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Protection of neo-natal pigs from virus disease: targeted intra-cellular immunisation with defective viral genomes

Contract nr: FAIR-CT96-1666
Project nr: 1666
Project type: SC
Starting date: 01/02/1997
Duration: 36 months
Total cost: 1,207,000 EUR
EC Contribution: 900,000 EUR
Scientific Officer: Isabel MINGUEZ-TUDELA
Research topic: Animal health
Acronym: INTRACELLULAR IMMUNISATION

Background:
Gastro-intestinal infections are common in new-born animals in intensive animal husbandry and pose a serious economic threat. While anti-bacterial agents have been very effective, anti-virals have met with limited success.

Objectives:
This project proposes to use a procedure known as intra-cellular immunisation, in which neonatal pigs will be inoculated with defective (non-infectious) viral genomes in order to prevent enteric infections.

These defective genomes are small (<=10 kb) and have the unique ability of interfering at the molecular level with (i.e. inhibiting) the multiplication of homologous infectious virus, and are known as defective interfering (DI) genomes. In vivo they need be present only in a sufficient number of cells to reduce infection and hence allow the host's defence mechanisms to overcome the virus. Intracellular immunisation will be specifically targeted to tissues normally infected by the viral pathogen, using virus-like particles (VLPS) with envelope proteins that confer the required cell tropism.

Description:

The benefit of this will be mediated by two complementary mechanisms:

1) direct interference with virus multiplication in the cell which is effective immediately after administration;

2) induction of a conventional protective immune response which is effective after a few days, in a manner analogous to human neonates who can be successively immunised by gut infection with oral poliovirus vaccine.

Intracellular immunisation will be developed for viruses of neonatal pigs, transmissible gast- enteritis virus (TGEV) and porcine epidemic diarrhoea virus (PEDV). In 1994 these viruses were major contributors to enteric and respiratory infections which cost the EU about 766 million euro (51% of an industry worth 1500 million euro, and comparable to that of North America). In addition, there is a major social impact as these losses are borne by small farmers rather than powerful industrial concerns. Virus like particles (VLPs) will be developed to target cloned TGEV DI RNA to pig enteric tract epithelial cells in culture. The final goal is the successful oral administration of this material to combat TGEV-induced disease in neonatal pigs. Successful development requires a trans-national combination of the complementary expertise of four research groups and a company to:

1) understand the molecular basis of intracellular immunity induced by DI virus genomes;

2) design VLPs to target the interfering molecules to tissues infected by the pathogen;

3) express interference with TGEV replication that is active in porcine gut epithelial cells;

4) evaluate protection in neonatal pigs against TGEV and PEDV. This technology will have many applications as a novel, practical, effective, and safe means for controlling and preventing virus diseases of live-stock in the EU, in North America and worldwide.

Current situation/results:
Two systems are being developed in parallel. One based on SFV that is used as a model since with this system in vivo protection has already been shown by interference with DI genomes. Progress in understanding the molecular basis of this interference has been shown to be related to sequences that contain the packaging signal of the viral RNA. The viral interference in this system provides 75-80% protection in vivo against the infection by the virulent virus. The system is very promising. In addition, it has been shown that the same type of protection has been shown with the influenza virus. Progress towards the development of a procedure to generate DIs free of helper virus is underway and most likely also will meet success.

The second system developed is the interference on TGEV. Two main achievements have been consolidated. One is the identification of a molecularly cloned DI-C RNA minigenome that interferes with the growth of wt virus by 90%. This minigenome has 9.7 kb and it has not been possible to reduce its size without losing the interference. The extent of the interference observed (90%) may not be enough for in vivo protection and a second mechanism of interference is being generated, based on a dominant negative mutant of the N protein, to increase the interference level and to avoid the selection of interference escaping mutants.

A helper-dependent expression system based on TGEV genome has been obtained that expressed between 5-10 µg/106 cells of ß-glucuronidase. These expression levels are considered, in principle, sufficient to interfere with virus replication when the vector is used to express an interfering molecule. Therefore, we consider the development of this expression system a success and the vector developed as a useful deliverable tool.
Two other expression systems with potential application in livestock have been developed based on infectious cDNA clones for Arteriviruses and Coronaviruses (TGEV). The expression of heterologous genes is being optimised with the new vectors. The infectious cDNA clones are being engineered for the expression of antigens and interfering molecules in farm animals.


Coordinator
Luis ENJUANES
CSIC - Consejo Superior de Investigaciones Cientificas
Campus de la Universidad Autonoma
E-28049 Cantoblanco (Madrid)
Tel.: +34 91 585 45 55
Fax: +34 91 585 49 15
E-mail: l.enjuanes@cnb.uam.es


Partners

  • Willy J.M. SPAAN
    University of Leiden
    P.O. Box 9600
    NL-2300 RC Leiden
    Tel.: +31 71 26 16 52
    Fax: +31 71 52 66 761
    E-mail: wspaan@microbiology.leidenuniv.nl

  • Juan PLANA-DURAN
    Laboratorios Sobrino SA
    Ctra. de Camprodon S/N
    E-17813 Vall de Bianya - Gerona
    Tel.: +34 97 229 00 01
    Fax: +34 97 229 01 02
    E-mail: JuanPlana@mx2.redestb.es

  • Nigel John DIMMOCK
    University of Warwick
    Gibbet Hill Road
    UK-CV4 7AL Coventry
    Tel.: +44 1203 52 35 93
    Fax: +44 1203 52 35 68
    E-mail: nd@dna.bio.warwickukc.uk

  • Reinhard VLASAK
    Austrian Academy of Sciences
    Billrothstr. 11
    A-5020 Salzburg
    Tel.: +43 662 639 61 24
    Fax: +43 662 639 61 29
    E-mail: rvlasak@oeaw.ac.at

 
 
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