Abstract
The development of alternative subunit based-vaccines against tuberculosis is necessary due to variable efficiency and some security concerns of the BCG vaccine. The aim of this work was evaluate the production of the Mycobacterium tuberculosis Ag85B antigen fused to Potato Virus X Coat Protein (PVX-CP) by transient expression in Nicotiana benthamiana for subunit-based tuberculosis vaccine formulation. A codon-optimized M. tuberculosis Ag85B gene was fused to PVX-CP and expressed both as a full length precursor and as a mature version lacking the leader peptide. Signal peptides of N. tabacum genes were added to precursor and mature Ag85B-CP to compare the efficiency of cytoplasmic and apoplastic expression. Constructs were agroinfiltrated into N. benthamiana leaves and the yield and integrity of recombinant proteins were analysed. Glycosylation status was determined by treatment with peptide N-glycosidase F. The highest amounts of fusion protein were obtained by expressing mature Ag85B lacking its leader sequence directed to the apoplast, which reached a yield of 100 mg of antigen per kg of fresh leaf. Glycosylated and non-glycosylated fusion proteins were obtained in the apoplastic and cytoplasmic space, respectively. We showed the feasibility of producing Ag85B-CP protein in N. benthamiana leaves for application as a subunit vaccine and demonstrated the importance of expressing mature Ag85B to increase yield and to avoid the production of degraded protein fragments unsuitable for a pharmaceutical product.
Highlights
Tuberculosis (TB) is a re-emerging disease that represents one of the leading causes of death from a single infectious agent
We studied the expression of both precursor (Ag85B) and mature antigen 85B (Ag85B) (Ag85Bm) sequences in N. benthamiana
To maximize codon usage in a plant expression system, the DNA sequence encoding precursor Ag85B was subjected to codon optimization aided by the OPTIMIZER software (Puigbò et al, 2007), resulting in 78% of codons being altered while maintaining the amino acid sequence
Summary
Tuberculosis (TB) is a re-emerging disease that represents one of the leading causes of death from a single infectious agent. It is a disease of poverty, affecting mostly young adults in their most productive years with the vast majority of TB cases occurring in developing countries. Interest in the development of alternative TB vaccines has increased in past years, including protein subunit-based vaccines attempting to induce immunity by one or a combination of antigens. Protein subunit-based vaccines are an interesting strategy for TB because, in contrast to BCG, they are not compromised by a previous exposure to mycobacteria and can be retained as a booster to BCG priming to prolong immunity in adults (Andersen and Woodworth, 2014). Efficient protection against bovine tuberculosis has been shown in the context of DNA vaccines (Teixeira et al, 2006) and as a subunit vaccine in a murine and guinea pig model
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