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THE USE OF F17 AND CS31A FIMBRISE AS CARRIERS FOR VIRAL EPI- TOPES AND THEIR APPLICATIONS AS VACCINES

Objective

The objectives of the project are as follows:

Identification and/or more accurate mapping of the epitopes of rotavirus and TGE virus strains.

The cloning and sequencing of CS31A and F17 subunit genes. These domains found will then be used for the introduction of the viral epitopes characterized.

Introduction of suitable restriction enzyme sites in the CS31A and F17 subunit genes using in vitro mutagenesis techniques. These restriction enzyme sites will allow the replacement of fimbrial unit sequences by synthetic oligonucleotides encoding the desired epitope.

The recognition and study of the viral epitopes present on the fusion proteins containing the viral epitopes, after vaccination in mice.

The constructs inducing antibodies in mice, which recognize the virus, will be further tested in protection studies (active or passive) in mice as a model system. The resistance of vaccinated mice to a challenge with a virulent rotavirus strain or of vaccinated piglets to a challenge with a virulent TGE virus strain will be compared to nonvaccinated animals as a control.
One difficulty of obtaining virus vaccine is the production of vaccinating antigens in large enough quantities. This aspect is particularly important for a virus which cannot be produced in vitro. Vaccinating epitopes are the minimal antigenic sites eliciting protective antibodies. Once these epitopes are identified, they can be introduced into a carrier protein. The fimbrial subunits F17 and CS3IA from Escherichia coli may be used as carrier proteins for the introduction of epitopes identified on bovine rotavirus strains and on transmissible gastroenteritis virus of piglets.
This group has been responsible for results in the following areas:
cloning, sequencing and genetic organisation of the fimbriae CS31A genes;
construction of the complementation system;
identification and localization of the sites in CS31A which allows insertion of heterologous peptides;
obtention of CS31A fimbrae with CS31A major subunit carrying up to 4 viral epitopes;
intraperitoneal immunization of mice with purified CS31A hybrid proteind (native or denatured) carrying epitope C and/or A of the spike protein S of TGE virus induced antibodies which recognise the viral peptides and the TGE virus.
These antibodies neutralized the infectivity of the TGE virus in vitro. The mice immunized with purified CS31A hybrid proteins carrying epitopes C and A of the S protein of TGE virus, an anamnestic response was demonstrated when mice were later innoculated with TGE virus
In mice orally innoculated with recombinant E coli producing CS31A with TGE virus epitope intestinal IgAs were produced against the viral peptide.

In addition complementary work was conducted to obtain a baby mouse model for testing rotavirus infection and vaccine protection and to study the gut colonizing ability of CS31A E coli in mice, and invivo stability of CS31A plasmids and genes.
gut colonizing ability of CS31A Escherichia coli and in vivo stability of the CS31A plasmids and genes.

One difficulty of obtaining virus vaccine is the production of vaccinating antigens in large enough quantities. This aspect is particularly important for a virus which cannot be produced in vitro. Vaccinating epitopes are the minimal antigenic sites eliciting protective antibodies. Once these epitopes are identified, they can be introduced into a carrier protein. The fimbrial subunits F17 and CS3IA from Escherichia coli may be used as carrier proteins for the introduction of epitopes identified on bovine rotavirus strains and on transmissible gastroenteritis virus of piglets.
A major common group epitope has been identified using a set of monoclonal antibodies and synthesis of all heptapeptides derived from the amino terminus of the capsid protein VP6. To test its application in protection a recombinant vaccinia virus expressing VP6 has been constructed and used in experiment of passive protection in the mice model.
The spike S protein (220K) is potentially the best candidate as protective antigen since it is the sole viral component capable to induce highly neutralizing antibodies.

One difficulty of obtaining virus vaccine is the production of vaccinating antigens in large enough quantities. This aspect is particularly important for a virus which cannot be produced in vitro. Vaccinating epitopes are the minimal antigenic sites eliciting protective antibodies. Once these epitopes are identified, they can be introduced into a carrier protein. The fimbrial subunits F17 and CS3IA from Escherichia coli may be used as carrier proteins for the introduction of epitopes identified on bovine rotavirus strains and on transmissible gastroenteritis virus of piglets.
A comparison has been made between the amino acid sequences of the fimbrial subunits F111-A and F17-A, identifying the 2 main variable regions of interest for viral epitope insertion.

One difficulty of obtaining virus vaccine is the production of vaccinating antigens in large enough quantities. This aspect is particularly important for a virus which cannot be produced in vitro. Vaccinating epitopes are the minimal antigenic sites eliciting protective antibodies. Once these epitopes are identified, they can be introduced into a carrier protein. The fimbrial subunits F17 and CS3IA from Escherichia coli may be used as carrier proteins for the introduction of epitopes identified on bovine rotavirus strains and on transmissible gastroenteritis virus of piglets.
Domains suitable for insertion of viral epitopes, have to be localized in the external regions of the fimbrial structure. These external regions can be epitopes of the F17 fimbriae, or can be detected by specific chemical modification reactions of nonburied amino acids. The peptides showing the strongest reactivity were synthesized, purified and linked to bovine serum albumin and will now be injected into rabbits in order to find out if it is possible to raise antibodies against these peptides and if these antisera will also react with the complete fimbriae. 3 peptides, potential neutralizing epitopes of VP4, from the sequence of the bovine rotavirus strain C 486 were synthesized. 5 mice were injected with each of the peptides, linked with bovine serum albumin. Immunoprecipitation showed that the mice used in our experiments were free of rotavirus infection.
Certain bacteria produce proteinaceous surface appendages (fimbriae). These proteins exposed at the surface of the bacteria are made of the association of a high number of protein subunits. CS31A and F17 for example are two fimbriae identified in bovine E.coli. Protein and DNA engineering enables the introduction of modifications in protein sequences, thus modified fimbriae can be produced and exposed in large quantities at the surface of the bacteria.

One difficulty of obtaining virus vaccine is the production of vaccinating antigens in large enough quantities. This aspect is particularly important for a virus which cannot be produced in vitro. Vaccinating epitopes are the minimal antigenic sites eliciting protective antibodies. Once these epitopes are identified they can be introduced into a carrier protein. The fimbrial subunits F17 and CS31A will be used as carrier proteins for the introduction of epitopes identified on bovine rotavirus strains and on transmissible gastroenteritis (TGE) virus of piglets. Both viruses are responsible for diarrhoea with high economic losses in farm animals. Vaccination with the hybrid proteins could be achieved either using the fusion proteins produced by bacteria in vitro (fermenter) or directly in vivo with bacteria multiplying in the gut. The intestinal vaccination could be particularly suitable for the induction of a protective local active immunity.

Coordinator

Institut National de la Recherche Agronomique (INRA)
Address
147 Rue De L'université
75341 Paris
France

Participants (3)

CENTRE D'ETUDE ET DE RECHERCHES VETERINAIRES ET AGROCHIMIQUES
Belgium
Address
99,Rue Groeselenberg 99
1180 Bruxelles
Rhône Merieux SA
France
Address
29 Avenue Tony Garnier
69348 Lyon
VRIJE UNIVERSITEIT BRUSSEL
Belgium
Address
Pleinlaan 2
1050 Bruxelles