Research On Attenuated Influenza A Vaccine Based On The Ubiquitin Proteasome System

Influenza viruses pose a significant threat to global health.However,the emergence of vaccines has greatly reduced the chances of humans being infected by viruses.Active vaccination can effectively prevent viruses from invading the body and prevent potential complications caused by the viruses,which is one of the most effective and economical methods to prevent influenza.The essence of the vaccine is to prepare the whole virus or its important components by inactivation,attenuation or genetic engineering.The main goal is to be harmless to the body or to minimize the damage to the body,and to retain its characteristics of stimulating the body to produce an immune response,and to ensure its safety,efficacy and consistency.Traditional vaccine strategies have certain limitations,such as loss or incomplete match of the natural antigens of circulating influenza strains,resulting in poor efficacy.Current virus attenuation strategies are often accompanied by a decline or loss of safety,efficacy,and productivity.In addition,influenza viruses generally undergo antigen drift and/or antigen conversion,so the protective ability of influenza vaccines is limited.It is necessary to develop a new generation of vaccines to achieve a balance and compatibility between safety and immunogenicity.This is a key scientific issue that needs to be addressed in innovative research on influenza vaccines,and it is expected that vaccination in a simpler way.Protein is one of the main components necessary for the formation of virus structure and the maintenance of its normal life activities,which provides an important entry point for the study of virus attenuation.The concept of PROTAC was first proposed in 2001.It’s a bifunctional small molecule,the overall molecular structure is divided into three parts,the first part can be connected to the ligand that binds the target protein,the second part is a linker,and the third part can be connected to the ligand of E3 ligase.Converting the entire life cycle of the virus to a live attenuated vaccine allows for a complete antigenic match between the vaccine and circulating virus strains to yield sufficient potency,a promising approach to producing more effective vaccines.Conditional control of key steps in the viral life cycle is key to achieving this goal.Since every step of the virus life cycle must depend on the virus protein,controlling the stability of the virus protein can control the life process of the virus,and use this principle to develop a live attenuated vaccine.In this study,the PROTAC vaccines were designed and constructed based on the model of A/WSN/33(H1N1),mainly by hijacking the ubiquitin proteasome system to target viral proteins for degradation.The design core of PROTAC vaccines were to introduce protein hydrolysis targeted degradation determinant(PTD)into influenza viral protein.The first part of PTD related to degraded viral protein,and the third part was combined with specific E3 ubiquitin ligase,so viral protein could be ubiquitinated and degraded by ubiquitin proteasome.PROTAC vaccines w prepared by over-expressing TEVp cells,and the safety,immunogenicity,protective effect and mechanism of PROTAC vaccines as a live attenuated vaccine were systematically studied at the cellular level and in animal models.The main research contents are as follows:1.Establishment and identification of overexpressed TEVp cell linesBased on the PROTAC design concept,in order to successfully prepare a live attenuated influenza vaccine,it is priority to establish cell lines that stably express TEVp.TEVp protease can selectively cleave the specific amino acid link ENLYFQG introduced between PTD and viral proteins,thereby removing PTD from the labeled viral proteins.Co-cultivation of HEK293 T and MDCK.2 is a standard method for generating influenza virus.In this study,a cell line stably expressing TEVp was constructed using a lentiviral vector.In HEK293 T cells and MDCK cell lines,PTD could be selectively cleaved from the labeled viral protein to ensure that the PTD-labeled viral protein would not be degraded,so that PROTAC vaccine could be prepared efficiently.The overexpression cell lines were verified by q PCR,Western blotting and plaque formation experiments.The constructed cell lines HEK293T-TEVp and MDCK.2-TEVp both successfully expressed TEVp protein,and the expression level of TEVp was significantly higher than that of conventional cells,about 5-fold and 6-fold higher than that of the original cell lines respectively.Virus infection experiments proved that HEK293T-TEVp cell lines and MDCK.2-TEVp cell lines had the same virus proliferation efficiency as HEK293 T cell lines and MDCK.2 cell lines.This experiment established the basis for the preparation of subsequent attenuated influenza vaccines.2.Construction of influenza virus reverse genetics system and viral protein screeningThe establishment of reverse genetic system is a novel and ingenious method for studying viruses,which plays a great role in many virological studies,such as the function of viral proteins,the relationship between viruses and cell host factors,the pathogenic mechanism of viruses and the mechanism of virus transmission,etc.In this experiment,the influenza virus WSN was used as a model.PTD was designed with a peptide recognizing E3 ubiquitin ligase VHL by genetic engineering.PTD was introduced into viral proteins M1,M2,NP,PB1,PB2,PA,NS1 and NS2.The virus plasmid containing PTD was co-transfected with other plasmids of the influenza virus rescue system into TEVp overexpressing cell lines to prepare eight PTD vaccine strains(PTDM1-VHL、PTDM2-VHL、PTDNP-VHL、PTDPB1-VHL、PTDPB2-VHL、PTDPA-VHL、PTDNS1-VHL and PTDNS2-VHL).M1 protein of influenza virus was successfully screened by cell infection experiment as a targeted degradation protein to prepare attenuated vaccine.At the same time,WSN virus was successfully prepared by twelve-plasmid reverse genetic system.The immunofluorescence and plaque purification experiments showed that PTDM1-VHL had a high productivity in MDCK.2-TEVp cells,while its replication was significantly reduced in MDCK.2 cells,suggesting that PTDM1-VHL had a certain safety.The genetic stability of PTDM1-VHL was confirmed by sequencing after 20 passages in MDCK.2-TEVp cells,which laid the foundation for efficient preparation,process optimization,safety and immunogenicity of the vaccine.After identification,IIV and CAIV were successfully prepared in this part of the experiment,which established a material basis for subsequent comparisons between different vaccines.3.Construction and mechanism verification of PROTACs vaccinesAt present,there are more than 600 ubiquitin ligases in the human body,which provides a variety of possibilities for the selection and design of PTDs.Five E3 ubiquitin ligases were selected in this study,including β-Tr CP,CRL4,KLHDC3,KLHL12 and KLHL20.Five amino acid sequences that could be specifically recognized by E3 ubiquitin ligases were introduced into the C terminus of influenza virus M1 protein by genetic engineering technology,and an ENLYFQG linker that could be specifically cleaved by TEVp protease was introduced between the sequence and the viral protein to remove PTD.After PROTACs vaccines were prepared by reverse genetics,the effects of PTD types on the production efficiency,safety,genetic stability and action mechanism of PROTACs vaccines were investigated at the cellular level.Cytopathic effect assay,plaque assay,TCID50 assay,immunofluorescence assay,western blotting,cell growth curve assay,viral genome sequencing was performed.According to the replication kinetics curves of PROTACs vaccines in TEVp overexpressing cells and normal cells,the results showed that all the five PROTACs vaccines could replicate effectively in MDCK.2-TEVp cells,which makes it possible to achieve large-scale expansion in the production process of vaccines.Compared with TEVp overexpressing cells,the PROTACs vaccines had a decreased ability to replicate on normal cells,ensuring a certain proliferation efficiency and safety.By western blotting and immunofluorescence experiments,the proteasome inhibitor MG132 was used to treat the infected cells,and the results showed that the degradation of PROTACs vaccines were dependent on the proteasome pathway.No mutations were detected in the PROTACs vaccines by gene sequencing after passage.In this study,PROTACs vaccines for five PTDs were successfully prepared,which were highly effective,genetically stable and safe.4.Animal experiment evaluation of PTDM1-VHL vaccineThere are three types of influenza vaccines platforms currently in production: LAIV,IIV,and recombinant influenza vaccines.In terms of requiring long-term protection and strong immune effect of vaccines,LAIV has absolute advantages,and the developed vaccine is safe,which provides a strategy for the prevention and control of influenza.In this chapter,the safety,immunogenicity,and immunoprotected efficacy of the PTDM1-VHL vaccine were studied at the animal level,mainly using mice and ferrets as models.Experiments were carried out by measuring the virus titer in animal tissues and organs,HI,NT,ELISpot,ELISA,challenge protection test,monitoring the body weight and mortality of animals.In the safety evaluation,the PTDM1-VHL vaccine showed excellent safety in animal models,consistent with the safety in cells,and the influenza virus could be attenuated in vivo.In the immunogenicity evaluation,it was found that the immune effect of the vaccine was dose-dependent by vaccination with different doses of PTDM1-VHL,so the optimal immunization dose of the mice was finally determined to be 105 PFU.Immunization with the PTDM1-VHL vaccine against CAIV and IIV induced T-cell responses in the lung of mice against the viral M1 antigen,and modest levels of mucosal Ig A,HI,NT and Ig G antibodies were generated in mice and ferrets.In conclusion,PTDM1-VHL could induce strong and broad humoral,mucosal,and cellular immune responses,which were much more effective than the widely used inactivated influenza vaccine in clinical practice and better than the currently used CAIV in clinical practice.The PTDM1-VHL vaccine showed cross protection against WSN and A/Holland/602/2009(pdm H1N1)influenza viruses in the challenge assay.5.Animal experiment evaluation of PROTACs vaccinesIn this part,three PROTACs vaccines,PTD-VHL,PTD-CRL4 and PTD-KLHL12,were selected to study their safety,immunogenicity and protective effect.The PROTACs vaccines were evaluated using a mouse model.In safety experiments,compared with WSN virus,infection with 105 PFU of PROTACs vaccines did not cause death,weight loss or any other disease symptoms in mice.On day 3 post-infection,the lung viral titers of the three PROTACs vaccines were below the detection line compared with WSN virus.These data indicated that PROTACs vaccines could significantly reduce influenza virus in vivo,indicated that PTD-VHL,PTD-CRL4 and PTD-KLHL12 had good attenuation ability and high safety in mice.In the study of immunogenicity,the sera of immunized mice were determined by HI,NT and ELISA,the results proved that PROTACs vaccines could induce strong antibody responses.PROTACs vaccines and CAIV detected high levels of Ig A antibodies in the lungs of immunized mice,indicating that PROTACs vaccines had a strong induction effect on mucosal immune response.PTD-VHL,PTD-CRL4 and PTD-KLHL12 induced strong T cell immune responses against influenza virus NP and M1 antigens in the lungs of mice.By studying the antigen presentation level of PROTACs vaccines on Raw264.7 cells,the results showed that the M1 antigen presentation ability of PTD-VHL,PTD-CRL4,PTD-KLHDC3 and PTD-KLHL12 was significantly higher than that of CAIV,IIV,WSN and blank group.These results indicated that PROTACs vaccine strain could induce stronger M1 protein antigen presentation than other vaccines,which might be due to PTD-mediated degradation of M1 protein to enhance M1 antigen peptide presentation.These results indicated that PROTACs vaccines strain could induce stronger M1 protein antigen presentation than other vaccines,which might be due to PTD-mediated degradation of M1 protein to enhance M1 antigen peptide presentation.In the challenge protection experiment,in order to prove the protective effect of PROTACs vaccines,PTD-CRL4 was selected to immunize mice after the challenge experiment,and the results showed that the PTD-CRL4 vaccine had a certain protective effect against both homologous WSN and heterologous A/X-31(H3N2).Based on all the above research results,this study systematically explored the relationship between the "life switch" elements of viral protein homeostasis regulation and the safety,immunogenicity,protective effect,and action mechanism of PROTAC influenza vaccine,so as to establish a perfect design theory of PROTAC influenza vaccine.In this study,influenza virus was used as a model virus to prepare an attenuated influenza vaccine by targeted degradation of viral proteins,which provided a new idea for the research and development of influenza vaccines.