Construction And Immunocompetence Study Of Recombinant Dengue EDâ…¢ Bacterial MVs

Dengue virus (DV) is an enveloped positive-strand RNA virus belonging to theFlaviviridae family, which is transmitted to humans via infected Aedes mosquitoes intropical and sub-tropical areas. There are four distinct serotypes of DV and each of theseserotypes can cause disease symptoms ranging from self-limited febrile illness calleddengue fever (DF) to dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS).About2.5~3billion people worldwide live in dengue fever epidemic areas and more than100million people suffer from infection each year.This will translate to around500,000to1million DHF and DSS,and the mortality rate is approximately5–20%。The southeasterncoast of our country is the dominant epidemic area, where cases of dengue fever are oftenreported every year. In rencent years, dengue viral infection has become an increasinghealth concern in our country due to the changing demographic and mosquitoes. Currently,there is no effective treatment to combat the dengue fever.Vaccine is the ideal candidate tocontrol dengue diseases.However, the available dengue vaccine strategies focous on onesingle serotype and the protection is limited, or just simply combine the certain peptidesderived from four types of DV, leading to higher molecular weight, incomplete structureand poor treatment. Furthermore, the non-neutralizing antibodies against a certain DV typewill result in the enhanced infection involving DHF and DSS when another type infectionoccurred (ADE), which particularly hinder the dengue vaccine development.Our previous work has analyzed the antigen epitope of four DV types and selected theneutralization epitope EDIII. Using amino acid degeneracy methods, we have designed andprepared a set of antigen molecules that can stimulate the body to produce high degreeprotective antibodies (1:204800) and protect more than95%animals from DV infections. From these molecules, we selected a vaccine candidate named as DV-EDIII， which hascross protection roles and has been applied for an invention patent (No.201110300602.3).Bacterial membrane vesicles (MVs) are spherical lipid bliayer vesicles that areextruded naturally from bacterial membrane.The size of the MVs ranges from20~200nm indiameter.The formation of MVs is one of the most important secretion system. As a newvaccine delivery system, the MVs can not only present the effective antigen epitope on thesuface but also simulate the protective immunity similar to native virus.The MVs arenaturally released from the surface of bacteria and have the advantages of efficiency andstability.The potential of MVs as vaccines against various infectious diseases such asmeningitis, tuberculosis (TB) and cholera give an insight into the OMV-based strategy.Although the exact immunological mechanisms underlying MVs still need to be elucidated,the already existing OMV vaccines against N.meningitidis strongly support the feasibilityof MVs vaccine approach and the immune protection raised by MVs challenge is mainlybased on the antibodies against one dominant or multiple antigens delivered by bacterialMVs. In2009, Lee et al. found for the first time that Gram-positive S.aureus can alsonaturally produce MVs. Taking the advantage of Gram-positive bacterial MVs as vaccinedelivery system, we can avoid the virulent effect of LPS delivered by MVs ofGram-negative bacteria. If an exogenous gene was inserted into that encodes a proteindeliverd by MVs, we can easily construct the recombinant MVs carrying the immunogensof interest to combat specific diseases. In this study, we constructed the recombinantstaphylococcal MVs producing bacteria by fusing the previous designed dengue virusprotective antigen DV-EDIII into the pdhB gene of S. aureus strain RN4220, and theimmunocompetence of OMVs-based dengue vaccine was also evaluated. The mainexperiments and results are as followed:1. Preparation of staphylococcal MVs and selection of target proteins for fusion ofDV-EDIII in S. aureusS. aureus strain RN4220was cultured with TSB broth and the filtrate was concentrated6-fold with ultrafiltration. And then MVs were prepared by density gradient centrifugation.TEM examnations revealed that the purified MVs were spherical bilayered and closedmembranous structures with20~200nm in diameter. SDS-PAGE analysis of MVs showedthat the S.aureus MVs contain different kinds of proteins. Five abundant bands appeared in the gel of SDS-PAGE were selected for mass spectrometry analysis, and a37kDa protein,named pyruvate dehydrogenase E1beta subunit (PdhB), was characterised and selected forthe fusion of target molecule DV-EDIII.2. Construction of engineered strain producing recombinant MVs with DV-EDIII①In order to fuse the protective DV-EDIII to the C-terminal of PdhB, the homologousarms were designed and amplified by PCR using the S. aureus RN4220genome as template,the DV-EDIII degenerate sequence was amplified from plasmid pET22b-EDIII, and aselection marker, the chloramphenicol gene, was amplified from plasmid pSET16. Arecombinant fragment of ‘left homologous arm-DV-EDIII-chloramphenicol gene-righthomologous arm’ was generated by SQE-PCR and inserted into the E. coli-S. aureus shuttlevector pYT3to construct pYT3-EDIII. According to the temperature sensitive property ofthe pYT3, the chloramphenicol-resistant clones were screened after S. aureus RN4220wastransformed with pYT3-EDIII and plated on selective BHI paltes. The engineered strainthat carrying DV-EDIII gene identified by PCR analysis and DNA sequencing wasdesigned as RN4220-EDIII+. Western blot analysis demonstrated that PdhB-EDIII fusionprotein can not only be correctly expressed in RN4220-EDIII+but also be secretedextracelluarily in the forms of MVs.②Construction of engineering S. aureus strainRN4220-Δagr/EDIII+. The MVs prepared from the engineered S. aureus strainRN4220-EDIII+were toxic to the mice challenged with50μg of recombinant dengue MVsper mouse. In order to reduce the toxicity and improve the safty of recombinant dengueMVs, we set out to knock out the bacterial virulence regulator gene (agr) and observe thetoxicity of MVs derived from the agr deletion strain. Two about900bp of homologousarms flanking the agr operon were amplified by PCR using the RN4220-EDIII+genome astemplate, and ligated directly to insert into the shuttle vector pYT3to produce pYT3-Δagr,then electrotransformed pYT3-Δagr to the RN4220-EDIII+to select RN4220-Δagr/EDIII+based on pYT3temperature sensitive propertiy amd tetracycline sensitivity. The resultingagr deletion clone was verified by PCR.The MVs were prepared from RN4220-Δagr/EDIII+and immunized the mice with50μg per mouse to check the toxicity. The results showedthat the toxicity of the MVs derived from agr deletion RN4220-Δagr/EDIII+has bensignificantly reduced. 3. Protective evaluation of dengue MVs antimouse sera against DV-2①Prepartion of MVs. The high-cell density fermentation of engineeredRN4220-Δagr/EDIII+was conducted in a3.7-L fermenter (Type KLF2000, BioengineeringCo., Switzerland) and the MVs were prepared from the supernate.The over-all yield wasabout23mg/L as measued by BCA.②Preparation the antimouse sera against recombinantMVs. The mice were challenged with MVs for three times and the antibody titer of theimmunized sera was determined by ELISA.③Potective evaluation of MVs sera. IFA andPRNT tests showed that the1:20~1:40diluted MVs sera can completely protect Vero cellsfrom DV infection, and even1:320diluted the MVs sera, around60%of Vero cells can stillbe protected. Thus, the recombinant dengue EDIII bacterial MVs sera prepared in this studyshow good protective immunocompetence.In summary, with genetic recombination approaches, we have successfully knockedthe dengue protective antigen EDIII degenerate sequence into the3’-terminus ofstaphylococcal pdhB and the fusion protein can be correctly expressed in the S. aureus andsecreted through bacterial MVs. We also demonstrated that the deletion of bacterial agrsystem will significantly reduce the toxicity of MVs and improve the usage safy ofrecombinant MVs. The antisera produced by MVs immunized mice can protect Vero cellsfrom DV-2infection and show great protective immunocompetence. We make a solidfoundation for the safe and effective dengue vaccine. Our data suggest that a degeneratemolecule based on the small EDIII of DV envelope E protein can be fused to a MV carryingprotein (such as PdhB) in S. aureus and secrete to the culture medium throngh MVs. Therecombinant dengue MVs with protective immunocompetence have great implications indengue vaccine development.