Development And Immune Effect Evaluation Of Three Genetic Engineering Vaccines Against Grass Carp Reovirus

Grass carp（Ctenopharyngodon idella）is one of the most important aquaculture species,and the grass carp hemorrhagic disease,caused by grass carp reovirus（GCRV）,is one of the most severe infectious diseases in aquaculture.GCRV is a member of Aquareovirus C type in the family Reoviridae that was first found in aquatic animal in China in 1983.A comparative study of sequences revealed that GCRV could be divided into three distinct subtypes:Type I,Type II and Type III.Among these types,hemorrhagic disease caused by GCRV of type II is prevalent in many provinces and is the most serious infectious disease that causes gravely death of fingerlings during rearing.However,given that vaccines of GCRV type II are currently limitedly available,the existing vaccines are unable to meet the needs of local aquaculture.Therefore,a novel and effective antigen with low cost and high security that used for vaccines needs to be developed,which is important for application in aquaculture.In this study,a strain of GCRV（GCRV-HN14）was recently isolated from local diseased fish in our laboratory,which was identified as belonging to the type II GCRV.The S11 and S7 segment in GCRV type II could encode protein VP35 and VP56,respectively.VP35 protein contained a conserved putative zinc-binding motif CxxC-n16-Hx C sequence,which also presented in the VP7 protein and MRVσ3.Therefore,VP35 was predicted to be an outer-clamp protein.Meanwhile,VP56 protein was characterized by its structure-based fiber motif,and thus VP56 protein was an outer-fiber protein,which was similar to fiber protein in adenovirus and had ability to neutralize the virus.It was speculated that VP35 and VP56had good immunogenicity.Based on the study above,we cloned the ORF of segment S11 into the eukaryotic expression vector pcDNA3.1（+）to evaluate its potency as a DNA vaccine.And the recombinant plasmids pET32a-VP35 and pET32a-VP56 were constructed to express recombinant VP35 and VP56proteins in prokaryotic cells,which were used to create subunit vaccines.To verify the protective effect of these vaccines against GCRV infection,a series of experiments were performed.1.DNA vaccine based on the S11 segment of GCRV provides immune protectionIn the present study,the open reading frame of S11 segment was cloned and transferred into the eukaryotic expression vector pcDNA3.1（+）to construct the recombinant plasmid pcDNA3.1-s11,which was then transfected into CIK cells.The VP35 protein was observed to be successfully expressed in the transfected CIK cells.The plasmid pcDNA3.1-s11（10μg）was intramuscularly injected into grass carp for immunity.（1）The in vivo distribution and expression of the pcDNA3.1-s11 plasmids were explored by the PCR and Western blot.Recombinant plasmids were detected in the blood,muscle,spleen,kidney and liver;however,protein expression could only be detected in the muscle.（2）The number of red blood cells（RBCs）has no significant difference between the two groups（p>0.05）.Compared with the control,the population of white blood cells（WBCs）significantly increased at1,7,and 14 days post-immunization（dpi）（p<0.01 or p<0.05）;The percentage of neutrophils in the immunized group was significantly increased and peaked at 7 dpi[（24.13±2.38）%;p<0.01],whereas the lymphocytes increased significantly and attained a peak with（93.30±4.71）%at 14 dpi（p<0.05）.（3）Serum antibody titer was detected using ELISA assay,and results showed the antibody titer of pcDNA3.1-s11 increased significantly at 7 dpi（p<0.05）,and antibody of anti-vp35 protein was produced in the immuned grass carp.At day 28 after immunization,the pcDNA3.1-s11 demonstrated the highest antibody titer with OD_450nm of 0.79±0.06（p<0.01）.（4）The mRNA expression levels of immune-related genes IFN1,TLR22,MHC I,and IgM were detected in the head kidney and spleen through qPCR.The relative expression levels of these genes were initially up-regulated and then returned to a normal level in the immunized group.The mRNA expression of IFN1 in the head kidney began to rise significantly at 7 dpi and reached a peak at 14 dpi,being about 12fold higher than the control（p<0.01）,while in the spleen,it began to rise significantly at 14 dpi and attained a peak at 21 dpi,being about 18 fold higher than the control（p<0.01）.The relative expression levels of TLR22 in the head kidney and spleen were considerably up-regulated on the first day following immunization（p<0.01）,and reached a peak at 14 dpi,being about 6 and 125 fold higher,respectively,than the control（p<0.01）.The expression of MHC I in the head kidney and spleen increased significantly（p<0.01）at 1 dpi,and rose continuously to attain a peak at 21 dpi（p<0.01）.The relative mRNA expression levels of IgM in the spleen and kidney in the immunized group increased at 7 dpi（p<0.01）,and attained a peak in the head kidney at 35 days（being about 12 fold,p<0.05）,and reached the highest in the spleen at28 days（being about 18 fold,p<0.01）.（5）To evaluate the protection efficacy of the DNA vaccine,grass carp was immunized with the pcDNA3.1-s11,following challenged by GCRV infection at 21 dpi.The pcDNA3.1-s11 vaccine enhanced the survival rate in the vaccinated fish with a survival rate 70.4⁷3.3%at day 14 post-infection,compared with 5%¹3%in the pcDNA3.1 or PBS control groups.Thus,the relative survival percentage was approximately 70%.2.Subunit vaccine based on grass carp reovirus VP35 protein provides protective immunity against grass carp hemorrhagic diseaseIn this study,the recombinant plasmid pET32a-VP35 was constructed to express recombinant VP35proteins in prokaryotic cells,which was prepared for a novel subunit vaccine.SDS-PAGE showed that the target protein was mainly presented in the inclusion bodies of the induced E.coli and the highest protein expression level was observed at 1 mM IPTG for 6 h after induction.Then recombinant proteins were purified through affinity chromatography in a nickel–iminodiacetic acid（Ni-IDA）column.The rVP35（30μg）was intramuscularly injected into grass carp for immunity.The immune protection of recombinant VP35 protein was estimated through a series of experiments in grass carp.（1）Results in change of blood cell number and differential leukocyte counts showed that the number of RBCs remained at a stable level（2.03±0.07）×10⁹/mL in the immunized fish.The number of WBCs increased to peak with（7.92±0.72）×10⁷/mL at 5 dpi（p<0.05）.The percentage of neutrophils and monocytes in WBCs were significantly higher at 3 dpi,than those of the control（p<0.05）,and reached（12.22±1.28）%and（18.70±1.78）%,respectively.The percentage of lymphocytes increased significantly to（92.37±2.10）%at 14 dpi（p<0.01）,whereas the percentages of neutrophils and monocytes declined significantly（p<0.01）,compared with the control.（2）Results in serum antibody titer of the immunized fish showed that antibodies of anti-vp35 protein were produced at 14 dpi.Furthermore,antibody titer reached a peak with OD_450nm of 0.65±0.07 at 21 dpi（p<0.01）and was higher than the control until 5 weeks post vaccination.（3）The results of the mRNA expression levels of IFN1,TLR22,MHC I and IgM showed that these genes had larger increases in the head kidney and spleen of grass carp compared with the control（p<0.05 or p<0.01）.The expression levels of IFN1 in the head kidney and spleen started to increase significantly at 5dpi and reached their peak values at 14 and 7 dpi（p<0.01）,respectively,being about 30 fold to 40 fold higher than the control.The relative expression levels of TLR22 were reached their peak values at 3 dpi（p<0.01）,being about 10 fold and 4 fold in the spleen and head kidney.The expression levels of the MHC I gene started to increase on the first dpi and reached their peak values（8 fold to 10 fold）at 5 dpi in the head kidney and spleen（p<0.01）.The relative mRNA expression levels of IgM in both organs of immunized fish started to increase at 14 dpi and 5 dpi（p<0.05 or p<0.01）,respectively,reached its peak with the 30 fold of control in the head kidney at 21 dpi（p<0.01）,and was 20 fold to 40 fold of control in the spleen at 28 dpi（p<0.01）.（4）Grass carp immunized with recombinant VP35 protein showed that the survival percent in the immunized group was 66.7%at 15 dpi and was significantly higher than that of the control（16.7%）（p<0.01）.Thus,the the relative percentage survival was about 60%after it was challenged with GCRV.3.Subunit vaccine based on grass carp reovirus VP56 protein provides immune protectionIn this study,the recombinant plasmid pET32a-VP56 was constructed to express recombinant VP56proteins in prokaryotic cells,and then r VP56 was purified and used for subunit vaccine.Subsequently,each fish was injected intraperitoneally with 30μg rVP56.The protective immunity of recombinant VP56protein was assessed by a series of experiments in grass carp.（1）Changes in the peripheral blood cells indicated that the number of RBCs started to increase significantly at 5 dpi and reached a peak at 7 dpi[（2.98±0.17）×10⁹/mL,p<0.05]in the immunized group.And the WBCs numbers in immunizied fish were significantly higher than those of the control at 5,7 and14 dpi and reached a peak[（8.42±1.01）×10⁷/mL,p<0.05]at 7 dpi.Additionally,the differential leukocyte count of monocytes and neutrophils attained a peak at 5 dpi,（9.05±0.92）%and（25.93±2.60）%respectively,than the control（p<0.05 or p<0.01）.The percentage of lymphocytes reached the highest in the immunized group at 14 dpi[（85.81±2.73）%;p<0.01].（2）Serum antibody titer was detected via ELISA assay,and results showed the antibody titer of rVP56 in the immunized fish increased with the extension of the immunized time.The antibody titer increased significantly and reached a peak with OD_450nm50nm of 0.34±0.01 at 21 dpi（p<0.01）.（3）According to the mRNA expression levels of immune-related genes detected by qPCR,the relative mRNA expression levels of IFN1 and MHC I reached their peak values at 5 dpi in head kidney and spleen（p<0.05 or p<0.01）.The relative mRNA expression levels of TLR22 in both tissues of immunized fish peaked at 5 dpi（p<0.05）and 3 dpi（p<0.01）,respectively.The relative mRNA expression levels of IgM in head kidney and spleen attained a peak at 21 dpi（p<0.05 or p<0.01）.（4）A challenge test was conducted at 21 dpi,which showed the rVP56 vaccine increased the survival rate,and the relative survival percentage was 71⁷5%.In conclusion,three genetic engineering vaccines were created based on a strain of GCRV（GCRV-HN14）isolated from grass carp in local farm through genetic engineering methods in this study.The different immune indexes were adopted to evaluate the protective effects of these vaccines in grass carp,which showed that three vaccines had the better immune protection,and the RPS of vaccines were60%⁷5%.These results indicated that three genetic engineering vaccines would be applied in aquaculture of grass carp to prevent from the disease outbreak in the future.However,the optimazation of vaccine,such as the preparation method,immune dose,and vaccination way,need to be further investigated and to improve the immune protective effect.