Results

The first 18 months of scientific activities have largely focussed on the definition and design of vaccine and diagnostic assay on the basis of (i) the type of immune response conferring protection against the pathogen, (ii) the knowledge of pathogen components (antigens) against which protective and non-protective immune responses are directed and (iii) the structural features of these components:

• First steps have been accomplished towards the identification of PRRSV antigens targeted by virus neutralising immune responses. Pigs were identified with antibodies capable of broadly neutralising PRRSV. A panel of recombinant PRRSV glycoproteins has been constructed to isolate broadly neutralising monoclonal antibodies. To help define virus structures targeted by these antibodies, a high-resolution model of the PRRSV virion by cryo-electron microscopy is being constructed. Recombinant pseudorabies virus vectors expressing PRRSV glycoprotein sequences have been constructed and characterised in vitro.
• Live attenuated vaccine strains of B. suis have been designed and produced by the targeted mutation of genes and antigens have been identified for development of diagnostic assays.
• A protocol has been optimised for the preparation of an antigen formulation based on C. abortus outer membrane protein complex for exploitation by one of the industrial partners.
• Diversified bioinformatics approaches have been adopted to identify candidate antigens for the development of C. abortus and C. burnetii DIVA subunit vaccines and companion DIVA diagnostic assays. Some of the identified antigens have been produced in recombinant form by “traditional” expression systems (i.e. Escherichia coli) or in association with “immunopotentiating” moieties using an “unconventional” expression system (i.e. Nicotiana benthamiana plants) and the scale up is underway to allow in vitro/in vivo testing.
• Polyclonal antibodies have been generated to candidate C. burnetii antigens and an in vitro infection assay developed to test their neutralising capacity.
• A validated workflow for antigen testing has been developed using a candidate C. burnetii antigen as test compound.

Between month 19 and month 36, activities have focused on the: i) production of selected antigens; ii) isolation and production of neutralising antibodies for the refinement of antigenic targets; iii) experiments to assess the antigenicity and immunogenicity of selected antigens and vaccine candidates; and iv) further development and evaluation of diagnostic assays.

• Single B cells have been sorted from pigs, whose sera broadly neutralise PRRSV and recombinant monoclonal antibodies generated and expressed to be characterised for specificity and neutralising capacity. Experiments are ongoing to provide a high-resolution model of the virion by cryo-electron tomography, and a new associated partner involved to support structure-guided vaccine design. Recombinant pseudorabies virus vectoring PRRSV glycoprotein or epitopes have been tested in pigs for immunogenicity, as a pilot proof-of-principle study.
• Live attenuated B. suis vaccine candidates were further characterised in THP-1 macrophages and evaluated in a mouse model for cross-protection against the three biovars causing porcine brucellosis. Selected antigens for delayed-type hypersensitivity (DTH) skin test and serological diagnosis were produced and characterised. Preliminary in vivo DTH and serological tests were performed.
• Rationally selected C. burnetii antigens have been produced and used to immunise sheep and polyclonal antibodies generated against these vaccine candidates tested for their ability to neutralize the bacteria through a novel assay developed within REPRODIVAC. In addition, candidate diagnostic antigens have been evaluated for serodiagnostic potential using an indirect ELISA platform and a panel of serum samples from ruminant livestock with known C. burnetii infection and vaccination status.
• Recombinant C. abortus antigens have been produced and their immunoreactivity evaluated using convalescent serum and peripheral blood cells from immune sheep. Selected antigens have been screened in the surrogate C. abortus mouse models. The protocol to produce COMC has been provided to one of the industry partners to support the commercialisation of this new vaccine. A molecular point-of-care loop-mediated isothermal assay (LAMP) has been developed and optimised for the detection of C. abortus DNA compatible with a direct extraction method and is currently undergoing validation using samples from experimental challenge studies.