Effect Of Amino Acid Mutation At Site333on Immunogenicity Of The Glycoprotein Of Rabies Virus SRV9Strain

Rabies, causing lethal encephalitis in animals and humans, is induced by rabiesvirus belonging to the Lyssavirus genus of the rhabdoviridae family. Among the fivestructural proteins of rabies virions, the transmembrane glycoprotein G wasdemonstrated as the exclusive component that resulted in the continuously generationof neutralizing antibodies by the host immune system and was comprehensivelyconsidered to be the efficient antigen of the rabies vaccines. In recent years, DNAvaccines and recombinant virus vectored vaccines become hotspot in vaccine studies.Moreover, in some countries and areas, rabies vaccines based on vectors includingpoxvirus, pseudorabies virus and adenovirus have already been used for animal rabiescontrol and prevention. The rabies virus glycoproteins used abroad for the preparationof genetically engineering vaccines are mainly sourced from ERA, SAD B19and PMstrains. Research on gene engineering rabies vaccines in China is relatively later thanthat in developed countries. Excitingly, in our laboratory, using the canine adenovirustype2(CAV-2) and pseudorabies virus Bartha-K61as vectors, we have successfullyconstructed a series of vaccines expressing the SRV9glycoprotein that wasdemonstrated to show evident immunogenicity. Recent research shows thatmodification of the glycoprotei ns in the gene engineering vaccines may alter itsimmunogenicity, which was illustrated by Bankovskiy’s work in2008when theyfound the alteration of the amino acid333of the glycoprotein could either enhance orattenuate its immunogenicity.In this study, the glycoprotein gene of SRV9was cloned by RT-PCR using thetemplate RNA isolated from the SRV9infected BHK-21cell cultures and wassubsequently inserted into the pUC19plasmid followed by the identification throughsequencing. The sequence responsible for the333Ser was altered into those encodingeither333Arg or333Glu by site-specific mutagenesis based on previously constructed recombinant plasmid. After identification, the genes of interest weretransferred into the shuttle vector plasmid pacAd5CMV K-NpA by a doubleendonuclease digestion with EcoRI and BamHI, forming recombinant plasmidsrespectively named as pacAd5-SRV9-G333S, pacAd5-SRV9-G333R andpacAd5-SRV9-G333E. These plasmids as well as the backbone plasmid pacAd59.2-100were subsequently linearized with PacI and transfected into the modifiedhuman embryo kidney cell line HEK293AD for package. After homologousrecombination of the plasmids transfected into the cells, the recombinant adenoviruseswere recovered and responsibly designated as rAd5-SRV9-G333S,rAd5-SRV9-G333R and rAd5-SRV9-G333E. Relatively, the recombinant virusstemming from homologous recombination of the plasmid pacAd5CMV K-NpA andpacAd59.2-100was designated as wt-rAd5employed as control. After4to9passages, the titers of all viruses could reach over107TCID50/ml.The recombinant viruses presented typical adenovirus shape when observed withelectron microscope. It was confirmed that the genes of interest were successfullyintegrated into the recombinant adenovirus according to the detection with PCR usingspecially designed primer and supernatant of the eighth generation virus cultures asthe templates. Besides, the target genes were demonstrated to be transcribed becausethe target sequences could be amplified from the total RNA isolated from the virusinfected cell cultures by RT-PCR using the oligo dT15as reverse transcription primerand to be translated due to the performance of direct fluorescence assay and westernblotting.The neutralization antibody titers of the sera respectively separated in14d and28d from the Kunming mice (n=20) immunized with recombinant adenovirus at106TCID50by way of bilateral gastrocnemius injection were determined byfluorescence antibody virus neutralization test. Mice presented rabies virusneutralization antibody productions on the14thday in spite of a significant individualdifference. The statistical analysis of the RV NA levels on the28thday showed that themedians of the three groups (rAd5-SRV9-G333S, rAd5-SRV9-G333R andrAd5-SRV9-G333E) were0.44IU/ml,1.32IU/ml and3.96IU/ml respectively with the seroconversion rates of50%,70%and85%responsibly and that the rank testresults of intergroup RVNA levels were0.017(rAd5-SRV9-G333S,rAd5-SRV9-G333E),0.239(rAd5-SRV9-G333S, rAd5-SRV9-G333R) and0.277(rAd5-SRV9-G333R, rAd5-SRV9-G333E) with the chi square test values of0.043,0.333and0.449. Moreover, the protection rates of the mice immunized withrAd5-SRV9-G333S, rAd5-SRV9-G333R and rAd5-SRV9-G333E were respectivelydetermined to be30%,40%and60%after a20-day continuous detection of the mice(n=10) inoculated with standard virulent strain CVS-24at50LD50.To sum up, in this study, recombinant replication-defective adenovirusesexpressing either SRV9-G or its mutant were constructed and identified bothmorphologically and molecular biologically. The estimation of the immunizationefficacy of the recombinant adenoviruses for the Kunming mice reflected theimmunogenicity rAd5-SRV9-G333R was weaker than that of rAd5-SRV9-G333E butstronger than that of rAd5-SRV9-G333S in spite of no statistical significance, whichstill reflected the immunogenicity variance between SRV9-G protein and itscorresponding mutants, providing a reference for screening more effective rabiesgenetically engineering vaccine candidate genes.