Recombinant baculoviruses will be constructed containing cDNA cassettes of foot-and-mouth disease virus (FMDV) which produce the procapsid proteins. The baculovirus system will be optimized for expression of these FMDV proteins and procapsid assembly. The antigenicity and immunogenicity of these products will be compared with conventional FMDV vaccines.
The general objectives of the project are construction of foot-and-mouth disease virus (FMDV) complementary deoxyribonucleic acid (cDNA) cassettes encoding the structural protein precursor P1-2A and the 3C protease which will produce the procapsid components 1AB, 1C and 1D. Preliminary characterization of the properties of the cassettes to ensure correct protein production and processing should also be undertaken;
insertion of cassettes into baculovirus transfer vectors and production of recombinant baculoviruses;
construction of deletion mutants for analysis of gene toxicity accounting for low level expression from previous constructs. Analysis of transcription and translation levels;
structural analysis of expressed procapsids. Results of the project to date are:
cDNA cassettes for all 3 European serotypes A, O and C of FMDV have been synthesized in the form ATG-P1-2A-3C. Each cassette has been shown to synthesize correctly processed 1AB, 1C and 1D when analysed in a transient expression system;
Each of these cassettes have been introduced into baculovirus transfer vectors and transfected into insect cells to allow the isolation of recombinant viruses;
Other studies on the level of ribonucleic acid on the level of (RNA) transcripts being produced from the polyhedrin promoter in baculovirus FMDV recombinants indicate that the rate of transcription is reduced by the presence of the 3C FMDV sequences. Translation of the P1-2A precursor in the absence of 3C is also low. This suggests that the low level of FMDV protein production seen with existing baculovirus/FMDV genome is due to effect of 3C on mRNA transcription superimposed on a low level efficiency of translation. In order to analyse which section of the FMDV genome adversely affecting the level of translation within baculovirus infected cells specific regions of the cassette are being deleted. The latest cassettes all lack the L protein. Further truncated versions of the L deleted cassette MM51 have been i solated and are being introduced into transfer vectors for analysis. The 3C protease has also been constructed individually which may be useful for independent expression of the structural protein precursor and the protease. Constructs aimed at expressing reduced yields of 3C are also being produced;
Characterization of FMDV procapsid proteins expressed in mammalian cells using vaccinia virus vectors containing the type O and type A cassettes has been performed and indicates that assembly to particles sedimenting at 70S in sucrose gradients occurs. Empty 30nm particles have been observed when extracts have been examined using electron microscopy;
An aspect of work not anticipated has been the determination of the internal potential of hydrogen (pH) of insect cells infected with baculovirus. The importance of this study is due to the lability of FMDV at low pH; new transfer vectors allowing the ready production of baculovirus recombinants containing foreign genes at 2 separate sites.
Recombinant deoxyribonucleic acid (DNA) techniques have been used to construct vectors containing regions of cDNA (termed cassettes) which can express proteins from foot-and-mouth disease virus which self-assemble into virus-like particles but which are completely non-infectious. This has been achieved for each of the three different serotypes of foot-and-mouth disease virus which threaten Europe. It has been established that the intracellular environment of insect cells should not adversely affect the synthesis of these particles. Hence current work on the isolation of recombinant baculoviruses carrying the cassettes mentioned above will proceed, these viruses are capable of expressing foreign proteins at high level, If successful the potential of these non-infectious virus like particles as vaccines will be assessed.
Foot-and-mouth disease virus (FMDV) is a pathogen of world-wide economic importance whose molecular and antigenic structure has been determined in fine detail. Within the BAP project we have constructed and characterized FMDV-baculovirus recombinants. These contain cassettes of type O or A FMDV cDNA encoding the capsid protein precursor linked to the viral proteases required for processing of this protein. Expression, processing and
assembly of the mature capsid proteins has been observed in
recombinant infected insect cells. The object of this proposal is to build on this knowledge to develop improved cassettes
enabling higher level expression, to include all three European serotypes of FMDV (A,O,C) and to study the structure and the immunogenicity of the expressed products. In the later phases of the study we will use protein engineering to generate capsid structures with improved characteristics, eg with respect to stability or mixed serotype specificity. These studies will
advance our aim of an effective, safe and stable vaccine against FMDV. They will increase our knowledge of the morphogenesis of FMDV capsids which should be applicable to other picornaviruses. These viruses are responsible for many animal and human
diseases. Furthermore the work will aid our understanding of the mechanisms controlling the production of foreign proteins by baculovirus Which are of considerable interest in biotechnology.