A Single Intranasal Administration Of Virus-Like Particle Vaccine Induces An Efficient Protection For Mice Against Human Respiratory Syncytial Virus

Human respiratory syncytial virus(RSV)is the most important pediatric pathogen causing acute lower respiratory illness(ALRI)in infants and young children,and also infects the elderly and the immunocompromised adults.However,no licensed vaccines are currently available.Since the clinical trials of formalin-inactivated RSV(FI-RSV)vaccine,the development of RSV vaccines has been faced with a huge challenge of either the poor safety,or the weak immunogenicity.Virus-like particles(VLPs)are formed by self-assembling viral proteins,can mimic the particle structure of virions,contain a large number of repetitive and conformational epitopes in a single structure protein and have no genetic material,which may bring new hope to produce RSV VLP vaccine with high immunogenicity and safety.Recently,some studies show RSV VLPcould be produced in the expression systems of baculovirus-insect cell or avian cell,and by the combination of fusion glycoprotein(F)or attachment glycoprotein(G)with matrix protein(M),as core of the VLPs,from Newcastle disease virus(NDV)or influenza virus other than RSV.And,these RSV VLP have been proved that they improved the capacity to induce neutralizing antibodies in mice via intramuscular route compared to RSV infection.There are many problems to be explored and improved for the development of RSV VLP vaccines in the purpose of clinical application.For example,compared with the expression system of insect or avian cell,mammalian cells have the potential advantages including specific glycosylation and correct folding of the expressed viral antigens,which are beneficial to improve the immunogenicity of VLP vaccines,and less risk to incur an allergic response in the vcaccinee.Given the potential advantages to RSV vaccine candidate produced by VLPs and the likely risks related with the expression system of non-mammalian cells,we test the feasibility of viral vaccine manufacturing for human application by constructing the recombinants of RSV fusion protein(F)and matrix protein(M)protein with first-generation adenoviral vector(FGAd),respectively,and coinfecting FGAd-F and FGAd-M into a simian-derived,continuous cell line Vero.We characterize the function and immunoreactivity of the assembled VLPs in vitro and the ability to induce immune responses and protective immunity against RSV infection in mice via intranasal(i.n.)immunization of the VLPs;and then perform the systemic comparisons on the induced immune efficacy in mice between the VLPs and RSV,and between the immunization routes intranasally and intramuscularly.The main innovations and works of this dissertation are as follows:1.RS V M protein is sufficient for the budding and assembling of RSV VLP.As the anticipated,RSV M protein is qualified for budding of RSV VLP.Additionally,RSV M protein has been thought to beimportant to induce CTL mediated immune response and get rid of the virus.We found RSV VLP composed of RSV F and M could mount RSV M-specific serum IgG antibody responsein CB6F1 mice,which showed the immunogenicity of M protein in the VLPs is strong enough.2.Functional and immunological RSV VLP are assembled successfully from Vero cells co-infected with F and M encoded-adenovirus vectors,FGAd-F and FGAd-F.Vero cells are a simian-derived,continuous cell lines for viral vaccine manufactures for human application.FGAd is one of the most commonly used gene delivery systems for the gene-based vaccines or therapeutics in clinical application and trials,because of its relatively large cloning capacity,high replication titer in HEK293 cells and highly efficient expression of a desired transgene in most mammalian cells.Unlike HEK293 package cell lines,Vero cells are susceptible but not permissive for FGAd.In other words,the FGAd-infected Vero cells can express F and M proteins efficiently,but would not be lysed by the replication of FGAd,which is beneficial for the purification of the assembled and released VLP in the supernatant.We found the assembled VLP from the FGAd-Vero cell expressing system were similar to RSV virion in immunoreactivity and morphology by the analyses of Western blot and transmission electron microscope(TEM),etc.3.The multifaceted and systemic comparison was finished by investigation on the immune efficacy induced by RSV VLP via i.n.and intramuscular(i.m.)routes.We found that a single administration of RSV VLP by either i.n.or i.m.route induced same extent and duration of protective immune responses against RSV infection with Thl-dominant immune type.Different from the immune response in mice evoked by i.m.immunization RSV VLP,the i.n.RSV VLP generated not only systemic and lung local immunity,but also secretary IgA antibody(SIgA)specific for RSV F,and CD8+T-cell responses against RSV F protein in bronchoalveolar lavage fluids(BALF).4.The multifaceted and systemic comparison was also finished by investigation on the immune efficacy induced by RSV VLP and RSV.A single i.n.RSV VLP was capable of generating immune responses and protective immunity in mice as effectively as RSV natural infection.Moreover,a single i.n.RSV VLP vaccine could induce neutralizing antibody responses observable up to 15 months,and were same long-lasting as and stronger than i.n.immunization RSV.This study demonstrates FGAd-infected Vero cells are a qualified platform for VLP production.Following the immunization via i.n.route in mice,the RSV VLP displayes same immunogenicity as i.m.immunization RSV VLP and i.n.immuinization RSV.Furthermore,a single i.n.immunization RSV VLP can induce extra mucosal immunity compared to i.m.immunization RSV VLP,and higher titers of neutralizing antibody than RSV following a single i.n.application.