Listeria Monocytogenes as Vaccine-vectors

Listeria Monocytogenes is a facultative intracellular bacterium that enters various non-professional mammalian cells by triggered phagocytosis and replicates in the cytosol of infected host cells. It is effective in presenting passenger antigens to MHC class II, especially class I pathways, and is therefore considered a promising vaccine vector.

Listeria Monocytogenes as Vaccine-vectors

Listeria Monocytogenes as Vaccine-vectors– Creative Biolabs

Listeria monocytogenes (L. monocytogenes) is a Gram-positive bacterium rod that is ubiquitous in nature and is an opportunistic pathogen for humans and various other vertebrates. The bacterium is an intracellular pathogen that expresses hemolysin (LLO), enabling it to invade and replicate in the cytosol of eukaryotic cells. Similar to proteins produced by viral infection, proteins produced by the cytosolic L. monocytogenes are processed and presented to the vertebrate immune system by the endogenous major histocompatibility complex (MHC) class I antigen presentation pathway. The phagocytosed foreign proteins can also be presented directly to MHC class II molecules. This bacterium can induce both CD4 and CD8 antigen-specific immune response, and the genomes of various L. monocytogenes strains have been sequenced and many well-defined virulence-attenuated mutants have been constructed. Owing to these properties, recombinant L. monocytogenes has been shown to be a useful vector for the delivery of antigens in a vaccine system.

The Features of Listeria Monocytogenes as Vaccine-vectors

  • With multiple routes of administration
  • Can deliver intracellular DNA, RNA or protein to cancer cells both in vitro and in vivo
  • The ability to infect and survive in antigen presenting cells
  • The potential to be used as a vector for expression of tumor-specific antigens

Our Design for Listeria Monocytogenes as Vaccine-vectors 


  • The highly attenuated strain of Listeria, L. Monocytogenes ΔdalΔdat strain (Lmdd), lacks alanine racemase (dal) and D-amino acid aminotransferase (dat), which are responsible for the synthesis of D-alanine, an important component of the bacterial cell wall. Therefore, Lmdd is heavily dependent on the exogenous supply of this amino acid. This stain is able to secrete the CD24 protein after parenteral administration and elicit specific immune responses which reduced tumor size.
  • The mutant strains L. Monocytogenes ΔactAplcB deleted actA (responsible for actin polymerization and resultant movement within eukaryotic cells and intercellular spread) and plcB gene (encoding a phospholipase or lecithinase). This attenuated ΔactAplcB mutant strain can be applied in high enough doses to elicit humoral, mucosal, and cellular immune responses without serious long-term health sequelae.

Antigen Expression Based on Plasmid Vector

Plasmid DNA must be equipped with two origins of replication, one for plasmid construction and propagation in E. coli, the other for vaccine vector L. monocytogenes. To ensure the stable maintenance of the plasmid in L. monocytogenes, we design a balanced-lethal system by deletion of the essential tryptophanyl-tRNA synthetase (trpS) gene in the genome of L. monocytogenes and in-trans expression of trpS from the DNA-delivery plasmid. This plasmid contains trpS, an erythromycin resistance marker and the eukaryotic expression cassette, and shows stable replication in L. monocytogenes strains. Loss of the vaccine plasmid causes the death of the carrier strain because deletion of trpS results in the strain is unviable. Therefore, even in the absence of antibiotic selection, only those bacteria can survive which carry the DNA delivery plasmid.

Antigen Integration into The Genome of L. monocytogenes

The vector, pPL2, has previously been used to stably integrate multiple genes into specific sites in the chromosome of L. monocytogenes. Based on this foundation, Creative Biolabs developed a novel integration vector, pPL2dalGlnA, which completely lacks drug resistance genes and allows chromosome insertion in a single step. The selectable markers in pPL2dalGlnA are glutamine synthetase (GlnA) and alanine racemase (Dal). This novel vector is maintained in an auxotrophic L. monocytogenes strain that normally requires D-alanine. The pPL2dalGlnA vector also partially restored the ability of the L. monocytogenes ΔdalΔdat strain to colonize the spleen and liver of infected mice.

Delivery of Secreted and Surface-Located Proteins in L. monocytogenes

Localization of heterologous antigens on the surface of bacterial cells implies important biotechnological applications, such as the concentration of antigens or specific enzymes at high density or expression of protein ligands recognizing specific host cell receptors. Thus, a system for anchoring heterologous antigens in the cell wall of L. monocytogenes is designed in Creative Biolabs based on the 3’ terminal cell wall anchoring sequences (LPXTG) of listerial internalin A (InlA). InlA and other members of the internalin family are covalently linked to the cell wall of L. monocytogenes by aspartase, and the C-terminus of InlA is exposed to the outside of the listerial cells.

With years of experience and advanced vaccine technology platform, Creative Biolabs has developed engineered and attenuated L. monocytogenes vectors that deliver foreign antigen to host cells. If you have any need for L. monocytogenes as a delivery vehicle, we are your best choice.

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