10.4225/03/58b3a1d7bd808 Gottimukkala, Chitti babu Chitti babu Gottimukkala Recombinant production, characterization, and immunogenicity of malaria vaccine candidates expressed in Bacillus subtilis Monash University 2017 Malaria Open access and full embargo Bacillus subtilis Plasmodium falciparum merozoite surface proteins thesis(doctorate) 1959.1/1059920 2014 monash:131124 ethesis-20140930-112023 2017-02-27 03:49:41 Thesis https://bridges.monash.edu/articles/thesis/Recombinant_production_characterization_and_immunogenicity_of_malaria_vaccine_candidates_expressed_in_Bacillus_subtilis/4697089 Malaria is an infectious disease transmitted by mosquitoes. This disease is endemic to tropical countries. Approximately three billion people (living in more than 100 countries) are at risk of malaria with 200 million cases per year and nearly 700,000 deaths-mainly in children under the age of five. The parasite species P. falciparum is the predominant cause of malaria deaths in humans. Mortality and morbidity due to the disease have been showing detrimental effect on economies. Although drugs are available for the treatment of malaria, they are expensive; furthermore resistance to these drugs is widespread. Various control measures such as environmental modifications to prevent mosquito breeding, impregnated bed nets, artemisinin combination therapy have reduced the burden of malaria in the past decade. However, sustained control requires the development of additional tools including a protective vaccine. Various proteins taken from the parasite’s life cycle have been demonstrated to be potential vaccine candidates, and are in various stages of clinical trials. Asexual blood stage of parasite’s life cycle is the reason for morbidity and mortality. Therefore vaccines against this stage are necessary to control the infection. Previous work suggested that a vaccine including multiple antigens from the parasite’s life cycle can be more effective than vaccine formulations containing a single antigen type. In this work, a set of potential vaccine candidates has been chosen to develop an efficacious vaccine. They are Plasmodium falciparum merozoite surface protein 119 (PfMSP119), 4 (PfMSP4), and 5 (PfMSP5). The selected vaccine candidates play an important role in erythrocyte invasion. Specific antibodies against these proteins inhibited parasite growth in in vitro experiments, and passively transferred antibodies showed in vivo growth inhibition. These proteins contain one or more cysteine rich EGF-like domains, which are conserved in all malaria species. Therefore, the functional form— vaccine candidates containing correctly folded EGF-like domains, are essential to induce specific antibodies which can inhibit parasite growth, and thus, malaria. One more candidate Plasmodium yoelii merozoite surface protein 4/5 (PyMSP4/5) has been selected. PyMSP4/5 is a single homologue of two potential human malaria vaccine candidates and it causes malaria in rodents. Therefore, it is a useful target to study the protection efficacy of the vaccine using mouse model. Falciparum malaria is most prevalent in developing countries and this in turn demands the requirement for safe and low-cost vaccines. A majority of available expression hosts: prokaryotic as well as eukaryotic systems failed to produce or yielded very low amounts. We have been seeking alternative expression systems that can overcome some of the bottlenecks and allow subunit production at low cost and large scale. In the present study, we have attempted to use an alternative expression host, Bacillus subtilis, for the production of malaria vaccine candidates. B. subtilis is a non-pathogenic, gram positive organism, which is known for its ability to secrete target proteins and has the advantage of well established methods for scale-up. Vaccine candidates were secreted into the culture medium at higher yields. Characterization studies including SDS-PAGE, mass spectrometry and N-terminal sequencing indicated the secretion of near full-length protein. Recombinant proteins as vaccines are poor in eliciting an immune response and they are required to be presented with an adjuvant, which can boost both humoral and cellular immune responses. Currently, alum is the only approved adjuvant for human, induces a good Ab (Th2) response, but poorly stimulates the strong cellular (Th1) immune response. Bacterial flagellin (FljB) is one of the potential protein adjuvants that has been chosen to boost the malarial vaccine formulation. Purified proteins emulsified with Freund’s adjuvant and mixed/conjugated with flagellin induced strong immune responses in mice following intraperitoneal injection. The mean titer values obtained were higher than those obtained with an equivalent dose of purified E. coli-derived proteins, suggesting that B. subtilis expressed malarial antigens are more immunogenic than those produced by E. coli. The mouse antibodies raised against B. subtilis produced malaria vaccine candidates were reactive to native parasite proteins as evident from immunoblotting on parasite lysate and indirect immunofluorescence assays of fixed parasites. Other advantages of this system over traditional expression methods included a higher level of full-length protein, decreased multimer formation and a simplified downstream processing procedure. All the above features are essential to formulate an effective malaria vaccine or any other high quality clinical material. Therefore, B. subtilis seems to be a valuable advance in the quest to produce an efficacious and cost effective malaria vaccine. Thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy of the Indian Institute of Technology, Bombay, India and Monash University, Australia.