Construction Of "Hu191" Recombinant Measles Virus And Its Oncolytic Effect And Mechanism To Non-small Lung Cancer

Part I Construction of "Hu191" Recombinant Measles Virus Expressing Fluorescent ProteinBACKGROUND and OBJECTIVESModern molecular technologies enable great insights into exploring diverse RNA virus vector systems by handling the virus anti-genome.Mealsles virus(MV),belonging to the family of Paramyxoviridae,are negative single RNA virus.Oncolytic MV has potential in restraining multiple cancer types progression in both animal models and clinical trials.The strategies for further improving the efficacy of MV are intensively explored.Arming MV with marker or therapeutic genes has emerged as a promising method to track viral replication and spread,even to enhance their oncolytic effect majorly based on the mechanism of recruiting immune cells into tumor environment and reverting inmmunosuppression situation.Attenuated live MV of Hu191 strain has been used in China since more than 50 years ago.We have constructed full length of cDNA clone from Hu191(recombinant MV-Hu191,rMV-Hu191)in previous research.In this part,we will explore that whether rMV-Hu191 could be used as viral vector to express foreign genes for immunizing patients against infectious diseases and enhancing oncolytic virustherapy to multiple cancers in consideration of its safety and efficiency.METHODS:Open reading frame(ORF)of enhanced green fluorescentprotein(EGFP)was inserted before nucleoprotein(N),between phosphoprotein(P)and membrane protein(M)or between hemagglutinin protein(H)and large protein(L)in MV anti-genome within seamless cloning kit.The DNA pieces containing EGFP and DNA pieces encoding structural protein were amplified by PCR.The PCR products were involved in ligation reaction by T4 DNase ligase and following the digestion with restriction endonucleases,and then were assembled into a full length cDNA clone.Identification of rMVs-Hu191-P-EGFP depends on the strategies of RT-PCR,sequencing and observing fluorescence.Replication ability of viruses was determined by plaque assay.The expression of foreign genes was detected by fluorescent microscope technique.Collected the total RNA of rMVs-Hu191-EGFP after 10 passages for Sanger sequencing.There are no any changes in rMVs-Hu191-EGFP anti-genome sequence.RESULTS:1.We successfully introduced marker genes coding EGFP into cloned viral cDNA from biological MV-Hu191.The results of RT-PCR,DNA electrophoresis and sequencing showed the sequences of three recombinant MVs-Hu191(rMVs-Hu191)carrying foreign gene sequences(rMVs-Hu191-EGFP)were correct.2.We transfected three full length plamids of rMVs-Hu191-EGFP into BHK-T7 cells,after 72 hours,transfected BHK-T7 cells were co-cultured with Vero cells.After 24 hours,Vero cells exhibited CPE(cytopathic effect),and under the fluorescence microscopy,the green fluorescence was observed.3.In the light of growth kinetic assay results,rMVs-Hu191 with additional insertions had some variations in viral titers at each time points to parental rMV-Hu191.The recombinant virus strain rMV-Hu191-N-EGFP,which expressed foreign gene before N protein,has weak viral titer compared with parental strain and other recombinant virus strains.While rMV-Hu191-P-EGFP with insertion between P and M protein arrived at peak viral titer delayed after 60 hours post infection,the CPE and viral titer induced by rMV-Hu191-H-EGFP were both similar to the parental rMV-Hu191.4.After Vero cells in six-well paltes infected with three rMVs-Hu191-EGFP respectively,we observed the fluorescence expression at different time points.The fluorescence expression of rMV-Hu 191-N-EGFP and rMV-Hu191-P-EGFP was much higher than rMV-Hu191-H-EGFP.Vero cells in 96-well paltes infected with three recombinant strains and tested the fluorescence intensity with microplate reader,the results were identical with the finding with fluorescence microscopy.5.Stability of EGFP expression was confirmed after 10 passages in Vero cells by immunofluorescence examination and sequencing.There are any mutations in recombinant viral strains compare with parental strain after 10 passages.CONCLUSIONS:1.rMV-Hu191 is able to be engineered as virus vectors expressing gene sequence of green fluorescent protein.2.The replication kinetics of three recombinant strains expressing EGFP gene are various compared with each other and parental strain.3.The EGFP expression level of rMVs-Hu191-EGFPs are different.4.The replication capacity of rMV-Hu191-P-EGFP with highest expression level of foreign gene exhibited no weakness compared with parental srain.5.The recombinant DNA technology and reverse genetic strategy applied in constructing rMVs-Hu191-EGFP are suitable for other paramyxovirus.Part Ⅱ Oncolytic Effect and Mechanisms of Recombinant Measles Virus of Hu191 Expressing Fluorescent Protein against Non-small Lung CancerBACKGROUND and OBJECTIVESLung cancer is one of the most common malignancies in adults,with high motality.Although it is rare in children,the children diagnosed with lung adenocarcinoma have poor prognosis.Seeking for effective therapy strategy is still the key point in future medical and life science research.Oncolytic measles viruse(MV)exhibited significant antitumoral potential.In previous study,attenuated live virus vaccine of Hu191 showed oncolytic effect on non-small lung cancer(NSCLC)with the risk of reverse mutations.We found that recombinant MV-Hu191 with green fluorescent protein gene insertion between phosphoprotein and membrane protein(rMV-Hu191-P-EGFP)could express foreign genes efficiently and stablely,which overcomed the problem of reverse mutations and could be used to observe viral replication and spread.The objective of the current study was to explore the oncolytic effect and mechanisms of rMV-Hu191-P-EGFP with fluorescent protein gene insertion,on NSCLC and test its therapeutic effect and mechanisms against lung cancer by rMV-Hu191.METHODS:The expression level of CD46 and Nectin-4 was tested by flow cytometry.The fluorescence microscopy was used to observe infectivity of rMVs-Hu191-EGFP to NSCLC(H460 is large cell lung cancer cell,and H1975 is lung adenocarcinoma cell).The CCK-8 kit was used to evaluate the cell viability after infection with rMV-Hu191-P-EGFP.Cytopathic effect on tumor cells,and cell viability assay were utilized to illustrate the antitumor effects in vitro.Viral replication kinetics were tested by growth curve.Annexin V-FITC staining analysis,Tunel assay and Western blot analysis were used to test cell apoptosis.Analysis of cell-surface calreticulin(CRT),heat shock protein 90(HSP90)exposure and passive release of ATP and high-mobility group protein 1(HMGB1)were used to evaluate immunogenic cell death.The mRNA levels of proinflammatory cytokines were detected by quantitative real-time PCR(RT-qPCR).In vivo assays were performed to evaluated the antitumor ability of rMV-Hu191-P-EGFP.The fluorescence microscopy and frozen tissue section were used to observe the expression of rMV-Hu191-P-EGFP in normal and tumoral tissues.RT-qPCR was applied to test the replication of rMV-Hu191-P-EGFP in normal and tumoral tissues.Western blot was used to test expression level of activated Caspase 3 in tumor tissue.RESULTS:1.The expression level of CD46 and Nectin-4 was confirmed by flow cytometry.2.rMV-Hu191-P-EGFP infected NSCLC H460 and H1975 at MOI=0.1 respectively.We can observe the green fluorescence syncytia were formed in infected tumor cell,and rMV-Hu191-P-EGFP show highest fluorescence intensity in both tumor cells,in concert with the results in Vero cells.3.NSCLCs H460 and H11975 were infected with rMV-Hu191-P-EGFP at MOI=0.1.We tested the viral titers at different time points and the growth curve demonstrated that rMV-Hu191-P-EGFP could efficiently infect and replicate in non-small lung cancer cell lines.While rMV-Hu191-P-EGFP arrived at peak viral titer delayed after 60 hours post infection(hpi)compared with parental strain rMV-Hu191,high level of viral titer last for a long time.4.NSCLC cells were infected with rMV-Hu191-P-EGFP at different MOI.At 24,48,72,and 96 hours post infection,we test the cell viability of unfected tumor cells.The results showed antitumor effect in a MOI and time-dependent manner both on tumor cells H460 and H1975.The cell viability of infected H460 and H1975 cells with rMV-Hu191-P-EGFP after 144 hours were compared with rMV-Hu191 infected group.The oncolytic effect of rMV-Hu191-P-EGFP to H460 is better than rMV-Hu191,while this advantage in oncolyitc effect is not obvious.Even at MOI=0.1,rMV-Hu191-P-EGFP also could inducing significant cytopathic effects and formed plaques in cell plates stained by crystal violet.5.NSCLCs H460 and H11975 were infected with rMV-Hu191-P-EGFP at MOI=0.1 We tested the viral titers at different time points and the growth curve demonstrated that rMV-Hu191-P-EGFP could efficiently infect and replicate in non-small lung cancer cell lines.While rMV-Hu191-P-EGFP arrived at peak viral titer delayed after 60 hours post infection(hpi)compared with parental strain rMV-Hu191,high level of viral titer last for a long time.6.Both rMV-Hu191-P-EGFP and rMV-Hu191 could induce apoptosis of NSCLCs H460 and H1975 cells.The percentages of apoptotic H460 cells were 89.8 and 80.6 respectively.The percentages of apoptotic H1975 cells were 36.33 and 41.39 respectively.NSCLCs H460 and H1975 cells were infected with rMV-Hu191-P-EGFP at different MOI of 0.1(low dose),0.5 and 1(high dose).After 72 hours post infection,infected cells were collected for fluorescence-activated cell sorting(FACS)analysis after staining with Annexin V and propidium iodide to detect apoptosis.Apoptosis was induced followed infection with rMV-Hu191-P-EGFP at a MOI-dependent manner.The percentage of apoptotic H460 cells at MOI=0.1,0.5 and 1 were 17.9%、36.6%and 38.1%,respectively.The percentage of apoptotic H1975 at MOI=0.1,0.5 and I were 16.3%、31.3%and 34.9%.Subsequently,apoptosis pathways were identified by Western blot,detecting expression of the key proteins caspase-3 and PARP,a substrate for caspase activity.The expression levels of cleaved PARP,cleaved caspase 3 increased at MOI-dependent manner.Total DNA fragmentation assay was realized by TUNEL assay.The red fluorescence in infected H460 and H1975 cell with MOI=1 was stronger than MOI=0.1,indicated a ladder-like pattern of DNA fragments consistent with internucleosomal fragmentation in apoptotic death.And this result proved the apoptosis degree is dependent on dose of rMV-Hu191-P-EGFP.7.Damage associated molecular patterns(DAMPs)released by rMV-Hu 191-P-EGFP-infeted cells were tested.We found both rMV-Hu191-P-EGFP and rMV-Hu191 could increase CRT expression in H460 and H1975 cells surface.Two NSCLC cell lines were also inoculated with rMV-Hu191-P-EGFP at different MOIs.After 48hpi,the cells were collected for detecting CRT expression by flow cytometry.The surface CRT expression increased in infected H460 and H1975 cells more than in uninfected control groups.H460 and H1975 cells were inoculated with rMV-Hu191-P-EGFP at different MOIs.The amount of ATP and HMGB1 in the supernatants then were determined at various time points.There were low levels of extracellular ATP after 24h infection in both cell lines,but the amount of extracellular ATP released by H460 and H1975 cells rose rapidly,reaching a peak at 48hpi in infected-cells.The levels of HMGB1 were much higher in each group of infected H460 and H1975 in a MOI-dependent manner,with a significant difference between the groups.rMV-Hu 191-P-EGFP induced increased expression of heat shock protein(HSP90),which was tested by immunofluorescence.8.We collected total RNA of rMV-Hu191-P-EGFP-infected(rMV-Hu191 as control)tumor cell lines of H460 and H1975 with MOI of 0.5 after 36 hours post infection.The results of RT-qPCR demonstrated that the mRNA levels of proinflammatory cytokines interleukin-1β(IL-1β),interferon-γ(IFN-γ),tumor necrosis factor-α(TNF-α),and interleukin-6(IL-6)in tumor cells increased during oncolytic rMV-Hu191-P-EGFP infection.9.To evaluate the anti tumoral effects of rMV-Hu191-P-EGFP in vivo,H460 cells(5×106 cells/mouse)were implanted in the right flanks of nude mice,then mice were treated with intratumoral injections of rMV-Hu191-P-EGFP or supernatants of infected H460 cells every day for 6 days.rMV-Hu191-P-EGFP had significantly inhibited tumor growth compared with the Opti-MEM control group or uninfected supernatants.The difference in tumor volumes was statistically significant(P<0.0001)between mock and rMV-Hu191-P-EGFP or infected supernatants treatment.Treatment with rMV-Hu 191-P-EGFP obviously prolonged the survival time of tumor-bearing nude mice.The median survival time in rMV-Hu191-P-EGFP-infected group(26 days)was conspicuously longer compared to the median survival time(18 days)of Opti-MEM-treated group.The EGFP expression was observed in tumor tissue and there was no virus existing in normal tissue of nude mice in infected-group.Furthermore,rMV-Hu 191-P-EGFP could also induce apoptosis in tumor tissues extracted from tumor-bearing nude mice.apoptosis in tumor tissues extracted from tumor-bearing nude mice.CONCLUSIONS:1.CD46 and Nectin-4 were expressed in cell surface of NSCLCs H460 and H1975.2.rMVs-Hu191 with insertions could efficiently express exogenous genes in cancer cells.3.The oncolytic effect of rMV-Hu191-P-EGFP is in a MOI and time-dependent manner.4.rMV-Hu191-P-EGFP could efficiently infect and replicate in non-small lung cancer cell lines,inducing significant cytopathic effects.5.rMV-Hu191-P-EGFP induced cell apoptosis,immunogenic cell death and proimmflatory cytokines relesase in NSCLCs H460 and H1975.6.Intratumoral injection of rMV-Hu191-P-EGFP resulted in significant regression of tumors in a H460 xenograft model.Treatment with rMV-Hu191-P-EGFP could prolonged the survival time and the median survival time was 26 day,compared to the median survival time(17 days)of Opti-MEM group.7.rMV-Hu191-P-EGFP could stably express EGFP and induce apoptosis in tumor tissues extracted from tumor-bearing nude mice.There was no virus existing in normal tissue of nude mice in infected-group.