Parapoxvirus vectors containing rabies virus antigen

A rabies virus, pox virus technology, applied in the application field of recombinant parapox virus in diagnostics

Inactive Publication Date: 2013-08-21
ZOETIS SERVICE LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Parapoxvirus vectors containing rabies virus antigen
  • Parapoxvirus vectors containing rabies virus antigen
  • Parapoxvirus vectors containing rabies virus antigen

Examples

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[0116] The preparation of parenteral formulations under sterile conditions (eg, by lyophilization) is readily accomplished using standard pharmaceutical techniques well known to those skilled in the art.

[0117] The recombinant parapoxviruses and immunogenic compositions and vaccines described herein can be used in the preparation of medicaments for vaccinating animals against rabies.

[0118] The present invention provides methods for determining the source of a parapoxvirus present in an animal.

[0119] Vaccination with the DIVA vaccine, which distinguishes infected animals from vaccinated animals, provides a way to determine the source of parapoxviruses present in animals. This differentiation can be achieved via any of a variety of diagnostic methods including, but not limited to, ELISA, Western blot, and PCR. These and other methods are readily recognized and understood by those of ordinary skill in the art.

[0120] The parapoxviruses described herein can be distingu...

Embodiment 1

[0127] Example 1. Production of rabies G protein expressing recombinant virus D1701-V-RabG

[0128] Recombinantly produced ovine parapoxviruses containing the gene encoding the G protein of RV were generated. The expression of the G protein and the immunostimulatory and protective properties of the recombinant virus against RV were assessed.

[0129] Construction of the transfer plasmid

[0130] For the production of recombinant ovine parapoxvirus D1701-V-RabG, the ovine parapoxvirus (PPVO) vector system was used (US Pat. J. Virol. 77, 9312-9323; Henkel et al., 2005, J. Virol. 79, 314-325). The rabies virus G protein gene was chemically synthesized (Blue HeronBiotech; USA) and provided in a pUC plasmid. The intact G gene was separated into a BamHI-EcoRI DNA fragment with a size of 1,582 bp by agarose gel (0.8%, weight / volume ratio) electrophoresis and purified by Qiaex II gel extraction kit (Qiagen; Germany). Plasmid pdV-Recl (Fischer et al., 2003) was double digested wi...

Embodiment 2

[0138] Example 2. Characterization of D1701-V-RabG

[0139] Preparation of virus stock solution

[0140] In order to obtain a high-titer recombinant virus stock solution, 10-20 T150 culture flasks (Greiner; Germany) were simultaneously infected at an MOI of 0.5. After 3 days, approximately 80% cytopathic effect (CPE) was observed, and cells and supernatants in all flasks were collected for centrifugation (13,000 rpm at 4°C for 2 hours). Carefully remove the supernatant, and dissolve the virus pellet in 1ml~2ml PBS overnight at 4°C. The virus suspension was completely dispersed by ultrasound (Soniccell disruptor, Branson; Germany) using three 10-second pulses (100 W) on ice (10-second rest between each pulse), followed by centrifugation (500 × g ~ 700 × g, 10 min, 4°C) to remove cell debris. The supernatant was stored on ice while the cell pellet was resuspended in 1.0 ml PBS and sonicated on ice (2 x 20 sec with 10 sec rest in between, then 1 x 30 sec). After centrifugat...

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Abstract

The present invention relates to recombinant parapoxviruses which carry in their genomes comprising heterologous DNA derived from a rabies virus, to the preparation of such constructs, and to their use in immunogenic compositions and vaccines. It further relates to the use of recombinant parapoxviruses for diagnostics.

Description

technical field [0001] The present invention relates to recombinant parapoxviruses containing heterologous DNA derived from rabies virus (RV) and their use in immunogenic compositions and vaccines. The invention also relates to methods for vaccination, treatment or prophylaxis against diseases caused by rabies virus. The invention also relates to the use of recombinant parapoxviruses in diagnostics. Background technique [0002] Poxviridae viruses are oval-shaped very large double-stranded DNA viruses. Parapoxviruses (PPV) are included in this class of viruses. Its size is about 220nm~300nm long and 140nm~170nm wide. They possess a unique helical capsid that distinguishes them from other poxviruses. [0003] PPV is divided into three different species. However, it remains unclear whether these viruses are autonomous species within the Parapoxvirus genus or whether they are the same species. The first species, Parapoxvirus ovis, is considered the prototype of the genus....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/205C07K14/145C12N15/863A61K39/00
CPCA61K39/205C07K14/005C12N7/00C12N15/86C12N2710/24243C12N2760/20122A61K2039/5256A61K2039/541C12N2760/20134A61K39/12A61P31/12A61P31/14A61P31/20A61P37/04C12N15/863C12N2710/24211
Inventor O.M.马蒂诺N.K.D.雷迪
Owner ZOETIS SERVICE LLC
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