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Immunogenic compositions comprising venezuelan equine encephalitis virus replicon vectors and paramyxovirus protein antigens

A virus replication and immunogenicity technology, applied in the direction of viral antigen components, viruses/phages, introduction of foreign genetic material using vectors, etc., can solve the problem of the absence of preventive immunogenic compositions

Inactive Publication Date: 2006-07-05
WYETH HOLDINGS CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Despite the prevalence and severity of RSV and PIV disease, and numerous early attempts to produce vaccines, no immunogenic composition for the prevention of these infections currently exists

Method used

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  • Immunogenic compositions comprising venezuelan equine encephalitis virus replicon vectors and paramyxovirus protein antigens

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1: Construction of the VRP plasmid expressing PIV protein

[0057] The present invention uses human PIV-3 as a model parainfluenza virus. Human PIV3 virus stock (Washington 47885 / 57 strain) was prepared as previously described. See, Stokes, A. et al., Virus Research 25:91-103 (1992). Virus stocks were purified by polyethylene glycol (PEG) precipitation. RNA was extracted with Trizol-LS (Life Technologies), and reverse transcription PCR was performed using Titan One Tube RT-PCR system (Roche) as a template. Primers were used to amplify a fragment covering the entire open reading frame (ORF) of N, P, M, C, HN and F, including the 5' Kozak consensus sequence. The resulting fragment was then digested with the following restriction enzymes: Cla1 and HindIII for F, EcoR1 and BamH1 for HN, Pst1 and EcoR1 for NP, Acc1 and Xba1 for P and C, HindI11 and Xba1 for M. The resulting fragment was cloned into the shuttle plasmid pKSR1. Subsequently, the Apa1-ORF-Not1 ca...

Embodiment 2

[0059] Embodiment 2: VRP preparation

[0060] This example describes how to prepare VRP expressing each of the antigens of PIV3 by electroporating RNA from the plasmid constructed in Example 1 into BHK21 cells. The original VEE plasmid pVR100, and the two helper plasmids pHC (capsid) and pHC (gp E1 / E2) were obtained from AlphaVax (Durham, NC). See, Pushko et al. Virology 239:389-401 (1997), the entire contents of which are hereby incorporated by reference. PIV3 glycoprotein gene RT-PCR fragments were cloned individually into pVR200 or into pVR100 (HN together with F). The resulting plasmid is then transcribed in vitro to produce RNA. The RNA is then electroporated into BHK21 cells to obtain VRPs encoding NP, M, P, C, F and / or HN genes respectively (VRP-NP, -M, P, -C, -HN, -F, -F / HN).

[0061] After individual PIV genes were obtained and cloned into an appropriate expression vector, capped RNA transcripts were prepared in vitro using the Not1 linearized plasmid template and...

Embodiment 3

[0062] Example 3: Viral titers of VRP and PIV

[0063] The titer of VEE replicon (VRP) expressing PIV protein and glycoprotein was measured by immunohistochemistry. VRP was propagated in the BHK-21 cell line. In this example, BHK cells were obtained from the CCL-10 clone to distinguish it from other BHK clonal populations that do not share cryptic phenotypic features. As defined herein, the cells may be referred to as BHK21 or simply BHK cells. Infect BHK21 monolayer cells with serially diluted VEE replicons: VRPNP; VRP-P; VRP-M; VRP-C; VRP-HN; VRP-F; Cultivate for 16-20 hours. Cell monolayers were then mixed with 1:1 acetone / methanol for 5 minutes and stained with either bacterially expressed rabbit anti-VEE NSP1 protein, polyclonal antibody r835, or horse anti-PIV3 serum. Goat anti-rabbit antibody conjugated with cyTM3 (Jackson ImmunoResearch, West Grove, PA) or horseradish peroxidase (HOURP) conjugated anti-horse antibody (Kirkegaard & Perry, Maryland, MD) and aminoethy...

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Abstract

Self-propagating, fusogenic blebs are produced from cells infected with a population of Venezuelan Equine Encephalitis virus replicon particles (VRP). The self-propagating, fusogenic nature of the blebs is derived from expression of heterologous genes encoding viral fusion proteins that are incorporated into the replication defective replicon particles. The resulting blebs can be harvested from supernatants of cells displaying severe cytopathic effects. The blebs are used to make immunogenic compositions and devise methods of immunizing mammals against paramyxoviruses such as parainfluenza virus type 3.

Description

technical field [0001] The present invention relates to the field of immunogenic compositions for the treatment or prevention of infectious diseases caused by paramyxoviruses such as parainfluenza virus type 3. The invention also relates to the field of recombinant DNA and methods for expressing foreign genes in host cells. Background technique [0002] Paramyxoviruses are enveloped, negative polarity, non-segmented, single-stranded RNA viruses whose members can be highly infectious, highly prevalent, and highly pathogenic. Examples include measles virus, mumps virus, respiratory syncytial virus and parainfluenza virus. An agreement was signed under the organization and coordination of the World Health Organization (WHO) to work together to try to eradicate paramyxoviruses such as measles virus. Other paramyxoviruses, such as Newcastle disease virus, wreak havoc on livestock animal populations. [0003] Respiratory syncytial virus (RSV) and parainfluenza virus types 1, 2,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/18C07K14/135A61K39/155C07K14/115
CPCC12N2770/36143A61K39/155A61K2039/543C12N2760/18634A61K2039/5256C12N2760/18522C12N2770/36122C12N2760/18622C12N15/86C07K14/005A61K39/12A61P31/12A61P31/16A61P37/02C07K14/18
Inventor G·R·科瓦克斯X·莫N·瓦西拉基斯S·巴加瓦T·J·赞布S·A·乌德姆
Owner WYETH HOLDINGS CORP
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