The Development Of RSV VLP Vaccine And Investigation Of RSV-induced Lung Injury Mechanism

Human respiratory syncytial virus (hRSV) is recognized as the most conmon cause of neonatal and the elderly respiratory tract disease in the world. There are more than 70% RSV-infected patients aged <5years old and around 10% of those with Lower Respiratory Tract Infection (LRTI) in hospital. Since the 1960s, gobal scientists have been paying attempts to develop effective vaccines, but there is still no lisenced vaccine. Thus WHO suggested that every country should enhance minitor of RSV epdemic and endeavor for the prevention.Blount et.al first found new virus in sick chimpanzee coryza agent (termed CCA) in 1956. Chanock et al reported that pediatric patients infected with CCA having bronchiolitis. And then they isolated the virus casing cell fusion, so called it respiratory syncytial virus (RSV). RSV is a negative-sense, single-stranded RNA virus, belonging to paramyxoviridae family. Its 1.5 kb genome encodes 11 surface and structural proteins. The F and G surface proteins of RSV are the major antigenic determinants of protection. And full-length F and G contain all the neutralizing and several T-cell epitopes of RSV. The conserved F glycoprotein induces specific cytotoxic lymphocytes, transforming a primarily CD4+and CD8+ cell T-cell response into a Thl immune response. On the other hand, the G protein elicits a Th-2-dominant immune response. There are two serum subtypes RSV-A and RSV-B based on G variance. In addition, both A and B subtypes are epidemic strains casing severe clinical lung disease.In 1970s, the first vaccine, a formalin-inactivated whole virus (FI-RSV), was tested in infants. Unfortunately, it failed to protect vaccinated infants and led to severely exacerbation of diseases. There were 80% FI-RSV vaccined infants with Vaccine Enhanced Diseases (VED) in hospitial. Even worse, there were two cases of death after FI-RSV vaccine [1] In following years, many scientists studied on the live-attenuated RSV vaccine by virus passage, clinical isolation and genetic modify. They could be considered as a promising vaccine candidate because of the protective efficacy. However we need to rise to a major challenge to achieve a balance between over-attenuation immunogenicity and under-attenuation pathogenic potentiality. RSV subunit vaccine is based on genetic engineering, expressing of F or G protein, and having a certain protective efficacy. For example, the study of BBG2Na had already stopped at clinical phase III causing lack of comprehensive protection. In addition, vector RSV vaccines expressing RSV epitopes in virus (HPIV、ADV) vetor were in clinical test. However, the virus vectors had pre-exsting immunology (PEI) in human, leading to failure in clinical test. Therefore, there are greatest difficulties and challenges in the development of RSV vaccine. We urge to solve following questions: 1) Naturation of RSV epitopes; 2) imbalanced Thl and Th2 responses resulting in VED; 3) potential risk factors caused by live-attenuated virus; 4) CTL responsed weekend by PEI. Recently, with development with the preparation technology of Virus-Like particle (VLP), it becomes more and more popular. VLP is similar with the morphology of naive virus, miniking process of infection. But it lacks of viral genome resulting in non-replication, non-recombiant, and non-diffusion. Previous studies report that influenza M protein could help to form VLP. But it owes PEI in human, enhancing the risk of VED. As well known, human is hardly infected with Newcastle disease virus (NDV). Furthermore, it has been suggested that NDV M and NP were easier to form VLP. In addition, RSV and NDV are both Paramyxivirinae, which help to form VLPs. Therefore, NDV backbone could serve as a platform for the expression of RSV proteins.RSV infects human via the mucosal surface. Mucosal Secretory IgA (sIgA) could neutralize the virus to avoid manifestation and dissemination. Mucosal immunization also could evoke systemic immunology, inducing serum IgG antibodies and CTL response. Moreover, mucosal vaccination does not require needles and causes less pain, so there is a high acceptance of these vaccines among children.To date, there are only two clinical drugs including Palivizumb and Ribavirin which have efficiency in treatment for pneumonia and bronchitis. However, expensive Palivizumb could allevriate lung disease only via Multi-dose. Ribavirin could not cure the immunocompromised because it’s side effects. Therefore, devlepment of noval thereputic drugs and strategies have significant meanings to control RSV-induced disease.The mechanisms of pathogenesis are critical to the design of vaccines and drugs. To date, RSV induced lung injury mechanism foucs on immune evasion, demage of host immune and lung inflammatory. In primary infection, RSV(NS1、NS2、G) is likely to block the TLR signaling pathway, depressing IFN-I type immune reponses. RSV could also complete the replication by escaping the host anti-viral innate immunity. And a mount of RSV spread to lower tract airway causing lung disease, such as pneumina. At that time, activated TLRs、RIG-I pathway signaling could induce to secret pro-inflammatory cytokines, including IL-2、IL-6、TNF-α、CCL2、 CCL3、IP-10. Follwing, the cytokines and chemokines could recruit and activate immune cells, such as DC and NK cells. Finally, a large number of immune cells and pro-immatory cytokines were boosted in lung tissue, resulting in damage. Additionally, there is close relationship between RSV-induced host cell death and pathogenesis of lung diseases. RSV infection could casue airway epithelial cell apotosis and shedding. The increasing hemaleuin and mucin may form thrombus, leading obstructive pulmonary and aggregate severity of lung injury. So we knew that, the mechanism of pathogenesis is complicated progress. Here, we need urgently to figure out following unsolved science puzzles:1) what is the fuction of RSV-induced cell death (apotosis> autophagy、necrosis) in pathogenesis;2) what’s mediator in RSV-induced death. And we believed that RSV predisposing gene and lethal gene could pave a way in pathogenesis illumination and noval drug target study.In this study, we have efforts to develp noval VLP vaccine, figure out the mechanism of pathogenesis and point out innovation theory, guiding the design of RSV vaccines and drugs. There are two parts in the study as following:The study of recombint RSV VLP vaccine preparation and immune evaluationObjective:We developed and evaluated recombint RSV VLP vaccines which might provide powerful evidents to achieve RSV vaccine candidates.Method:We had prepared and evluated the recombint RSV VLP vaccines based on NDV backbone, containing RSV F or G protein. At first, we optimized codon target sequence. To improve the transfection of rNDV/RSV/F or rNDV/RSV/G, the RSV F or G gene, we incorporated them into the NDV genes order of NP and M to create a recombinant plasmid. The SV40 and polyhedron promoter were inserted at the junctions among the three genes. And then plasmids were transfected in sf9 cells, and VLPs were released successfully. We examined VLP using WB and Electron microscopy analysis. Next, we acquired the purified VLP by sucrose gradient centrifugation. In the evaluation test, we detected the levels of IL-4 and INF-y by Elisa and ELISPOT. Finally, we observed the mice weight change、lung histophagy and virus load in vaccined mice after challenged.Result:We detected the molecular weight of the F protein expressed from rNDV/RSV/F was 56 KD similar to that from the wild type mice (WT). Interestingly, the G protein expressed from rNDV/RSV/G was 60 KD, as indicated by Western blot. Both of the NDV proteins in VLPs were 40 KD and 51 KD. The results demonstrated that the total proteins of VLPs were expressed properly. Electron microscopy analysis of the purified VLPs revealed the morphologies of rNDV/RSV/F and rNDV/RSV/G The peak of the size distribution of rNDV/RSV/F was 91-110 nm; whereas that of rNDV/RSV/G was 51-130 nm.And then VLPs were purified by sucrose gradient centrifugation. BALB/c mice immunized intranasally (i.n.) with rNDV/RSV/F plus rNDV/RSV/G developed robust humoral, mucosal RSV-specific antibodies and cellular immune responses. Furthermore, rNDV/RSV/F plus rNDV/RSV/G provided better protection than rNDV/RSV/F or rNDV/RSV/G alone, as shown by an obvious decrease in viral replication together with alleviation of histopathological changes in the lungs of the challenged mice.Conclution:Our datas demonstrate that the intranasal vaccination of combined RSV virus-like particle vaccine candidates has great potential for protection against RSV infection.The study of pathogenesis of RSV-induced lung diseasesObjective:We attempted to find RSV predisposing gene and lethal gene and investigate their fuctions. We hope that our data could prove innovation theory system and point out noval vaccine and drug target.Method:We had achieved the target gene via siRNA libray screening. And then we should study on the fuction of target gene. At first, we detected cell viability on posted RSV via MTS in the siRNA libray. Then we successfully aquired ACE2 and MSK-2 gene, and continue to investigate further. Next, we used Westernblot and ELISA to measure the levels of Ang Ⅱ, ACE and ACE2 in clinical patients and exprimetal mice. And we also built the RSV-induced lung injury mouse model measured by wet to dry ratio, lung pathology and weight changes. Moreover, we investigated the fuction of ACE2 in RSV-induced lung injury. In addition, we built RSV-induced autophagy model in A549. We dected the ratio of LC3 Ⅰ/LC3Ⅱ and observed autophsome. Finally, we study on the MSK-2 fuction in siRNA and inhibition tests.Result:We successfully established the siRNA screening platform to selecte genes associated with RSV-induced cell death. There are 10 death genes (MSK-2) and 3 survial genes (ACE2) in the selected pool including 4,000 genes. Finally, we selected MSK-2 and ACE2 to study.We report that angiotensin-converting enzyme-2 (ACE2) protects against the severe lung injury induced by RSV infection in an experimental mouse model and in pediatric patients. Additionly, we provide evidence to porve RSV infection causes severe lung injury partly, by modulating the RAS system via down-regulating the ACE2-AT1R axis. Based on our findings in this work, we suggest that modulation of ACE2 expression and/or blocking lung injury response may represent potential pharmacological approaches to prevent or treat RSV-induced lung disease.To investigate the function of MSK2, we established the RSV-incuded autophagy in A549 cell lines. MSK2 is likely a regulator of RSV-induced autophagy via LC3. Also, we need to do further researches to find more evidences which can figure out the mechanisms of RSV infection.Conclution:We could find the RSV predisposing gene and lethal gene via human genome siRNA screening, proving experimental data of mechanism pathogensis and noval targent of vaccine and drugs. In this section, ACE2 could alleviate the symptoms of acute lung injury during RSV infection. In addition, MSK2 is likely a regulator of RSV-induced autophagy via LC3.