Mycobacterium smegmatis as Vaccine-vectors

Mycobacterium smegmatis is a non-pathogenic and rapidly growing bacterium that express both Mycobacterium tuberculosis antigens and heterologous antigens from other pathogens, and is therefore used as a viable vector for the development of live vaccines against some pathogens such as human immunodeficiency virus (HIV), hepatitis B virus, and Helicobacter pylori.

Mycobacterium smegmatis

Mycobacterium smegmatis (M. smegmatis), a non-pathogenic member of the genus Mycobacterium, was first discovered in 1884 as a rapidly growing saprophytic bacterium that can propagate one generation every 1-3 h. It is non-pathogenic and commensal in the human body, and it acts as a powerful cellular immune adjuvant. Different from other mycobacterial species, such as Mycobacterium tuberculosis (M. tuberculosis), that survive in host cells by inhibiting phagosome maturation, M. smegmatis is rapidly destroyed by phagolysosomal proteases in the phagosomes of infected cells. However, M. smegmatis induces production of cytokines by macrophage better than pathogenic mycobacterial species and can activate and induce maturation of major histocompatibility complex (MHC) class I and costimulatory molecules. M. smegmatis also contributes to rapid uptake of expressed antigens and cross-presentation of antigens.

Why is Mycobacterium smegmatis used as Vaccine-vectors?

This fast-growing M. smegmatis does not arrest phagolysosome maturation and does not escape intracellular killing. In addition, its rapid clearance by the host differs from M. tuberculosis and even the vaccine strain M. bovis bacillus Calmette-Guérin. Moreover, it activates dendritic cells and induces a CD8-mediated immune response. Finally, killed M. smegmatis has been shown to have the same adjuvant activity as M. tuberculosis during the induction of experimental autoimmune encephalomyelitis. Due to these characteristics, M. smegmatis is used as a vaccine carrier.

The Strategies for Mycobacterium smegmatis as Vaccine-vectors in Creative Biolabs

Antigen expression based on plasmid vector

We designed conjugative, thermosensitive shuttle plasmid that can be transferred from E. coli to M. smegmatis. They contain both an E. coli replicon and a thermosensitive replicon derivative of the pAL5000 (the replicon used most often in genetic manipulation of mycobacteria, which permits replication of only five copies per bacterium). Using a temperature shift protocol, the conjugative plasmid is used to deliver the foreign antigen from E. coli into the chromosome of M. smegmatis.

The pAL5000 copy number is limited by the use of the two plasmid-encoded proteins, RepA and RepB that recognize the plasmid origin of replication (oriM). To avoid the negative autoregulation of pAL5000, we overexpress these proteins in trans from M. smegmatis chromosomes. These M. smegmatis strains overexpressing the Rep protein significantly increased plasmid copy number and improved delivery efficiency.

Antigen integration into the genome of Mycobacterium smegmatis

• Specific site recombination has been widely used in mycobacteria, using integrase produced by phage L5. Mycobacteriophage L5, a temperate phage of mycobacteria, integrates site-specifically into the M. smegmatis chromosome. This protein catalyzes the insertion of DNA into the tRNA locus and produces recombinants at high frequency. Site-specific integration of mycobacteriophage L5 shares many features with the site-specific integration systems of other phages. We constructed plasmid vectors that efficiently transform M. smegmatis through stable site-specific integration of the plasmid into the bacterial genome.
• In addition to specific site recombination based on L5, we design a new set of large serine recombinases for site-specific recombination. Integrase of bacteriophage ϕC31 which infected with Streptomyces coelicolor catalyzes recombination between the phage attachment site (attP) and the attachment site on the bacterial chromosome (attB) independent of host factors or other phage-encoded products. Similar to the well-studied integrase-catalyzed reaction, the ϕC31 integrase catalyzes the recombination between non-identical attP and attB sites, resulting in a hybrid site for attR and attL. This specificity indicates that the integrated product produced by the ϕC31 integrase is stably maintained when no additional excision factor is present.

M. smegmatis vaccine strains expressing foreign antigens are an attractive option for mass vaccination programmes against various infectious diseases. With years of experience and advanced vaccine technology platform, Creative Biolabs provides several approaches to improve the ability of mycobacteria to transfer foreign genes to mammalian cells. If you have any needs in this regard, we are your best choice.

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