Novel expression and production of Foot-and-mouth disease virus vaccine candidates in Nicotiana benthamiana

Foot-and-mouth disease, also known as FMD, is caused by the aphthovirus Foot-and-mouth disease virus (FMDV), which is a highly contagious disease of cloven-hoofed animals. It is endemic in Africa, parts of South America and southern Asia. In South Africa, the disease is controlled essentially through prophylactic vaccination. Current vaccines on the market are chemically inactivated virus strains. However, these are not considered ideal due to the possibly insufficient inactivation which could fail to render the virus harmless. Research on recombinant vaccines which obviate the need for high biosafety requirements for vaccine preparation has shown that recombinant FMDV virus-like particles (VLPs), devoid of viral genetic material, are an ideal vaccine candidate as they are as immunogenic as the virions themselves when administered to animals. These VLPs are formed by the assembly of the FMDV capsid proteins VP0, VP1 and VP3, which are generated upon the proteolytic cleavage of the capsid precursor protein P1-2A, by the FMDV 3C-protease. The expression platforms used to co-express and produce the component capsid proteins and the protease are usually mammalian, insect or E. coli cells. The use of these expression systems requires extensive bioreactor infrastructure and sterile conditions for vaccine preparation which are costly. In addition, some studies have shown how the co-expression of the 3C-protease can prove to be deleterious when expressed at a high concentration in expression systems. In order to circumvent this, and encourage more efficient production of the capsid proteins and subsequent VLP assembly, researchers have shown that the levels of the 3C-protease can be down-regulated by introducing mutations in the 3C gene or a ribosomal frameshift in the gene sequence which subsequently reduce its deleterious effect. Our laboratory has previously shown that similar FMDV VLPs can be assembled by the expression of FMDV P1-2A (referred to as oP1-2A, in this study), in the absence of the 3C-protease in the plant Nicotiana benthamiana, albeit in low amounts. This platform does not require high biocontainment facilities for the production of recombinant proteins and VLPs and the process is easily scalable. This study centers mainly on optimisation of the FMDV capsid protein expression in N. benthamiana in order to increase VLP yields. I first used codon-optimisation as an approach to improve expression of the capsid proteins and compared expression in the presence and absence of the 3C-protease, using mP1-2A-3C (a new codon-optimised construct), mP1-2A and oP1-2A. Electron microscopy (EM) showed that VLPs resulting from the expression of both mP1-2A-3C and mP1-2A were very low in yield, and irregular in shape and size compared to those produced using oP1-2A. The stability of the plant-produced VLPs was assessed by counting numbers of VLPs, when it was seen that expression of mP1-2A-3C compared to oP1-2A produced an average of 1 VLP per field of view versus 3 VLPs per view, for the same magnification. Furthermore, maturation trials at room temperature was performed on the oP1-2A VLPs, whereby a time-point between 30 to 45 minutes was considered ideal to produce stable VLPs. It is also known that FMDV, unlike the other members of the Picornaviridae family, is acid and heat-labile. The second aim of this study was to promote the stability, and hence encourage the amount of the VLPs produced, by engineering acid and heat-resistant mutants, namely, VP1 N17D, VP2 H93C and VP1 N17D/VP4 S73N using site-directed mutagenesis. A fourth mutant, VP3 A118V which is acid sensitive was used as a control in downstream experiments. The mutants were subjected to a lower than normal pH and a higher than normal temperature. Expression of oP1-2A from the pH and heat assays was assessed to be better than its mutants. The optimum VLP count of 3 VLPs per field of view, was achieved from expression of oP1-2A, after treatment at pH 6.2, compared to 2 VLPs or 1 VLP per field of view for the other mutants tested under all the different conditions. The final aim of this study was to test the immunogenicity of the VLPs from expression of oP1-2A in Balb/C mice. Due to the low yields of VLPs obtained from purification through a continuous gradient, a partial purification method was adopted. Two experimental groups of animals were either vaccinated with P1-2A VLPs or with adjuvanted P1-2A VLPs. A control group was administered with partially-purified plant extract, previously infiltrated with pEAQ-HT. The two experimental groups elicited a marginal increase in humoral immune response at 41 days post vaccination (dpv), which increased significantly at 58 dpv. To my knowledge, this is the first study showing that VLPs produced from expression of FMDV P1-2A only, in tobacco plants, can withstand otherwise degradative acidic and heat conditions. This characteristic has potential for extending the shelf-life of such a candidate vaccine. I also implemented maturation steps to further promote the stability of such VLPs. Finally, the partially purified VLPs showed that they stimulate a significant FMDV P1-2A-specific immune response, particularly in combination with the adjuvant Montanide suggesting that it has potential as a candidate FMDV vaccine. ABBREVIATIONS A Alanine Ad5 Adenovirus serotype 5 AEC Animal Research Ethics Committee ANOVA One-way analysis of variance BCIP 4-chloro-3-indoxyl-phosphate bp base pairs BSA Bovine Serum Albumin BSL 2 Biosafety Level 2 C Cysteine CPMV Cowpea mosaic virus cre cis-acting replicative element CysPs cysteine proteinases D Aspartic acid DIVA Differentiating Infected from Vaccinated Animals DNA deoxyribonucleic acid dpi days post infiltration dpv days post vaccination EDTA Ethylenediaminetetraacetic acid ELISA enzyme-linked immunosorbent assay EM Electron Microscopy FMD Foot-and-mouth disease FMDV Foot-and-mouth disease virus g grams GFP green-fluorescent protein H Histidine h hour HBcAg hepatitis B virus core antigen gene His histidines I-ELISA Indirect ELISA HS heparan sulfate iFMDV inactivated virus IPTG isopropyl b-D-1-thiagalactopyranoside IRES internal ribosome entry site kb kilobase kDa kilo-Dalton KNP Kruger National Park LB Luria broth M Molar MES 2-morpholinoethanesulfonic acid mg miligram min minutes ml mililitres MM Molecular Marker N asparagine NBT nitroblue tetrazolium NFDM non-fat dairy milk nm nanometres NS non-structural NTE Sodium chloride, Tris, EDTA buffer OD Optical Density OIE Office International des Epizooties ORF open reading frame PBS phosphate-buffered saline PCR Polymerase Chain Reaction PKs pseudoknots pNPP p-Nitrophenyl phosphate PTGS posttranscriptional gene silencing RF replicative form RGD Arg-Gly-Asp RI replicative intermediate RNA Ribonucleic acid RT-PCR RNA using reversetranscription PCR S Serine S Svedberg units SAT South African Territories SAVP South African Veterinary Products sec seconds siRNA small interfering RNAs TBS Tris-Buffered Saline TEM Transmission Electron Microscopy TSP Total Soluble Protein TST TBS-Tween UTR Untranslated region UV ultraviolet V Valine V Volts VLPs virus-like particles VPg virus protein, genome linked VPs Viral Proteins WT wild-type μg micrograms μl Microliter TABLE OF CONTENTS CHAPTER 1: LITERATURE REVIEW (Foot-and-mouth disease vaccines: The past and the present) . 2 1.1. GENERAL INTRODUCTION . 2 1.2. FMDV SEROTYPES AND GEOGRAPHICAL DISTRIBUTION . 4 1.3. CLASSIFICATION AND STRUCTURE OF FMDV . 5 1.4. INFECTIOUS CYCLE OF THE VIRUS . 10 1.5. VACCINES AGAINST FMDV . 13 1.6. AIMS OF THIS STUDY . 23 CHAPTER 2: Transient expression of different codon-optimised FMDV constructs in planta and expression of FMDV capsid precursor in E. coli . 26 2.