To deliver foreign antigens or epitopes as vaccines to mucosal surfaces, both living and nonliving carriers can be attractive vehicles. Basically in this project two problems have to be solved: (a) construction of stable live recombinant vectors which present foreign epitopes; (b) proper folding and optimal exposure of foreign epitopes on carrier proteins with favourable immunogenic properties.
Two immungens are under study as potential epitope carriers: maltose binding protein (MalE) and hepatitis B core protein (HepB). Epitopes from transmissible gastroenteritis coronavirus (neutralization site D of spike protein of TGEV) or canine parvovirus (CPV) are being cloned into these proteins. It is hoped to deliver these immunogens directly to the mucosal surface as non-living vaccines and as carried antigens in live attenuated strains of Salmonella. To boost the mucosal immunogenicity of purified MalE, cell binding motifs from filamentous haemagglutinin (FHA) are being investigated which may target the MalE to antigen presenting cells in the respiratory tract.
As alternative live vehicle, two attenuated strains of TGEV have been created which show tissue tropism to mucosal surfaces: strain PTV to gastrointestinal tract, strain PUR46 to respiratory tract. Together with infection-defective TGEV minigenomes to be created it is thought that vaccine antigens can be selectively targeted to either intestines or lung.
Anti-TGE viral activity induced by epitope site D inserted in MalE. Site D of TGEV was inserted either as monomeric or dimeric epitope at two different sites (site 133 and 303) of MalE. Initial products were generated in E. coli and yielded anti-site D and antiviral acitivity upon intraperitoneal (ip) injection in mice, the best and most reproducible being the hybrid with site D in dimeric form at position 303 (named MalE303[TGEV]2 hereafter). The introduction of the epitope in MalE yielded indications for a changed overall MalE structure: the hybrids elicited antibodies to a broad set of highly antigenic linear sites, while controls - wild type MalE or the MalE with dedicated cloning site - did not. Yet purification of the hybrid products is still straightforward. Addition of an oil like adjuvant also broadens the response of native MalE. The significance of this finding in mice ie. inverse relation between structural integrity and immunogenicity, was confirmed in other species like guinea pigs, but not evident in rabbits.
Transfer of MalE303[TGEV]2 into S. typhimurium SL3261 using a range of different candidate plasmids initially yielded recombinant Salmonella strains which were poorly able to induce anti-site D responses upon ip. injection. At this stage, by manipulating with promotors, signal sequences and transcription terminators, expression with recombinant Salmonella strains has improved. Moreover, strains with cytoplasmic expression after i.p. injection as well as upon oral administration induce anti-site D antibody titers. In order to assess the quality of the strains for in vivo application, both the recovery from spleens and the immune responses are currently under further investigation. In addition, when in vitro intracellularly present in macrophages, expression of MalE303[TGEV]2 protein by Salmonella strains appeared consistent with proper folding and expression at the periplasmic level. Thus, major hurdles in obtaining favourable expression of the desired hybrid immunogens by Salmonella have now been overcome.
Chimeric MalE-FHA products carrying inserted site D. A chimeric protein resulting from a fusion between wt MalE and the +/- 800 codons long BamH1 fragment of FHA appeared a good inducer of systemic and local immune responses in mice to both MalE and FHA notwithstanding the route of immunization (intranasally or subcutaneously). Responses to linear epitopes were respectively very limited upon i.n. and considerable upon s.c. administration. The expectation is that the FHA fragment contributes to the local immune response due to its cell binding motifs. To have stronger in vitro attachment by FHA, effort has been invested to make the FHA portion of the candidate MalE-FHA carrier longer with defined cellular attachment domains such that ultimately an even better cell adhesion function would be acquired in the fusion product. This was combined with the addition of the above defined MalE303[TGEV]2 hybrid. Unfortunately, these constructions of stable clones appeared to be unsuccessfull after several rounds of trials. Then, a fusion of the MalE303[TGEV]2 hybrid with the BamH1 fragment of FHA appeared productive but the protein appeared very insoluble and impossible to be purified.
Construction of infection defective TGEV mutants with tissue tropism. In the preparation of an avirulent viral vector with tissue tropism, an infection defective TGEV RNA species of +/-9.7 kb (DI-C) was selected for transfection of helper virus-infected cells. It further appeared possible to rescue this as a recombinant RNA minigenome using as helper virus avirulent TGEV strains PTV or PUR46 (which have respective tropism for gastrointestinal and respiratory tract). These constructions could further be reduced to 3.3 kb by introducing internal deletions. This minigenome can still be encapsidated in the presence of helper virus. In this way the basis has further been improved for a family of safe vectors specific for swine. The TGEV DI rescue system was shown capable to express heterologous proteins using the luciferase reporter protein. Also, respiratory strain helper virus together with recombinant minigenome containing the enteric spike gene resulted in chimeric viruses expressing the spike protein from both enteric and respiratory isolates; a change in tropism of the respiratory isolate became obvious. Therefore, a new family of eukariotic vectors has been obtained based on chimeric viruses which allow tissue specific expression of an antigen of choice in the enteric or respiratory tract.
HepB core fusions with foreign epitopes. While previoulsy the insertion and expression of site D of TGEV in HepB core protein was partially succesfull, now the well established CPV epitope (N-terminus of capsid protein VP2) has been succesfully introduced in the Nhe1 site of HepB core protein, resulting into the new plasmid pGAR1 containing this CPV epitope. The system appeared capable for preparing recombinant core particles for immunization experiments in mice, rabbits and mink, the latter being the target animals for mink enteritis virus, a host range variant of CPV.
MAJOR SCIENTIFIC BREAKTHROUGHS:
MalE is a favourable carrier for a foreign epitope with respect to immunogenicity properties for the inserted viral epitope and to induce virus neutralization acitivity; codon `303' of MalE is the preferred site for insertion and the epitope as a dimer appears to be advantageous for immunogenicity. A Salmonella strain has been created which induces the required site directed antibody response upon oral administration. A new family of eukariotic vectors has been obtained based on TGEV which allow tissue specific expression of an antigen of choice in the enteric or respiratory tract, to induce mucosal immunity.
Funding SchemeCSC - Cost-sharing contracts
SW7 2AY London