Construction And Protective Efficacy Of Recombinant Influenza A Virus As A Vector For FLU-RSV Vaccine

So far, respiratory tract infectious disease is still one of the main reasons todeath in the world, and influenza virus and respiratory syncytial virus (RSV) arethe important respiratory pathogens. At present, the different types of safe andeffective influenza vaccines have provided a guarantee for influenza preventionand control. So far, there is no approved RSV vaccine for RSV prevention andcontrol because of the RSV immune self-limiting. As we all known， RSV is themost important cause of viral lower respiratory tract illness in infants, meanwhileit is an important reason of hospitalization and death from pneumonia in theelderly and immunodeficient adults worldwide. According to the statistics, theratio of hospitalization within six-month age was70%in infants and youngchildren, and it was up to99%within two years old children. Because of widepathogenic range, high pathogenic condition and causing serious consequences,RSV poses a serious threat to human health and life safety.RSV is a non-segmental, single-stranded and negative chain RNA virus,which is a member of the family Paramyxoviridae in the order Mononegavirales,and contains two serotypes of A and B. The length of RSV genome isapproximately15Kb. There are ten major proteins coded by RSV, which isrespectively constituted by three transmembrane proteins (G, F and SH), twomatrix proteins (M and M2), three nucleocapsid proteins (N, P, and L) and twonon-structural proteins (NS1and NS2). The RSV fusion glycoprotein (F) andattchment glycoprotein (G) are the most important viral proteins of RSV togenerate immune protective antibodies. The G protein is not the necessary toinfection and cell fusion because of easy to variation. However, F protein could be stably genetic and causes cell fusion lesions separately. The systemic neutralizingantibodies against RSV F and G glycoproteins could effectively prevent RSVreinfection, so the RSV F and G glycoproteins have been recognized as theprotective antigens and pathogenic molecules of virulence.Vaccination is one of the best ways to control the infectious disease. For RSV,a FI-RSV vaccine, developed by Fnlginiti VA in the1960’s, was ended in failurebecause of the TH2-type immune ultra, which led to death of two children andhospitalization of80%child-patients. Sereval types of RSV vaccine were beingdeveloped, such as vector vaccine, live attenuated vaccine, subunit vaccine, DNAvaccine, VLPs vaccine and so on, but still no approved RSV vaccine was availableup to now. The research of RSV vaccine had always been the focus in internationand society. However, the existing RSV vaccine was still not break through thedevelopment because of facing with those bottleneck problems as no effectivemucosal and cellular immune responses from the immune of injection, restrictionon the immune protection, potential security of DNA vaccine and potentialTh1/Th2imbalance in the overall length of the RSV vaccine of F and G proteins.In view of the maturity of the influenza virus reverse genetics technology and theunique advantages of influenza virus vector, the RSV vaccine candidates, preparedby influenza virus as a delivery system, could achieve double efficacy on theimmune protection, broaden the development prospect and provide new train ofthought for the research of RSV vaccine because of high security and easy tooperation.The object of this thesis is to construct and rescue recombinant FLU-RSVvaccine using reverse genetics techniques, and to conduct the comprehensiveidentification of the recombinant FLU-RSV vaccine. Then the immunogenicity andimmune protection of recombinant FLU-RSV vaccine were evaluated by animal immunization and challenge, and finally provide new ideas and experimental basisfor the development of RSV vaccine and realizing "seedlings of dual-use". Thisstudy is made up of two parts:Part one: Rescue and characterization of recombinant influenza A virusas a vector for FLU-RSV vaccineThe RSV F protective antigen epitopes (F205-223： PIVNKQSCRISNIETVIEF; F255-278： SELLSLIN DMPITNDQKK LMSNNV) and RSV Gprotective antigen epitopes (G142-204: QPSKPTTKQR QNKPPNKPNNDFHFEVFNFV PCSICSNNPT CWAICKRIPN KKPGKKTTTK PTK) wereconfirmated by bioinformatics analysis and literature survey. Base sequencesgotten by analysis were connected by a GPG linker and inserted into the NS1geneof influenza virus. In these sequences, F205-223-GPG-F255-278was named as Fand G142-204was named as G. Next, the recombinant plasmids of NS1-F, NS1-Gand NS1-F+G, and the two-way transcriptional expressing carrier of pHW2000were constructed by BSmBⅠ enzyme digestion, agarose gel electrophoresis (AGE),gel recycling, objective plasmids connection with pHW2000carrier,transformation with DH5α competence, pick monoclonal colony to wave andbacteria liquid PCR. When positive bandings from PCR products were identifiedby AGE, and the results of Shanghai Sangon sequencing was consistent tooriginally design, it showed that the recombinant plasmids of NS1-F, NS1-G andNS1-F+G were successfully constructed.Recombinant plasmids of NS1-F, NS1-G and NS1-F+G, and influenza virusseven plasmids of PB2, PB1, PA, HA, NP, NA and M were combined with “7+1”for viral transfection. The8plasmids of influenza virus were co-cultured in COS-1cells and MDCK cells by Effectene, and NS plasmid was instead of recombinantplasmid.24hours later, the Effectene-DNA complexes in6-well plates were removed and added fresh DMEM medium with0.8μg/mL TPCK trypsin. Initialinspection of hemagglutination was finished from supernatant after48hours.When a hemagglutination positive result was found, the virus liquid and the cellswere inoculated into9-day-old SPF chicken embryos. Allantoic fluids werecollected3days later and proceeded hemagglutination retest, which showedpositive again.The recombinant FLU-RSV vaccine, inoculated into9-day-old SPFchicken embryos for three generations, could stable inheritance, it indicated thatthe rescue of recombinant influenza virus was successful, and we respectivelynamed those recombinant FLU-RSV vaccines as rFLU/RSV/F, rFLU/RSV/G andrFLU/RSV/F+G.The recombinant FLU-RSV vaccines were amplified by chicken embryos,concentrated by30KD membrane package, and purified by30%-60%sucrosegradient. Then hemagglutination titers and virulence titers (CCID50) of those threerecombinant vaccines were measured and the results were recorded. Meanwhile,expression of RSV-specific proteins were detected by Western blotting, and sizedistribution and particle characteristics of the recombinant virus consistent withinfluenza virus particles were observed by electron microscopy. What’s more, thegrowth of recombinant FLU-RSV vaccines was determined in MDCK, A549andCNE-2Z cells and could be effectively replicated. These results suggested that therecombinant FLU-RSV vaccines were successfully rescued.Part two: Immunogenicity and immune protection of recombinantinfluenza A virus as a vector for FLU-RSV vaccineSix-week-old female BALB/c mice (30mice per group) were randomlygrouped in seven groups. We established two experimental dose groups as105CCID50group and104CCID50group of rFLU/RSV/F, rFLU/RSV/G andrFLU/RSV/F+G vaccines, and PBS group as control. Mice were intranasally immunized follow the above doses in the volume of20μL. The boost immunizationwas operated four weeks after the prime immunization, and the sera, lung andnasal lavage fluids, and spleen cell suspensions of mice were collected beforeimmunization, four weeks after prime immunization and two weeks after boostimmunization. To evaluate the system immune response in mice, neutralizingantibody titers against PR8influenza virus and RSV, and the antibody subtypetiters of IgG, IgG1and IgG2a against RSV were measured in mice serum samples.Meanwhile, the cytokines of IL-2, IL-4, IL-5, IFN-γ and TNF-α against RSV weredetected in spleen cell suspension of mice for the system immune evaluation. Inaddition, mucosal immunity was evaluated by sIgA titers in mice lung and nasallavage fluids. It suggested that immunized with recombinant FLU-RSV vaccine inmice was capable of producing neutralizing antibodies against the influenza virusand RSV, a Th1-type cellular immune response and mucosal immune responseagainst RSV. Meanwhile, the immune results showed that immunogenicity ofrFLU/RSV/F+G vaccine strain was superior to rFLU/RSV/F and rFLU/RSV/G,whereby the initial inference was that rFLU/RSV/F+G vaccine strain was the mostpotential of influenza viral as a vector for RSV vaccine candidate.To evaluate the protection against influenza virus and RSV infectionfollowing rFLU/RSV/F+G immunization, BALB/c mice (20mice per group) werechallenged with live PR8influenza virus (100LD50) and RSV A2(2×106PFU/mL)by intranasal inoculation two weeks after boost immunization. Five mice weremonitored for weight change and survival ratio throughout a14-day observationperiod, others were sacrificed four days against influenza virus or six days againstRSV and the samples of BALF, nasal turbinate and lung tissues were obtained.Lung samples were divided into two portions, one for pathological examination,and another for recovering the influenza virus or RSV infective particles. The pulmonary tissues were isolated, fixed with10%neutral buffered formalinimmediately and stained using standard histological techniques by Hemotoxylinand Eosin (H&E). Virus titers of influenza in tissue homogenates were determinedby TCID50in MDCK cells, and virus titers of RSV in tissue homogenates weredetermined by the50%plaque reduction assay using HEp-2cells. What’s more,total inflammatory cells and cell differentials against RSV were respectivelycounted in BAL. The increase of total cell numbers was associated witheosinophils (EOS), neutrophils (NEUT), small mononuclear cells (MONO),macrophages (MAC) and lymphocytes (LYMP). The indexes showed that theimmune protection was able to effective generate in mice immunized with therecombinant FLU-RSV vaccines, and rFLU/RSV/F+G vaccine was the best onethan rFLU/RSV/F and rFLU/RSV/G. It was suggested that rFLU/RSV/F+G vaccinecandidate strain was the most potential recombinant influenza virus as a vector forFLU-RSV vaccine candidate strain, because it had significant immunogenicity andprotective efficacy, and could effectively improve humoral, cellular and mucosalimmune response.In conclusion, the recombinant FLU-RSV vaccines were were successfullygenerated using RG and influenza A virus as a vector. The resuits of identificationshowed that rFLU/RSV/F, rFLU/RSV/G and rFLU/RSV/F+G vaccines could beeffectively replicated in MDCK, A549and CNE-2Z cells, the size distribution andparticle characteristics of the recombinant FLU-RSV vaccines were consistentwith influenza virus particles, and they could express the RSV-specific proteins.The results of immunogenicity assessment suggested that immunized withrecombinant FLU-RSV vaccine in mice was capable of producing neutralizingantibodies against the influenza virus and RSV, a Th1-type cellular immuneresponse and mucosal immune response against RSV. The results of challenge showed that recombinant FLU-RSV vaccines could induce simultaneouslyprotective immunity against backbone-FLU and inserted-RSV infections, and therFLU/RSV/F+G vaccine highlighted a greater advantage. The above researchprovided experimental basis for the development of RSV vaccine and realizing"seedlings of dual-use".