Abstract
BackgroundDelivery of antigens by live bacterial carriers can elicit effective humoral and cellular responses and may be an attractive strategy for live bacterial vaccine production through introduction of a vector that expresses an exogenous protective antigen. To overcome the instability and metabolic burden associated with plasmid introduction, alternative strategies, such as the use of in vivo-inducible promoters, have been proposed. However, screening an ideal in vivo-activated promoter with high efficiency and low leak expression in a particular strain poses great challenges to many researchers.ResultsIn this work, we constructed an in vivo antigen-expressing vector suitable for Edwardsiella tarda, an enteric Gram-negative invasive intracellular pathogen of both animals and humans. By combining quorum sensing genes from Vibrio fischeri with iron uptake regulons, a synthetic binary regulation system (ironQS) for E. tarda was designed. In vitro expression assay demonstrated that the ironQS system is only initiated in the absence of Fe2+ in the medium when the cell density reaches its threshold. The ironQS system was further confirmed in vivo to present an in vivo-triggered and cell density-dependent expression pattern in larvae and adult zebrafish. A recombinant E. tarda vector vaccine candidate WED(ironQS-G) was established by introducing gapA34, which encodes the protective antigen glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the fish pathogen Aeromonas hydrophila LSA34 into ironQS system, and the immune protection afforded by this vaccine was assessed in turbot (Scophtalmus maximus). Most of the vaccinated fish survived under the challenge with A. hydrophila LSA34 (RPS = 67.0%) or E. tarda EIB202 (RPS = 72.3%).ConclusionsQuorum sensing system has been extensively used in various gene structures in synthetic biology as a well-functioning and population-dependent gene circuit. In this work, the in vivo expression system, ironQS, maintained the high expression efficiency of the quorum sensing circuit and achieved excellent expression regulation of the Fur box. The ironQS system has great potential in applications requiring in vivo protein expression, such as vector vaccines. Considering its high compatibility, ironQS system could function as a universal expression platform for a variety of bacterial hosts.
Highlights
Delivery of antigens by live bacterial carriers can elicit effective humoral and cellular responses and may be an attractive strategy for live bacterial vaccine production through introduction of a vector that expresses an exogenous protective antigen
Construction of quorum sensing-based in vivo expression systems The quorum sensing components including luxI, luxR and their respective promoters were cloned from V. fischeri
A stable plasmid pUTat [35] containing this circuit was named pQS (Figure 1A), and four quorum sensing-based in vivo expression systems ironQS1-4 were designed on this basis (Figure 1B)
Summary
Delivery of antigens by live bacterial carriers can elicit effective humoral and cellular responses and may be an attractive strategy for live bacterial vaccine production through introduction of a vector that expresses an exogenous protective antigen. Selecting an appropriate expression strategy to optimize the production of the recombinant antigen is one of the most important issues relevant to the use of bacterial strains as vaccine carriers [3]. High copy number vectors achieve sufficient antigen expression but can cause over-attenuation of the carrier and lack of immunogenicity [8]. To circumvent these problems, optimal and controllable expression, such as with the use of in vivo-activated expression systems, is a crucial step in multivalent vaccine design
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