SEQ ID NO: 7).

[0084] sNGAS: 5'-CTCGAGTAGCAGCCGGATCAAGCTTATGTACATTGCTCTGGTATC-3'. (SEQ ID NO: 8).

[0085] All PCR reactions were performed using Accupol DNA polymerase (PGS Scientifics Corp) using the following settings: first ...

Embodiment 2

[0094] Example 2: Protein production of soluble G protein using vaccinia virus

[0095] For protein production, genetic constructs containing codon-optimized sequences were used to generate recombinant poxviral vectors (vaccinia virus, WR strain). Recombinant poxviruses were then obtained using standard techniques using tk selection and GUS staining. Briefly, CV-1 cells were transfected with pMCO2sHeV G fusion or pMCO2sNiV G fusion using the calcium phosphate transfection kit (Promega). These monolayers were then infected with the Western Reserve (WR) wild-type strain of vaccinia virus at a multiplicity of infection (MOI) of 0.05 PFU / cell. After 2 days, the cell pellet was collected as crude recombinant virus stock. TK-cells were infected with recombinant crude stock in the presence of 25 μg / ml 5-bromo-2'-deoxyuridine (BrdU) (Calbiochem). After 2 hr, virus was replaced with an overlay of EMEM-10 containing 1% low melting point (LMP) agarose (Life Technologies) and 25 μg / ml ...

Embodiment 3

[0096] Example 3: Protein production of soluble G protein using 293F cells

[0097] 293F cells (Invitrogen) were transformed with a genetic construct containing a codon-optimized sequence to generate a stable cell line expressing HeV soluble G glycoprotein. CHO-S cells (Invitrogen) were also used for transformation and expression of HeV soluble G glycoprotein. Place the transformed cells in a 162 cm medium containing 35 ml DMEM-10 2 Plate in tissue culture flasks. Allow cells to attach and store at 37°C and 5-8% CO 2 Grow for a few days. When cells are confluent, split them into multiple culture flasks (30 ml / flask) containing DMEM-10 and 150 μg / ml hygromycin B. When the cells were 70-80% confluent, they were washed twice with 30 ml PBS, then 20 ml 293SFM II (Invitrogen) was added, and the cells were incubated at 37 °C and 5-8% CO 2 Incubate overnight. The next day, cells were transferred to Erlenmeyer flasks containing 200 ml of SFM II medium. Allow cells to cool at 37...

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Abstract

Immunogenic compositions directed against Hendra and / or Nipah viruses, and methods of its use, are provided. In addition, methods of distinguishing subjects vaccinated with the immunogenic compositions of the invention from those infected with Hendra and / or Nipah virus are provided.

Description

technical field [0001] The present invention relates to immunogenic and vaccine compositions comprising G glycoproteins from Hendra virus (HeV) and / or Nipah virus (NiV), and methods of use related thereto. Background technique [0002] Recently, recurring outbreaks of NiV causing mass human mortality have been problematic (see eg Butler (2000) Nature 429, 7). HeV is also known to cause lethality in humans and animals, and it is closely related genetically and immunogenically to NiV. There are currently no vaccines or treatments available to prevent infection or disease caused by Nipah or Hendra viruses. Both Nipah virus and Hendra virus are National Institute of Allergy and Infectious Diseases (United States, National Institute of Allergy and Infectious Disease) Category C Priority Biodefense Concern Agents. Furthermore, since these viruses are zoonotic Biosafety Level 4 agents (BSL-4), it is very costly and difficult to safely produce vaccines and / or diagnostics. Therefo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/155
CPCA61K2039/55577A61K2039/544A61K2039/55505A61K2039/55561C12N2760/18234A61K39/12G01N2469/20C07K14/005A61K2039/552C12N2710/24143C12N2760/18222A61K2039/575G01N33/56983G01N2333/115A61P31/12A61P31/14A61P31/16A61P37/00A61P37/04A61K39/155C12N7/00A61K2039/55511A61K2039/54A61K2039/545
Inventor 马丁·艾莱克里斯多佛·C·布勒德黄金安
Owner ZOETIS SERVICE LLC
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