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WORKPACKAGES
4.4. Work package descriptions
Workpackage 6 : Development of a new marker vaccine
PARTICIPANTS
Partners 2 and 10
Person-months per participant: 5 and 96
Start date or starting event: Month 1
OBJECTIVES
- To evaluate the protective potential of pre-constructed FMD marker vaccines using chemically inactivated FMD chimera viruses containing replacement FMDV or another foreign sequence in the immuno-dominant and antigenically important linear site of the structural protein VP1 (G-H loop region).
- To develop an assay method for evaluating the potential of positively and negatively discriminating the polyclonal antibody responses of such chimera marker vaccines with that elicited by the natural capsid structure (see WP2).
- To compare the antigenic spectrum of these chimera marker vaccines with that of the natural FMDV capsid structure normally used in a conventional vaccine.
- To establish the potency of these chimera marker vaccines and compare with that of the natural FMDV capsid structure normally used in a conventional vaccine.
- To specifically target DC which would be involved in promoting systemic plus mucosal immune responses.
- To determine the efficiency of the vaccines under study to induce mucosal immunity.
Description of work:
- 6.1 Development and evaluation of FMD marker vaccines.
- 6.1.1 To evaluate the protective potential of pre-constructed FMD marker vaccines - Two chimera constructs, based on the A12 FMDV serotype in which only the G-H loop region of VP1 has been exchanged with sequences representing either an O1 or a C3 serotype of different serotypes (A/O and A/C) (Rieder et al., 1994) and an additional chimera which has the VP1 G-H loop residues 142-157 replaced with sequences surrounding the RGD sequence of bovine vitronectin (Baxt et al., 1998) will be administered to groups of 5 cattle as a chemically inactivated and conventionally formulated vaccine and challenged 21 days later by homologous needle tongue challenge using unsolicited A12 FMDV. The vaccine will be formulated to a payload representative of an emergency FMD vaccine i.e high payload, and at the same amount regardless of construct used. A further group of 5 cattle will be vaccinated with a similar payload of the natural FMDV capsid structure to represent the conventional 'emergency' vaccine alongside a control group of unvaccinated animals to determine that virus challenge was accomplished. Protection will be determined by the lack of clinical signs and samples will be routinely taken for serological analyses to support the other 3 objectives.
- 6.1.2 To compare the antigenic spectrum of these chimera marker vaccines - Ideally vaccine strains are chosen for their broad antigenic coverage over field isolates within each serotype. Using a two-dimensional microneutralisation test a comparison will be made between the antigenic spectrum of the chimera constructs and the natural FMDV capsid structure to see what effect, if any, the exchange of G-H loop sequences has on its ability to cover the same candidate field isolates. The field isolates will be drawn from an extensive panel held within the World Reference Laboratory and will be chosen to represent extremes in relationship.
- 6.1.3 To establish the potency of these chimera marker vaccines - Using the well-established guinea pig model the same vaccines used in the cattle study will also be evaluated to determine a potency value (PD50). The vaccine will be diluted from neat to 1/81, administered into groups of 5 guinea pigs and subjected to virus challenge 28 days post vaccination. Animals will be examined for clinical signs and scored for protection. Along with quantitative analysis of the antibody response in the different cattle groups from objective 1, it will be determined whether exchange of the G-H loop region has any bearing on the potency of the vaccine.
- 6.1.4 To develop an assay method for evaluating the potential of positively and negatively discriminating the polyclonal antibody responses - See WP2.
- 6.2 Mucosal immune responses and dendritic cell targeting of the vaccines.
- 6.2.1 In vitro screening - This work will be initiated using the conventional FMDV vaccine, to form the basis against which subsequent analyses employing the new vaccines being developed under WP6.1 will be compared. The marker chimeric vaccines to be evaluated will be dependent on the results deriving form WP 6.1. By performing these experiments initially in vitro, the number of animal experiments can be reduced to a minimum. In vitro tests for vaccine efficacy should be forthcoming, to help reduce animals experimentation. Potential FMDV vaccine candidates will be "fed" to dendritic cells (DC), and their capacity to stimulate FMDV-specific immune responses monitored in terms of antigen processing, T cell proliferation, cytokine (IFNg and/or IL-4) and antibody production. The influence of the vaccine on monocytic cell chemotaxis, phagocytosis, and endosomal activity will also be monitored, due to their role in innate immune defences. It is possible to induce mucosal immunity by parental immunisation. Known modulators of mucosal immunity, including biosafe derivatives of cholera toxin or heat-labile enterotoxin, as well as CpG-ODN and mucosal-associated cytokines and chemokines, will be employed to enhance DC activity in vitro. DC targeting will focus on the role of MIP-3a interaction with CCR6+ DC (would traffic to the mucosal associated lymphoid tissues).
- 6.2.2 In vivo studies - The capacity of inactivated conventional or marker vaccines, plus the appropriate immunomodulators, identified through the screening under 6.2.1 as having the greatest potential for targeting DC towards promoting mucosal immune responses, will be tested in vivo in swine. Quantification of FMDV-specific antibodies in the serum and mucosal secretions at different time points post-vaccination will use neutralisation of infectivity and ELISA, and feed into the NSP-tests (WP2). Vaccinates developing specific immune responses, both systemic and mucosal, will be challenged by contact infection at 21 days post-vaccination. When mucosal immunity and protection are induced, this aspect of the work package will feed into WP4. Detection of virus secretion in saliva will be effected through WP3.
Deliverables:
- D.9 - Characteristics of dendritic cell targeting by conventional FMDV vaccine serotype O (18 months).
- D.19 - Characterised potential of marker vaccines to target dendritic cells (36 months).
- D.20 - Dendritic cell characteristics (chemokine expression, migration, immunostimulatory properties) associated with the FMDV vaccines targeting these cells (36 months).
- D.21 - Proposal of vaccine candidates with potential to promote mucosal immunity (36 months).
- D.38 - Samples for all assay validation and development during this project (48 months).
- D.43 - A chimera marker FMDV vaccine based upon A12 serotype but exchanged with a G-H loop sequence representing O1 capable of conferring protection in cattle against the A12 serotype virus strain (48 months).
- D.44 - A chimera marker FMDV vaccine based upon A12 serotype but exchanged with a G-H loop sequence representing C3 capable of conferring protection in cattle against the A12 serotype virus strain (48 months).
- D.45 - A chimera marker FMDV vaccine based upon A12 serotype but exchanged with a 'foreign' RGD sequence of bovine vitronectin capable of conferring protection in cattle against the A12 serotype virus strain (48 months).
- D.46 - The antigenic spectrum of these chimera marker vaccines compared to that representing the normal conventional vaccine (48 months).
- D.47 - Potency (PD50) value of the different chimera marker vaccines compared to the normal conventional vaccine (48 months).
- D.48 - Potency by serology of the different chimera marker vaccines and normal conventional vaccine administered in cattle (48 months).
- D.49 - Potency of dendritic cell targeting of vaccine at promoting mucosal immunity and protection against FMDV (48 months).
- D.50 - Reports: Scientific publications (48 months).
Milestones and expected result:
- M.36 - Assessed protection against the A12 serotype in the main target species (cattle) using chimera marker FMDV vaccines exchanged with sequences representing either an O1 or a C3 serotype of different serotypes.
- M.37 - Assessed protection against the A12 serotype in the main target species using chimera marker FMDV vaccines exchanged with a 'foreign' RGD sequence of bovine vitronectin.
- M.38 - Determined antigenic spectrum of these chimera marker vaccines and that representing the normal conventional vaccine.
- M.39 - Determined potency (PD50) value of the different chimera marker vaccines and normal conventional vaccine in guinea pigs.
- M.40 - Compared potency by serology of the different chimera marker vaccines and normal conventional vaccine in cattle.
- M.41 - Identified chemokines and adjuvant systems in vitro that modify DC migration and immunostimulatory properties permitting immunological homing to mucosal tissue and mucosal anti-FMDV immune responses (month 24).
- M.42 - In vitro efficacy of the vaccines under test (month 36).
- M.43 - Determined vaccine potential to induce mucosal immunity (month 36).
- M.44 - Assessed immune responses elicited in vivo by FMDV vaccines formulated for targeting DC and mucosal immunity (month 48).
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