Molecular Detection Of The Tick-borne Pathogens And The Study Of SFTSV Neutralizing Antibodies

BackgroundTicks are obligatory blood-sucking arthropods that are ectoparasite on the surface of animals,and are important transmission vectors of zoonotic diseases.Ticks and Tick-borne diseases(TBDs)are of great medical significance worldwide.Ticks transmit pathogens through blood-sucking and cause major harm to the health of humans.Tick-borne diseases have been reported around the world,and the number of new human TBDs and the number of reported cases has been increasing.For example,severe fever with thrombocytopenia syndrome(SFTS)is a new infectious disease found in China in recent years and Haemaphysalis longicornis is its major transmission vector.China’s geography is very complex,and the variety of landforms makes the ticks and tick-borne diseases diverse.As more and more tick borne pathogens affecting human health are discovered,the surveillance of tick-borne diseases has become increasingly important in China.SFTS was first discovered in China in 2010.The pathogen is SFTSV,which belongs to the Bunyaviridae family.In addition to China,the virus is also widely distributed in South Korea and Japan.SFTSV is a segmented RNA virus that is divided into three fragments: L,M,and S.The clinical symptoms of SFTS patients are not specific,mainly including fever,thrombocytopenia,gastrointestinal symptoms,and leukopenia.Very often severe clinical symptoms also occur,which progress rapidly and can develop into multiple organ dysfunction syndrome(MODS).The case fatality rate of SFTS is over 20%.At present,the pathogenic mechanism of the virus is unclear.The M fragment of the virus encodes two glycoproteins Gn and Gc.Glycoproteins play an important role in the process of virus invasion and release outside the cell.According to research on other bunyavirus,glycoproteins Gn and Gc are the two main antigenic components on the surface of the virus,and are the target of specific neutralizing antibodies.Previously only two monoclonal antibodies with neutralizing activity against Gn protein antigenic epitopes have been reported,and it is unclear whether Gc proteins have neutralizing antigenic epitopes.To date,there are no specific vaccines or therapeutics against SFTSV.Studies have found that SFTS patients produced neutralizing antibodies against SFTSV and the neutralizing antibodies can be maintained for a long period of time and provide protection.Therefore,humanized neutralizing antibodies can be used for emergency prevention and treatment of susceptible people and critically ill patients.ObjectiveThe aims of our study are to detect the bacterial and viral pathogens carried by ticks collected in Jiaonan City,Shandong Province to understand the epidemic situation of tick-borne diseases in China and to provide a basis for the prevention and control of new zoonotic diseases;to determine whether neutralizing epitopes present on glycoprotein Gc to provides guidance for subsequent antibody development;to construct humanized monoclonal antibodies and determined their neutralization activity against SFTSV to provide a basis for the development of vaccines and the treatment of SFTS.Methods1.Tick sample collectionTicks were collected on the grass by flagging and were classified morphologically and by PCR amplification and sequencing the mitochondrial 16 S rRNA gene of the ticks.2.Tick-borne pathogen detectionTick total nucleic acids were extracted,and the bacterial pathogens of rickettsioses,tularaemia,Lyme disease,and Q fever were amplified by PCR,and the Crimea-Congo hemorrhagic fever virus gene was amplified by RT-PCR.The positive PCR products were cloned into the 19 T vector,and the positive clones were sequenced in both directions.The sequences obtained in this study were analyzed with the BLAST in NCBI to determine the species of the organisms.3.Gene sequence analysisThe DNAstar software was used to analyze DNA sequence homology.The phylogenetic trees were constructed using the Maximum Likelihood(ML)of the MEGA7.0 software with bootstrap value of 1000.4.SFTSV glycoprotein expression and purification and immunization of miceThe p GAGGS eukaryotic expression vector was used to clone the Gn and Gc genes of SFTSV and the recombinant proteins were expressed in HEK 293 T cells.The expressed protein was purified by gel chromatography column and identified by SDS-PAGE experiment.The purified protein was mixed with the adjuvant to immunize mice.5.Detection of antibodies in mouse serumGn and Gc proteins were coated on ELISA plates,respectively,and the titer of antibodies in mouse serum was detected by enzyme-linked immunosorbent assay(ELISA).Immunofluorescence assay(IFA)was used to test reaction of the serum antibody with SFTSV and micro-neutralization assay was used to determine neutralization activity of the mouse sera.6.Construction of humanized monoclonal antibodiesUsing phage display technology,three single-chain fragment variables(scFv)against Gn proteins were newly obtained.The variable region sequences of the scFv light chains were searched through the website(https://www.imgt.org/IMGT＿vquest/input)to determine the corresponding constant region and primers were designed to build a complete humanized monoclonal antibody by overlapping PCR.The light and heavy chains were cloned into pCAGGS eukaryotic expression vectors,respectively,and expressed in HEK 293 T cells.The expression of the monoclonal antibody was identified by Western blot,and the monoclonal antibody was purified by a gel chromatography column,and analyzed by SDS-PAGE experiment.7.Biological activity of monoclonal antibodiesThe binding activity of the expressed monoclonal antibody and Gn protein was determined by ELISA;the affinity of the monoclonal antibodies to SFTSV was anlyzed by IFA;the neutralization activity of the monoclonal antibodies was detected by micro-neutralization assay.Results1.Based on morphological and molecular biological identification,all 2,560 ticks we collected were Haemaphysalis longicornis.2.The ticks were grouped and analyzed for Rickettsia by PCR for rrs、gltA、ompB and ompA genes.17 groups were positive for Rickettsia,and the minimum infection rate of Rickettsia was 0.66%(17/2,560).Sequence homology analysis and phylogenetic analysis showed that 17 Rickettsia positive samples were divided into three different genotypes.Sample J244 has the highest homology and in the same branch with Candidatus Rickettsia longicornii.Therefore it belongs to Candidatus Rickettsia longicornii.Three samples,J84,J85,and J217,have the highest homology with Rickettsia canadensis,but phylogenetically they are not in the same clade.It is a new species of Rickettsia and named as Candidatus Rickettsia jiaonani;the remaining13 samples have the highest sequence homology and in the same branch with R.japonica,belonging to R.japonica.3.The 16 S rRNA gene and tpiA gene of Francisella were amplified by PCR.Two groups of ticks were positive.The minimum infection rate of Francisella was 0.08%(2/2560).The 16 S rRNA gene sequence homology of the two positive samples with Francisella-like endosymbiont(FLE)in Haemaphysalis flava tick was 99.8% and99.4%,respectively,and the homology of the tpiA gene was 100% and 99.3%between the 2 samples and FLE.Phylogenetically,both positive samples were on the same branch as the FLE of H.flava,and far from pathogenic Francisella branches,indicating that the two strains of Francisella detected in the tick samples belong to the FLEs.4.Lyme disease and Q fever pathogens and Crimea-Congo hemorrhagic fever virus were also analyzed by PCR and RT-PCR,respectively,but none of them was positive.5.The SFTSV glycoprotein Gn and Gc were cloned into pCAGGS eukaryotic expression vector and expressed in HEK 293 T cells.The purified recombinant Gn and Gc were used to immunize mice.Micro-neutralization assay showed that the mouse sera immunized with Gn and Gc proteins can effectively neutralize the virus at a dilution of 1: 4 to 1:64,indicating that the mouse sera have neutralizing antibodies.6.Three humanized monoclonal antibodies(4-6,1B2,1F6)were successfully constructed,expressed and purified in eukaryotic cells.Micro-neutralization assay showed that all three humanized monoclonal antibodies could effectively neutralize SFTSV when the monoclonal antibody concentration was at 100μg/ml and 50μg//ml.Conclusion1.We conducted molecular investigations on some of the infectious disease pathogens in 2,560 Haemaphysalis longicornis.We found that these ticks carried Rickettsia and Francisella endosymbiont.The innovation of this study was the discovery of the existence of R.japonica and a new species of Rickettsia in Jiaonan,Shandong Province,providing a basis for the prevention and control of rickettsial diseases.2.We expressed and purified the Gn and Gc proteins of SFTSV,and immunized the mice with them to obtain the corresponding immune serum.Micro-neutralization assay showed that the antibodies produced by these two proteins can neutralize SFTSV in cell culture.The innovation of this research is to prove that glycoprotein Gc also exists neutralizing epitopes,which will guide subsequent neutralization antibody development.3.We successfully constructed three new single-chain antibodies(scFvs)against Gn protein of SFTSV,expressed and purified them in eukaryotic systems.We have demonstrated that all three monoclonal antibodies have virus-neutralizing activity in cell culture,which will provide a way for further vaccine research.