Cloning, expression and immunogenicity of plant-made mycobacterium tuberculosis antigens ESAT6, Ag85B and Ag85B-ESAT6

Abstract

Tuberculosis (TB), caused by intracellular bacteria Mycobacterium tuberculosis (Mtb), is one of the leading causes of deaths from a single infectious pathogen around the world, second only to human immunodeficiency virus (HIV). The only available vaccine against TB is Bacillus Calmette-Guerin (BCG) which is prepared by long term serial cultivation of attenuated strain of a related microorganism Mycobacterium bovis. With the appearance of extensive and multidrug resistant Mtb, the efficacy of BCG has reduced considerably, especially in adult pulmonary TB. The reducing efficacy of BCG vaccine has led to identification of several alternative antigen candidates for a more effective TB vaccine. Early secretory antigenic target (ESAT6) is a mycobacterial membrane protein which possesses cell lytic activity, thereby promoting bacterial virulence. Antigen 85B (Ag85B) is a fibronectin (a large adhesive glycoprotein) binding protein, present in mycobacterial cell wall and helps in attachment of Mtb to macrophages of the host organism. These antigen are the most potent and immunogenic alternative TB antigens, shown to be protective in animal models and highly immunogenic in humans. These antigens are being developed as subunit vaccines against TB as individual antigens and also as a fusion antigen Ag85B-ESAT6. Subunit vaccines are prepared from an antigen or specific parts of the antigen (epitopes) that are able to stimulate the host immune system. Plant-made vaccines (PMVs) are considered as safer alternative to produce subunit vaccine antigens compared to conventional bacterial, insect and mammalian expression systems in terms of cost, stability, absence of mammalian pathogens and endotoxins. This thesis aims to address the issues of antigen yield and stability by transiently producing these antigens in Nicotiana benthamiana leaves, targeting to endoplasmic reticulum (ER), vacuole and apoplast of the plant cell. It also investigates the immunogenicity of these subcellular targeted plant-made antigens, when administered subcutaneously to mice. In order to construct plant expression vectors, a four-fragment multisite Gateway® cloning technology was used that facilitated the assembly of up to four gene fragments (promoter, signal peptides and antigens) into a gene expression cassette. These plant expression vectors were used in Agrobacterium-mediated transient expression of TB antigens in N. benthamiana leaves. In order to obtain the optimal expression of the antigens, several factors including coinfiltration with silencing suppressor gene P19, acetosyringone concentration, Agrobacterium suspension density, optimum harvest time, mode of infiltration and optimal subcellular location were investigated. Highest accumulation of TB antigens was found in leaves coinfiltrated with P19, using 300 μM acetosyringone and Agrobacterium suspension of OD600 0.25. Leaves harvested 6 days after infiltration (dpi) showed higher accumulation of antigens than other harvest times from 2 to 10 dpi. The antigens were targeted to different subcellular locations under the optimized conditions, with ER and vacuole being the compartments with the highest and lowest accumulation of antigens respectively. ESAT6 antigen showed the highest accumulation in the N. benthamiana leaves (56 mg/Kg of fresh leaf weight) among all the antigens tested. Western blot and deglycosylation assay of all the plant-made TB antigens showed the presence of N-glycosylation which may be important in the stability and immunogenicity of these antigens. In order to investigate the immunogenicity of plant-made ESAT6, Ag85B and Ag85BESAT6, these antigens were subcutaneously administered in BALB/c mice in the form of crude leaf extract. The mice showed an antigen-sp c f c γ-immunoglobulin (IgG antibody) production, indicating that plant-made crude antigens retained their immunogenicity. The ratio of IgG subclasses (IgG1/IgG2a=1) indicates the involvement of both cell and antibodymediated immunity, which may be an important factor in protection against TB infection. To investigate the effect of presence of crude leaf extract on the type of immunity developed, mice were immunized with E. coli. derived purified TB antigens prepared in N. benthamiana crude leaf extract from wild type plant and phosphate buffered saline (PBS) respectively. showed a lower ratio than the mice immunized with antigens in phosphate buffered saline (PBS). These results suggest that crude leaf extract may have some role in shifting the immune response to Th1 from Th2.