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Rational design and delivery of vaccines against infectious diseases in pigs : rapid identification of immunoprotective prv antigens and optimal antigen delivery systems

Deliverables

A system was developed to screen large fragments of the PRV genome. First, two transactivator genes IE180 and EP0 were cloned into eukaryotic expression vectors and a set of four fragments, available as cosmid clones and constituting the whole PRV genome, were tested on integrity. The activity of these transactivator products in stimulation the expression of PRV genes was demonstrated. The sequence of PRV cosmids however was not complete and it was not possible to generate the complete PRV genome out of these fragments. Possibly due to the same reason, the expression of genes from the cosmids was not successful. However, using the substitute large fragments of cloned PRV DNA, the method of screening the genome for immunoprotective antigens was established and optimal ratio of plasmids/transactivator genes for efficient expression were established. Furthermore, baculovirus recombinants expressing IE180 and EPO were constructed and their recombinant antigens for screening the immune response were prepared.
For PRV, early reduction of virus shedding and prevention of virus transmission is strongly related to the strength and the quality of cell-mediated immune responses. In search of novel antigens potentially capable of inducing a protective cell mediated immune response (CMI), plasmids containing sequences coding for PRV proteins which have been identified as important target antigens in other herpes models (e.g. tegument proteins, viral dUTPAse, protein kinase) were constructed. These were plasmids coding for the PRV genes UL13, UL21, UL39, UL47 UL 48, UL 49 and UL50. The constructs were checked for expression in vitro and in mice, in vivo. Preliminary results indicate that UL 21 and Ul 47 induce CMI responses.
For PRV, early reduction of virus shedding and prevention of virus transmission is strongly related to the strength and the quality of cell-mediated immune responses. In addition to the search for immunorelevant antigens, ways to improve the efficacy of DNA vaccines were sought. We therefore evaluated the adjuvant properties of plasmids coding for IFN-g or IL-12, of CpG immunostimulatory motifs and of the conventional adjuvants dimethyldioctadecylammonium bromide-in-water (DDA) and sulfolipo-cyclodextrin in squalene-in-water (SL-CD). We demonstrated that a DNA vaccine combined with DDA but not with the other adjuvants, induced significantly stronger immune responses as compared to plasmid vaccination alone. Moreover, pigs vaccinated in the presence of DDA were protected against clinical disease and shed significantly less PRV after challenge infection.
The Orf virus (ORFV), a member of the genus parapoxvirus of Orthopoxviridae, was found a promising alternative human and animal vector vaccine. 1. For vector construction the highly attenuated, VEGF-E negative ORFV strain D1701-VrV is used, which can be propagated in cell lines. Despite a strong and rapid immune response, the ORFV-specific immune response is short-lived allowing multiple repeated immunisations. Even in non-permissive hosts, like mice or swine, ORFV vector induces a strong humoral and cell-mediated antigen-specific immune response. ORFV recombinants expressing the glycoproteins gC and gD of PRV (Pseudorabiesvirus; Suid herpes 1) were generated and evaluated in mice as a PRV challenge model. 2. Both recombinants conferred solid protection against lethal PRV infection, mediated even by a single application of the D1701-VrVgC alone. 3. Collaborative experiments with partners 1 and 2 of that project demonstrated the immunogenic potential of both ORFV recombinants against PRV infection of pigs. Particularly, their use in prime-boost vaccination in combination with pcDNA or pSIN plasmids encoding PRV glycoproteins improved the protection against Aujeszky's disease in swine. 4. In cooperation with partner 4, PRV gB and IE (immediate early protein) expressing recombinants were constructed. Collectively, the performed work substantiated the excellent potential of ORFV as a novel safe, efficacious and versatile vector vaccine.
Preliminary studies were performed to assess the protective immunity provided for by different prime/ boost combinations (nucleic acid + nucleic acid, nucleic acid + ORFV) in a pig challenge model. The different prime/ boost combinations based were based on gD and compared with the efficacy of modified live vaccine.A prime/boost ORFV –ORFV vaccination resulted in highest antibody responses followed by DNA-ORFV vaccination. DNA –DNA vaccination resulted in lowest antibody responses. Six weeks after second vaccination, pigs were challenged the virulent wild-type strain NIA-3 . There was no significant difference in virus excretion between the different prime-boost regimes and the conventional vaccine. Of the different prime-boost regimes, surprisingly pigs vaccinated with DNA-ORFV appeared to perform better than pigs vaccinated with DNA-DNA or ORFV-ORFV (clinical signs and MRDG7), reaching similar levels of protection as pigs vaccinated with a MLV vaccine. In addition, a preliminary prime – boost regime was assessed using Sindbis based DNA (pSIN)constructs and ORFV constructs based on gC and gD. Priming with pSIN followed with a booster vaccination with ORFV constructs, resulted in a clear booster response.
A sponge-implantation system was established for pigs to analyse ex vivo the local immune response after antigen application. To this end, gelatine sponges containing antigen or buffer as negative controls have been implanted subcutaneously, removed after vascularisation (day 5 post implantation), and subsequently phenotypes of the locally attracted T-lymphocytes were determined in two-colour flow-cytometry focusing on the activation markers CD25 and CD45RC. Comparative analyses were performed with matching PBMC derived from the same individuals.
Studies were performed to improving the efficacy of nucleic acid vaccines i) by using auto-replicative vectors (Sindbis based replicons) for enhanced foreign gene, ii) by using CpG motifs for adjuvation and iii) by redirecting antigen processing through ubiquitination to improve the cell-mediated immune response. Ad i) DNA vaccines constituting of pSIN (Sindbis) constructs encoding PRV glycoproteins gB, gC or gD of Pseudorabies virus (PRV) were constructed. The efficacy of these constructs were tested and compared with the conventional DNA vaccine constructs. A cocktail of DNA vaccines constituting of three pSIN encoding PRV glycoproteins gB, gC or gD was shown to induce a similar specific and protective immunity against PRV infection as compared to the conventional pcDNA3 vector-base vaccine. Furthermore, the dose could be lowered from 340µg to 13µg without loosing out on efficacy. Ad ii) The adjuvant activity of a porcine-specific type A CpG-containing oligodeoxynucleotide (CpG-ODN) was evaluated. CpG-ODN improved the PrV-specific humoral immune response and cell-mediated immune (CMI) response, and improved clinical protection against lethal PrV-infection. Ad iii) Plasmids encoding individually PRV gB, gC and gD fused to ubiquitin (Ubi-PRV-pcDNA3) were constructed and compared to or native glycoproteins constructs (PRV-pcDNA3). Whereas there were no differences in the PRV-specific IgG1 type antibody responses, vaccination with the ubiqituinated constructs resulted in a delay in the IgG2 response. In terms of the CMI response, ubiquitinated constructs resulted in a CMI response, which seemingly weaned off earlier than when using non-ubiquitinated constructs. Ubiquitinated constructs did not result in improved protection against challenge infection.

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