Research On The Vesicular Stomatitis Virus-based Oncolytic Viruses And COVID-19 Vaccines

Infectious diseases and cancer have caused a great burden on humankind,one of the hottest research topics in the biomedical field is the research and development of vaccines and therapeutics,which is full of requirements and challenges.With the development of molecular virology,a broad understanding of virus replication and gene expression,as well as pathogenic mechanisms,genetic modification like reverse genetic modification have got great development.Some viruses had been developed as vectors of vaccines or therapeutic drugs with specific druggability and become a class of biological products that could not be ignored.The viral vectors that had been successfully applied to the marketed vaccines include:Adenovirus type-5,Adenovirus type-26,Chimpanzee adenovirus and Vesicular stomatitis virus(VSV);in addition,Herpes simplex virus type 1,Enterovirus,VSV and many other viruses had also been modified for the development of oncolytic virus.Benefits of the VSV vector platform make it potentially to be both a vaccine vector and an oncolytic virus vector:(1)mild pathogenicity in humans;(2)inability of the vector’s viral RNA to integrate into the host genome;(3)fast production of high titers and its propagation in almost all mammalian cells,high expression efficiency of foreign genes;(4)simple genetic modification with the possibility to accommodate one or multiple antigenic inserts;(5)VSV envelope glycoprotein can be replaced by heterologous envelope glycoprotein from a variety of other viruses,which has been enabling wide accessibility to study properties of high containment level 3 or 4 viruses at a low containment level;(6)the low seroprevalence and without pre-existing immune in the human population.These advantages make the technology of construction and optimization of VSV vector platform as well as the research of related candidate molecules particularly valuable.The global pandemic caused by emerging Coronavirus Disease 2019(COVID-19)highlights the importance of practical and feasible platform technology and research system.Therefore,this study decided to develop oncolytic virus and COVID-19 vaccine research based on the VSV vector.Achieved a safer and more effective carrier through modification and optimization,which provided candidate molecules and accumulated technical and scientific basis at the same time.The first part of this study was the research of oncolytic virus based on VSV vector.Oncolytic viruses preferentially infect and replicate in cancer cells,spreading to other tumor tissues and killing them in the process,which also have the potential to induce an anti-tumor response indirectly by activating immune cells to help them target and kill cancer cells.Several studies had reported that VSV could be applied as an oncolytic virus,However,multi-dose virus intravenous injection was not recommended due to the strong neurotoxicity and high level of neutralizing antibodies induced by wild type VSV,VSV envelope glycoprotein plays a key role for neurotoxicity and inducing neutralizing antibodies.Therefore,this study constructed a kind of replicationcompetent Oncolytic virus rVSV-GP1 based on the modification of the VSV vector by replacing the wild-type VSV-G gene with the Lymphocytic choriomeningitis virus(LCMV)G gene.To explore the oncolytic efficacy of rVSV-GP1 in vitro and in vivo,we first examined the tumor cell-killing effects of rVSV-GP1 on 51 kinds of cultured human tumor cell lines.Our results showed that rVSV-GP1 remarkably induced cell death in most tumor cell lines(40/51),including hepatocellular carcinoma cell lines,lung cancer cell lines,ovarian cancer cell lines,and glioma cell lines showed high sensitivity to rVSV-GP1(MOI=1.0,cell-killing>70%),rVSV-GP1 exhibited over 20000-fold reduced neurovirulence compared to the unmodified VSV-WT after intracerebrally injection of rVSV-GP1 or VSV-WT in C57BL/6 mice The safety was significantly improved and especially no significant neutralizing antibodies were detected after rVSV-GP1 multiple intravenous injections,while the control wild type VSV rapidly induced high titer neutralizing antibodies in mice,which support the result that rVSV-GP1 may be beneficial for multi-dose treatment in cancer therapy especially in the intravenous inhection route.Secondly,in order to improve the effect of candidate viruses in specific tumor,our study adopted the directed evolution to obtain the adaptive mutant strain by passing rVSV-GP1 in HCC cell line Huh7 with multiple generations Huh7-P52,a mutant strain with significantly changed was obtained after 52 generation passing.Compared to rVSV-GP1,Huh7-P52 showed significant improvements in safety and tumor cell-killing efficacy.In addition,based on the ability that oncolytic viruses could alter the tumor microenvironment and promote immune cell infiltration,this study further explored the strategy of combining rVSV-GPl with tumor-specific T cells in melanoma models carrying specific tumor-specific antigens and transgenic mice with corresponding T cell receptors.our results showed that the combination of oncolytic virus rVSV-GP1 and tumor-specific T cell could significantly enhance the tumor destruction compared to using rVSV-GP1 or tumor-specific T cell alone,and it was found that rVSV-GP1 could promote tumor-specific T cell infiltration into the tumor,while the proportion of non-specific T cell infiltration was significantly lower.Therefore,VSV-based oncolytic virus therapy combined with tumor antigen specific T cell therapy may provide new ideas and strategies for cancer therapy.The second part of this study was the research on the COVID-19 vaccine based on the VSV vector.Firstly,in the early stage of the pandemic of COVID-19,this study constructed a robust neutralization assay based on SARS-CoV-2 S-protein-bearing vesicular stomatitis virus(VSV)pseudovirus and ACE2-overexpressing BHK21 cells.This pseudotype infection assay and wild type SARS-CoV-2 assay were highly correlated(R2=0.8396,P<0.0001),which could be applied to the screening of compounds that could inhibit infection by SARS-CoV-2 and neutralizing antibodies.More importantly,this method can be used to measure the neutralization titer of serum from vaccinated animals or humans,which is fast and convenient for the development of vaccines and drugs against COVID-19.Secondly,the wild-type VSV-G gene was replaced with SARS-CoV-2 Spike gene to construct a COVID-19 candicate vaccine based on the VSV vector,which showed strong immunogenicity in a variety of animal models.Protective efficacy evaluation was tested in the severe hamster model that mimic COVID-19 in human after intranasal SARS-CoV-2 challenging method.The results showed the vaccine based on the VSV vector immunized hamsters exhibited significantly marked viral RNA reductions(>3.3 log10)in turbinate,trachea,and lung tissues and could protect hamsters from SARS-CoV-2 infection and its related pneumonia.In addition,this study indicated that the immune protection provided by nasal inoculation may have unique advantages in preventing SARS-CoV-2 infection.Therefore,this study also successfully rescued VSVdG-INSARS-CoV-2(1N)which placed the SARS-CoV-2 Spike gene in the first sequence of the VSV genome.This modification provided a better choice for nasal inoculation with more stringent safety requirements,whether nasal inoculation or intramuscular inoculation.The candidate vaccines based VSV had shown safety and efficacy in hamster models and provided the potential to clinical transformation.With the spread pandemic of COVID-19 and the accumulation of variant,it is necessary to explore broad-spectrum antibodies and epitopes.In this study,we developed an antibody library composed of 39 monoclonal antibodies after immunization with SARS-CoV and SARS-CoV-2 pseudoviruses based on VSV vectors.These monoclonal antibodies can simultaneously cross-neutralize SARS-CoV and SARS-CoV-2,and three of the monoclonal antibodies showed high neutralization efficiency against SARS-CoV-2 variants of concern(IC50<3.0 ng/mL),providing candidate monoclonal antibodies for the development of broad-spectrum antibody-based therapies.At the same time,which provided a theoretical basis for the identification of epitope of SARS virus and the design of a broad-spectrum vaccine.In summary,as oncolytic viruses,this study had obtained a candidate oncolytic virus rVSV-GP1,the safety had been significantly improved with no neutralizing antibodies induced in mice,and the directed-evolved strain Huh7-P52,which shows a stronger cell-killing effects on HCC,this study provided a great platform for cancer therapy.The potential of oncolytic virotherapy combining with tumor-specific T cell could enhance the tumor destruction,which could form part of future multimodality treatment strategies for cancer therapy.As COVID-19 vaccines,this study obtained a COVID-19 candidate vaccine VSVdG-1NSARS-CoV-2(1N)based attenuated VSV that can be intranasally inoculated,showing significant safety,immunogenicity and efficacy in animals.This study constructed a robust neutralization assay based on SARS-CoV-2 S-protein-bearing vesicular stomatitis virus(VSV)pseudovirus,and a broad-spectrum neutralization monoclonal antibodies library for SARS-like virus was obtained.In conclusion,this study providing a candidate VSV-vector-based oncolytic virus and a COVID-19 candidate vaccine,at the same time the VSV-vector-based technology were well accumulated to prepare for emergency requirements.