Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are the causative agents of botulism and represents a family of seven structurally similar but antigenically different serotypes (A to G). The BoNTs are expressed in C. botulinum as a single polypeptide chain and then posttranslationally nicked, forming a di-chain polypeptide chain consisting of a 100-kDa heavy chain and a 50-kDa light chain held together by a disulfide bond. Topologically, the neurotoxins are composed of three domains, a binding domain (HC), a translocation domain (HN) and a catalytic domain. The BoNTs act preferentially on cholinergic nerve endings in both humans and animals and thus produce a flaccid paralysis that may result in death. In southern Africa, BoNT types C and D have been associated with botulism in cattle. To combat the disease, a bivalent vaccine consisting of formalin-inactivated type C and D holotoxins is currently available, and although it is efficacious, several concerns regarding its production has been raised, most notably its cost. The development of efficacious recombinant subunit vaccines may provide a means whereby many of the production problems may be eliminated or minimized. Consequently, the aim of this investigation was to produce a recombinant botulinum neurotoxin serotype D binding domain [BoNT/D(HC)] vaccine candidate for preventing BoNT/D intoxication. Towards this end, the gene fragment for the heavy chain (HC) of the BoNT produced by the C. botulinum type D vaccine strain D-50 was amplified, cloned in Escherichia coli and characterized by nucleotide sequence analyses. An alignment of the deduced amino acid sequence with that of characterized clostridial type C and D neurotoxins demonstrated that the heavy chains are composed of highly conserved domains interceded with tracts of amino acids exhibiting little overall relatedness, although considerable identity between the components ofa specific pair is apparent in certain of the regions. The deduced amino acid sequence exhibited 99, 66 and 73% identity with the reported amino acid sequences of BoNT/D-SA, BoNT/D and BoNT/C1, respectively. Attempts at expressing the native gene sequence for the HC from BoNT/D-50 in Brevibacillus brevis 47-5Q were unsuccessful. This may have been due to differences in codon bias between the heterologous gene and B. brevis. Consequently, a completely synthetic codonoptimized gene encoding the HC of BoNT/D-SA was constructed and expressed using a B. brevis 47-5Q mutant as expression host, obtained on mutagenesis with N-methyl-N’-nitro-Nnitrosoguanidine (NTG). Extracellular expression of the 48-kDa recombinant protein was verified by Western blot analyses with anti-BoNT/D antibodies. The recombinant BoNT/DSA(HC) protein was purified from the culture supernatant and used to vaccinate mice, after which their survival against challenge with active toxin was evaluated. Mice given two subcutaneous vaccinations were protected against intraperitoneal administration of 4 X 102 mouse lethal dosages (MLDs) of 16S BoNT/D-50 toxin. Antibody levels in mice surviving challenge were determined by enzyme-linked immunosorbent assays and confirmed that BoNT/D-SA(HC) was successful in evoking a protective immune response, whilst Western blot analyses indicated the presence of anti-16S BoNT/D-50 toxin antibodies in the serum. From these results it could be concluded that the recombinant BoNT/D-SA(HC) protein is an effective immunogen, able to protect against a high challenge dose of BoNT/D-50 neurotoxin. / Dissertation (MSc)--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

Amebiasis, also defined as invasive intestinal and extra intestinal amebiasis, is caused by Entameoba histolytica, an invasive protozoan parasite. World Health Organization (WHO) has reported that approximately 50 million people are infected each year causing an estimated 40 to 100 thousand deaths annually. Entameoba histolytica ranks only second to malaria as a protozoan cause of death. Amebiasis occurs world wide but people living in Central and South America, Africa and Asia are the majority to suffer from morbidity and mortality. The enteric parasite has no zoonotic reservoirs and insect vectors for its transmission and infects humans and non-human primates. Therefore, anti-amebic vaccine could completely eradicate the disease. Entamoeba histolytica invades tissue and causes the disease in series of events. The disease is caused when the cyst form of the parasite is ingested with contaminated food or water. After excysting in the small intestine to form the trophozoite, the parasite adheres to the colonic mucus and epithelial cells through interaction of Gal/GalNAc lectin, an amebic surface adhesin with the host glycoconjugates. The parasite then secrets the proteolytic enzymes that disrupt the intestinal mucus and epithelial barrier facilitating tissue penetration. The trophozoite then kills the host epithelial and immune cells. Also, it resists the host's immune response causing the prolonged infection called the invasive amebiasis and causes colon or liver abscess. The symptoms include gradual onset of abdominal pain, diarrhea and bloody stools. Also, it can form cysts that are excreted with stools to start new cycle. The parasite recognition of the host glycoconjugates plays an important role in the pathogenesis. Therefore, the Gal/GalNAc lectin could be a possible vaccine candidate. The Gal/GalNAc lectin is composed of a 260-kDa heterodimer of disulfide-linked heavy (170 kDa) and light (35 kDa) subunits, which is non-covalently associated with an intermediate sub-unit of 150 kDa. The only recognized Carbohydrate recognition domain (CRD) was found in the heavy sub-unit. The CRD of the lectin is the potential target for colonization blocking vaccines and drugs. Preliminary studies have shown that the recombinant fragments of cysteine-rich region of LecA (lectin) containing the CRD (carbohydrate recognition domain) of the GalNAc lectin conferred protection against amebiasis. Therefore, production of LecA in plants using chloroplast genetic engineering would result in low cost vaccine because of high expression levels of vaccine antigens, and elimination of the cold-chain (low temperature, storage & transportation), hospitals and health professionals for their delivery. The LecA protein was expressed in transgenic chloroplasts of Nicotiana tabacum var. Petit havana by transforming the chloroplast genome using the LecA gene (1755 bp) by homologous recombination. The pLD-CtV has trnI and trnA genes that are used as flanking sequences for homologous recombination and the constitutive 16s rRNA promoter to regulate transcription. The aadA gene conferring spectinomycin resistance has been used for selection and gene10 regulatory sequence from T7 bacteriophage to enhance translation. The chloroplast integration of LecA was confirmed by PCR and Southern blot analysis. The expression of LecA protein in transgenic chloroplasts was analyzed by immunoblot analysis using anti-LecA antibodies. Maximum expression levels of LecA up to 6.3 % of the total soluble protein were observed in the old leaves. The evaluation of the immune response in animal model is underway. This is the first report of expression of LecA in a plant system.