The Virosome As A Vaccine Candidate For DENV Vaccine Development And The Key Aspects Of DENV Virosome Production

Dengue virus comprises4closely related but genetically distinct serotypes(DENV1-4). All the four serotypes are endemic throughout the tropical andsubtropical regions. Dengue virus causes classic Dengue Fevers (DF) when peopleinfected by the virus for the first time. The DENV can induce both the humeral andcellular anti-dengue immune response. However, the antibodies do not protectsecondary infection by different serotypes. The sub-neutralizing concentration ofthose antibodies could enhance the infection in host cells. And it is postulated to bethe top one risk factor for DHF and DSS in secondary infection by differentserotypes. This phenomenon is known as Antibody-Dependent Enhancement.The potential ADE activity of the antibodies induced by DENV not onlyrelated to the severe disease, but also brings a great challenge in the development ofthe DENV vaccine. The mechanism of cross-protective immunity against DENV isnot fully understood and a vaccine candidate inducing ADE might be anotherproblem to concern. Wide ranges of vaccine technologies have been applied toDENV vaccines development. Fortunately, several candidates are currentlyundergoing either clinical evaluation or preclinical development, including liveattenuated virus, inactivated virus, recombinant subunit, and virus-like particles.Although these approaches have their advantages, but the potential risks of ADEunderscore the importance of the candidate DENV vaccines. The most ideal DENVvaccine is tetravalent, which induces both the humoral and cellular immuneresponses to infections of all serotypes and the preparation of monovalent candidatewith lower ADE activity is urgently needed. By this resone, our work focused on:1. The applications of DENV-2virosomes. Virosomes are the reconstitutedviral envelopes that lack the viral genetic materials. Since the virosome are closelymimicking the native virus with naturally configured surface proteins. The Eproteins on the virosomes will undergo a natural antigen processing andpresentation pathway. In this work, we prepared the virosomes from DENV-2andinvestigated whether the DENV-2virosome can induce the same immune responseas DENV-2does. We expect that the virosome of DENV-2could induce powerfulimmune response. Importantly, we hope the antibodies stimulated by the virosomeshave lower ADE activity. The result demonstrated that the DENV-2envelope could be reconstituted as a virosome, which contains the enveloped protein (E) but lacksthe genetic materials and membrane protein (M). The blocking activity experimentshowed that the0.0154mg/mL DENV-2virosome could reduce50%PFU ofDENV-2, suggesting that the reconstituted DENV-2envelopes are functionallymimicking native DENV-2in host cell binding properties.As for the immunogenicity, the average IgG titer (Log10) of the mice seraimmunized with DENV-2virosomes is3.56as same as that stimulated by DENV-2(LogTiter=3.62). The viral neutralizing activity assay by50%Focus ReductionNeutralizing Test (FRNT50) of those mice sera was following the same trend;LogFRNT50=1.717(against DENV-1) and LogFRNT50=2.389(against DENV-2)for anti-virus serum, which is a litter bit higher than LogFRNT50=1.696(againstDENV-1) and LogFRNT50=2.314(against DENV-2) for anti-virosome serum. Thevaccine-induced ADE is a risk in DENV vaccination, thus the enhancement fold ofthe heterologous serotype infection must be considered. Even the native DENVcould induce higher neutralizing antibodies titer, however, that anti-DENV-2serumwould enhance56%heterologous infections，which is10percentage higher thananti-DENV-2virosome serum dose (46%). These datas demonstrated that thereconstituted DENV enveloped with E protein could induce the same fully immuneresponse as the whole viral does, but the lack of non-neutralizing antigens resultedin lower ADE activity, which means the virosomes will be safe in vaccination.2. Quality virus produce and viral purification. After acclimate the cells togrowth in medium with5%,2%and1%FBS, we developed a Vero cell bank thatcan live in vitro with lower FBS concentrations (2%). The recovery efficiency ofthose cells in the bank were90.8%and with a3days life cycle. For DENV-2virusproduce, those Vero cells with a density in80%infected with0.05-0.1MOI of thevirus can gather4×105PFU/mL of quality virus product between72-96h. We haduse (NH4)2SO4and PEG1000to established an aqueous two-phase system andextract PRRSV or DENV-2from the virus-rich cell cultural medium into the firstphase of this two-phase system. After the virus was purified by this extract system,the virus solution was concentrated to20.9%of the original volume and there is noimpurities which can be detected by SDS-PAGE. The result is that (NH4)2SO4/PEG1000aqueous two-phase systems can be successfully applied to purify andconcentrate virus in large-scale.3. Virosome preparation. Adjust the virus protein concentration to2mg/mL use HNE buffer, and then the virus will lysised when0.3mg/ml was added. After beingchromatographed by Sepharose4FF, the viral cores can be removed and the virusenvelope will auto-reconstituted after Triton X-100removed by dialysis. In ourwork the virosomes can be reconstituted and look like a native virus. Thephospholipid recovery is68.5%, the envelope protein recovery is58.6%and theresidue of Triton X-100was5.3%. For DENV-2virosomes, the envelope ofDENV-2could be reconstituted as virosome devoid of the genetic materials. Using0.0154mg/mL of the prepared virosome could achieve50%reduction in PFU ofDENV-2infected Vero cells.4. Pilot production. In addressing the key technology DENV-2virosome afterpreparation carried out DENV Virosome pilot production. Seting up a productiontechnology roadmap will prepare process for DENV quantify cell cultureproduction in cell culture, Virus semi preparation, Virosomes semi preparation, andvaccine preparation5processes. After quantification of processes and process thesame period of co-ordination, it is responsible for the production of cell cultureaspects of recovery in Vero, each providing3×107cells. Ring production culturewith3×107cells were exactly cells that were seeded bottle15L spinner flasks, andfor the preparation of monolayer cultured cells transfected with a viral bottle. Afterthe semi-finished part,500mL of DENV can be produced (viral proteinconcentration of2mg/mL) of DENV, just for the preparation of a DENV-2virusome. Preparation virosome produced protein concentration of each part of2mg/mL of virosome100mL. Route designed every aspect of the productiontechnology can operate independently, providing a steady stream down a part of thedemand. The resulting final product was tested in the pilot production line with therequirements of Chinese Pharmacopoeia.