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New molecular approaches for improved vaccines to poultry diseases

Contract nr: FAIR-CT96-1502
Project nr: 1502
Project type: SC
Starting date: 01/02/1997
Duration: 42 months
Total cost: 1,629,000 EUR
EC Contribution: 1,092,000 EUR
Scientific Officer: Isabel MINGUEZ-TUDELA
Research topic: Animal health
Acronym: POUVAC

Background:
The poultry industry is facing increasing problems due to the emergence of progressively more virulent forms of pathogenic viruses, which require increasingly effective vaccines to prevent disease. However, vaccine strains aggressive enough to protect against these new viruses can themselves cause pathogenic effects.

Objectives:
The aim of this project is to overcome this problem by identifying the specific peptides within vaccines which cause the strongest vaccine response in animals of particular genotypes, so that combinations of vaccine and host genotype can be developed which maximise both the strength and consistency of protection.

Description:
There has been rapid progress in understanding the manner in which the immune system recognises and reacts to foreign molecules, and it is now clear that this process depends crucially on the ability of molecules of the Major Histocompatibility Complex (MHC) to present small peptide fragments of proteins to the immune system. Only a small proportion of the peptide fragments present in viral proteins are presented in this way, and animals with different MHC molecules present distinct, very specific subsets of these peptides. It is already known that chickens of some MHC types respond well to certain vaccines. By systematically extending this information it will be possible to identify animals of similar genotypes in commercial flocks. More dramatically, it will be possible to characterise the particular viral peptides these MHC types present, making it possible to improve vaccines both by increasing the proportion of these peptides in the vaccines and by matching particular vaccines to birds of particular MHC types. By identifying and selecting the MHC types present in flocks it will then be possible to optimise vaccination. Most significantly, this will make it possible to create vaccines in which different components are optimised to stimulate the maximum protection of a number of different host MHC types, creating a epidemiological situation much less favourable to the development of forms of the virus able to overcome the vaccine.In this project, work will concentrate on infectious bursal disease virus (IBDV) and Marek's disease virus (MDV), the viruses that cause two of the most serious diseases of European poultry. These diseases are ideal candidates for this approach since, in both cases, current vaccines suffer from the problems outlined above and MHC-related differences in response to current vaccines have already been shown.These viruses provide tests of this approach for a relatively small and simple virus (IBDV) and for a large and highly complex virus (MDV). Although this work is targeted at IBDV and MDV the methodology developed should apply equally to other vaccines both in poultry and in other farm animals, and may ultimately be of relevance to man.

Current situation/results:

Task 1 - Characterisation of vaccine responses

  • The specific antibody responses of 18 chicken lines (bearing at least 20 MHC haplotypes) to commercial vaccines which include killed IBDV (as well as other small disease viruses) was determined. A wide range of antibody responses to killed IBDV (and to other vaccine viruses) in different chicken lines was found, with clear evidence from some lines that the MHC is a major determinant of these responses.
  • Different responses to the killed IBDV, infectious bronchitis virus (IBV) and avian rhinotracheitis virus (ART) in the vaccine were found for all chickens within any particular line, suggesting that the response is determined (at least in part) by whether the MHC molecules in those chickens bound peptides from a vaccine virus or not.
  • Four F2 crosses between MDV-resistant and susceptible chicken lines were vaccinated with RISPENS vaccine, infected with the very virulent strain of MDV (RB1B) and examined for mortality and blood viral DNA levels. We found that protection increased for some MHC haplotypes but not in others, similar to the differences in efficiency of vaccination seen for the parental lines.
  • Comparison of the blood viral DNA levels of the RISPENS vaccine shows that the vaccine virus persists in some lines but is rapidly eliminated in other lines. The effect of these kinetics on protection (vaccine efficiency) is not yet clear

Task 2 - Molecular definition of MHC haplotypes

  • The microsatellite LEI258 has been tested extensively against all of the chicken lines examined in Task 1, and shows both high linkage to the MHC, a wide range of alleles and discrimination between most haplotypes. It should prove very useful for MHC typing.
  • The DNA sequence has been determined for a large portion (140 kB) of the chicken MHC (the B locus including the class I and class II genes analysed for Task 3.1) for the B12 haplotype. This work provides additional sequence that can be screened for further microsatellites which can be used to define extended haplotypes.

Task 3 - Molecular characterisation of MHC genes and molecules

  • The sequences of the important (polymorphic) regions of both the class I alpha and class II beta genes of the chicken MHC (B complex) have been determined for all of the chicken lines examined in Task 1. In addition, some sequences for genes from the near-by Rfp-Y region have been determined.
  • The sequences show that the genes are identical for most lines with the same MHC haplotype, but for one haplotype (B19), there is variation between the class II genes even within lines that appears to be the result of micro-recombination (gene conversion).
  • Analysis by two-dimensional gel electrophoresis of class I molecules from different lines shows that there is a single dominantly-expressed class I molecule in many haplotypes (B4, B12, B15, B19) but more than one such molecule in some haplotypes (B2, B21).
  • Clear peptide-binding motifs have been determined for the class I molecules of several more MHC haplotypes (B2 and B14), bringing the total to seven, including the ones (B2, B12, B15) necessary for Tasks 4 and 5.
  • The peptides bound to class II molecules of several more MHC haplotypes have been analysed, with the surprising finding that each has a dominant bound peptide, a finding never reported for any mammalian class II molecule. The peptide-binding motif for the class II molecule (B15) necessary for Tasks 4 and 5 has been refined for use in prediction.

Task 4 - Analysis of the immune response to vaccines in vitro.

  • An in vitro assay has been established for proliferation of cells from CB (B12) chickens inoculated with src-transformed tumor cells, which shows specific response upon presentation of the predicted peptide (B12-6) shown to bind to the class I molecule from B12 chickens, but not to a non-binding peptide.
  • Recombinant DNA expression systems have been used to express various soluble his-tagged IBDV proteins (VP1, VP2, VP3, VPX, polyprotein, and VP5). These have been used both as specific antigen in class II-dependent proliferation assays and to express IBDV proteins after vaccinia virus infection of target cells in order to function as specific antigen in class I-dependent cytotoxicity assays.
  • An in vitro assay for T cell proliferation has been established using his-tagged VP1, VPX and VP3 proteins from IBDV. For VP1F1 (a 150 amino acid long fragment from the N-terminus of VP1) there was no proliferative response. However for the VPX and VP3 proteins there were proliferative responses which were both dose dependent and which were MHC class II-restricted, with cells of the B15 haplotype showing a proliferative response while those of the B21 haplotype did not. Additional immunisations increased the observed proliferative response.
  • The peptides derived from certain IBDV proteins (VP1, polyprotein) that potentially bind the class II molecule(s) of the B15 haplotype have been predicted based on the peptide-binding motifs determined in Task 3, in order to synthesise and test in the in vitro proliferation assay.
  • A novel in vitro assay based on flow cytometry and REV-B transformed target cells has been developed to measure cellular cytotoxicity (so far using alloreactive cells from B15 and B21 haplotypes). This assay overcomes many of the limitations encountered with radioactive labeling of chicken target cells, such as poor incorporation, high spontaneous release and low specific release. Analysis of the cells responsible for the observed cytotoxicity showed these to be principally CD8 positive T cells, although other non-CD8 cells were also involved.
  • The results of the cytotoxicity assays for effector cells from B15 chickens show that VP3 and polyprotein can induce cell-mediated immune response. Target cells expressing IBDV polyprotein show a low (about 2.5%) but specific death, and VP3 expressing targets demonstrate a higher cytotoxicity with a mean of about 5%. The response is IBDV protein-specific since uninfected target cells, that do not express IBDV proteins, were not killed by effector cells. Levels of specific cytotoxicity were higher with effector cells from spleen than from peripheral blood.
  • The peptides derived from certain IBDV proteins (VP1, polyprotein) that potentially bind the class I molecule(s) of the various haplotype have been predicted based on the peptide-binding motifs determined in Task 3, allowing the possibility of synthesis and examination in the in vitro cytotoxicity assay.
  • The peptides derived from certain MDV proteins (ICP-4, meq) that potentially bind the class I molecule(s) of the various haplotype have been predicted based on the peptide-binding motifs determined in Task 3, allowing the possibility of synthesis and examination in the in vitro cytotoxicity assay.

Task 5 - Analysis of protection by peptides and defined antigens in vivo

  • Experiments testing the ability of the predicted peptide B12-6 to confer protection against tumour growth in CB (B12) chicken s in vivo have repeatedly demonstrated highly significant reduction or prevention of tumour growth in the vaccinated chickens. Chickens similarly vaccinated with the less well bound peptide B12-4 were less well protected. This result provides the crucial demonstration of principle of the POUVAC programme.
  • More extensive vaccination of CB chickens with the moderately binding B12-4 peptide was able to induce a level of protection. This indicates that it is possible to utilise less well bound peptides through repeated vaccination.
  • Different methodologies for vaccination using peptides have been examined, along with optimisation of viral dose and host age.B12-6 peptide inoculate in liposomes was more effective in preventing tumour growth than the same peptide inoculated in incomplete Freund's adjuvant or in microspheres.

Coordinator
Nathaniel BUMSTEAD
Institute for Animal Health
Compton Laboratory
UK-RG20 7NN Compton - Nr Newbury
Tel.: +44 1635 57 84 11
Fax: +44 1635 57 72 63
E-mail: Nat.bumstead@bbsrc.ac.uk


Partners

  • Rima ZOOROB
    CNRS - Centre National de la Recherche Scientifique
    Rue Guy Moauet 19
    P.O. Box 8
    F-94801 Villejuif
    Tel.: +33 1 49 58 35 00
    Fax: +33 1 49 58 35 09
    E-mail: zoorob@lovelace.infobiogen.fr

  • Karel HALA
    Leopold-Franzens Universität Innsbruck
    Fritz Pregl-Straße 3
    A-6020 Innsbruck
    Tel.: +43 512 507 31 03
    Fax: +43 512 507 28 67
    E-mail: karel.hala@uibk.ac.at

  • Anne-Marie CHAUSSE
    I.N.R.A.
    Centre de Recherche de Tours-Nouzilly
    F-37380 Nouzilly
    Tel.: +33 2 47 42 76 19
    Fax: +33 2 47 42 77 74
    E-mail: coudert@inra.tours.fr

  • Jim KAUFMAN
    Institute for Animal Health
    Compton Laboratory
    Compton
    UK-RG20 7NN Newbury
    Tel.: +44 1635 57 72 34
    Fax: +44 1635 57 72 63
    E-mail: jim.kaufman@bbsrc.ac.uk

  • Jan SALOMONSEN
    Royal Veterinary and Agricultural University
    Bülowsvej 13
    DK-1870 Frederiksberg C
    Tel.: +45 35 28 31 28
    Fax: +45 35 28 27 42
    E-mail: jans@ruc.dk

  • Helle JUUL-MADSEN
    Danish Institute of Animal Science
    Research Centre Foulum
    P.O. Box 39
    DK-8830 Tjele
    Tel.: +45 89 99 15 60
    Fax: +45 89 99 15 00
    E-mail: jeh@sh.dk
 
 
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