Development Of Human Monoclonal Antibodies Against Dengue Virus

Dengue virus?DENV?is an important mosquito-borne infectious virus.It spreads in the crowd by the bite of Aedes aegypti and Aedes albopictus.Dengue virus infection usually causes varying degrees of clinical symptoms ranging from moderate dengue fever?DF?to severe dengue hemorrhagic fever?DHF?and dengue shock syndrome?DSS?.There are 2.5 billion people from 128 countries who are at the risk of dengue virus infection.About 500,000 people need to be hospitalized every year because of dengue virus infection,and the mortality rate is about 2.5%.Tropical and sub-tropical regions are the major epidemic areas of dengue virus infection.Guangdong,Hainan,and Yunnan province in China all belong to the dengue area where dengue infections occur almost every year.The outbreak of dengue fever in Guangdong Province in 2014caused 46,864 infections and 6 deaths,resulting in serious public safety problems.There is no specific drugs defending against or treating dengue virus infection at present except that a live-attenuated tetravalent vaccine,Dengivaxia,developed by Sanofi-Pasteur has been approved for use in three countries including Mexico,Philippines and Brazil since the end of 2015.Unfortunately,the protection of Dengvaxia against DENV-2 infection was unsatisfactory and serious consequences might occur when Dengvaxia was used in DENV-negative individuals.At the end of2017,the Philippine health authorities had asked to stop the vaccination.Therefore,there is a desperate need to develop more effective anti-DENV infection measures.Therapeutic treatment with antibodies is now emerging as a effective treatment method against viral infections.It is shown that anti-dengue virus neutralizing antibodies can neutralize dengue virus in mice quickly and effectively and inhibit virus proliferation.Therefore,neutralizing antibodies should be an ideal and effective treatment strategy.The preparation of antibody basing on the single cell PCR is a fully human antibody preparation technique developed in recent years.The basic principle is to collect peripheral blood of patients or vaccine-immune population and isolate single B cell.RT-PCR and nested PCR were performed to amplify the genes of antibody from single B cell.Fully human antibodies which were affinity maturation in vivo can be quickly obtained by this technology.In order to obtain anti-dengue virus antibodies rapidly,antibody-secreting cells from the peripheral blood obtained from dengue fever patients during the recovery period were used to amplify the antibody genes and the development of neutralizing antibodies against dengue virus were carried out in this study.We collected 30 m L of peripheral blood from patients infected with DENV,and PBMCs were separated from the blood by Ficoll density gradient separation.As DENV-specific antibodies were generated from activated B cells.The PBMCs obtained were stained with anti-CD3,anti-CD19,anti-CD20,anti-CD27 and anti-CD38 antibodies.Following this,the activated antibody-secreting cells were classified as CD19^high,CD3^negative,CD20^{low to negative},CD27^high and CD38^high.A total of 1056 B cells were sorted and 496 H-chains,707?-chains,and 222?-chain variable region gene fragments were obtained by RT-PCR and nested PCR from the cells.Finally,a total of 285 gene pairs were obtained,accounting for about 27%of the total,of which 223 pairs genes for H and?,62 paired genes for H and?.261 of the antibodies above were successfully expressed through transfecting FreeStyle^TM293-F cells with equal parts of plasmids encoding corresponding heavy and light chains.The specificity of the expressed antibodies was tested by ELISA using an equal mixture of inactivated dengue viruses of four serotypes as antigens.The results showed that 88 antibodies accounting for 33.7%could specifically bind inactivated dengue virus.Further neutralizing experiment revealed that 24 of the 88 antibodies could neutralize more than 50%of DENV-1 at a concentration of 10?g/mL,of which1G5,3A6,6B1 and 9C7 behaved best,with 100%neutralizing activity for 1G5 and 9C7and 95%for 3A6 and 6B1.Using inactivated viruses as antigen,the affinity of the four antibodies with the highest neutralizing activity was evaluated by ELISA.The results showed that 1G5 has a high affinity for DENV-1,3A6,6B1,and 9C7 could bind to four serotypes of dengue virus,but the affinity of 9C7 to DENV-4 was weak and the affinity of 6B1 to DENV-3and DENV-4 was also weak.The protein antigens of Dengue viruses recognized by the four antibodies were identified by Western blotting.According to which 1G5 and 9C7recognized the E protein of dengue virus but not binding to the recombinant E protein expressed by E.coli.It was speculated that the epitopes recognized by the two antibodies may be conformational epitope or epitopes with modifications such as glycosylation.6B1 can recognize not only the E protein displayed on the virion but also the recombinant E protein,suggesting that 6B1 binding site may be a linear epitope on the E protein.3A6 recognized the prM protein.Evaluation of the in vitro neutralizing activity of these antibodies revealed that 1G5 could only effectively neutralize DENV-1,9C7,3A6 and 6B1 could neutralize four serotypes of dengue virus,while 3A6 and 6B1had much weaker effect.9C7 could effectively protect mice from charging with a lethal dose of all four serotype dengue virus before or after administration of antibody.But1G5 only had effect on DENV-1.The antibody dependent enhancement?ADE?induced by dengue virus antibodies is an important factor restricting its clinical application.Since it was shown that 1G5and 9C7 can increase dengue virus infection at a certain sub-neutral concentrations in K562 cells expressing Fc?R through ADE.Leu to Ala mutations?positions 234 and 235?or deletion of 9 amino acids?positions 231-239?at the Fc domain of 1G5 and 9C7 was introduced to eliminate binding to Fc?R.ADE was completely abrogated for1G5-LALA and 1G5-9 del.The mouse model with knockout of type I and type II interferon receptor genes(Ifnar1^-/-Ifngr1^-/-)is the preferred model animal for dengue virus infection and the assessment of antiviral activity of antibody in vivo.We successfully constructed this immune-deficient mouse model using CRISPR-Cas9.It is of great significance for the further development of anti-dengue virus neutralizing antibodies.In summary,we established the method to generate antibody from single B cell.The method made it is possible to prepare prophylactic and therapeutic antibodies from the peripheral blood of infected patients or immunized population rapidly,which is effective for developing neutralizing antibodies.The two neutralizing antibodies 1G5and 9C7 will be promising drug candidates against dengue virus infection after further optimization.