Preparation And Identification Of Mouse Monoclonal Antibodies To Dengue Virus Type 2

Dengue virus (DV) is a member of mosquito-borne flaviviruses and possesses a positive-stranded RNA genome. And DV has 4 distinct serotypes. All of them can result in classical dengue fever (DF) and/or dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Every year there are about 100 million DF patients and 500 000 DHF/DSS patients in the tropics and subtropics worldwide. Now WHO already put DHF/DSS, Hepatitis, Malaria and Tuberculosis into the list of the most four serious infectious disease of the whole world. However, the pathogenesis of DV infection is not clear, and there is not an efficient way to deal with the disease in the clinic therapy.There are the main three theories about the pathogenesis of DHF/DSS: (1) antibody dependent enhancement (ADE) infection hypothesis: DV specific IgG was thought to induce the immune disorder; (2) the factor of virus itself: the virulence or replication ability of virus is the main reason of the serious disease; (3)immuno-pathogenesis theory: the cross-reactive T cell results in the immuno-damage. Recently the high titer of virus, secondary infection with different serotype DV and DV2 were thought to be the three main factors to cause the DHF/DSS. In the past years, the vaccine development focuses on tetravalencea vaccine to defend four serologic types. Although the live attenuated tetravalencea vaccine had been regarded as the most potential vaccine, unfortunately there is no an available and safe vaccine up to now.Based on above-mentioned situation of DV infection and their clinic therapy, researchers think about passive immunity or therapy-antibodies once more. This technique was reported first in 1890's, and wildly used in 1930's. However it was forbidden in 1940's because of the limitation of antibody technology and background of immunology. With the development of the antibody technology including preparation, purification andhumanization, many researchers pay attention to the specific antibodies for passive immunity or the strategy of therapy-antibodies. The neutralization antibodies can defend DV infection that has been already verified in vitro and vivo tests in recent years. So to obtain the high quality antibodies is useful for investigation to both the pathogenesis of DV infection and vaccine development.In the present study, we prepare the mouse monoclonal antibodies (mAb) to dengue virus type 2 (DV2, Trl751) using the standard way and identify their characteristics. Several mAb were obtained and it will be helpful for further studying the pathogenesis of DV infection and development of DV vaccines as the immunological tools.RESULTS1. Obtaining three hybridoma cells which secrete the specific mAb to DV2In our experiment, the suckling Blab/c mouse brain homogenate which contains high titer of virus was used as the antigen. Blab/c mice were immunized for several times and the spleen cells were fused with Sp2/0 cells by PEG4000. Then the hybridoma was selected by HAT culture medium. The living hybridoma was cloned by dilution-culture, and positive clone was determined by ELISA.As a result, three hybridoma cells were found to secret DV2 specific mAb after five cycles of dilution-culture clone and ELISA to each hybridoma, and they were be named B152, I1 and I9.2. Specificity of the three mAb were testified by indirect immuno-fluorescence staininghi the experiment, ECV304 cells infected with DV2 or normal ECV304 cells were fixed using 4% paraformaldehyde and then immuno-fluorescence staining was performed as follows. Ascites mAb and goat-anti-mouse IgG-FITC were used as the first antibody and second antibody respectively. The cells were observed under fluorescence microscope. As results, virus antigen often concentrated at one side of cytoplasma just like the fire or meteor in infected cells. And sometimes the antigen of virus was seen around the nucleus. There is no staining in the normal ECV304 cells.Interestingly, special antigen liking the cell constituent was observed besides virusantigen when mAb B152 was used as the first antibody. The stained cell constituent is as linear and reticulate structure as actin in the cytoplasma, and can be found in normal ECV304 cells. So double staining was performed to further confirm special antigen using mAb B152 and the Phallodin-TRITC (special for actin). The comma-like actin which appears after DV infected usually was shown by mAb B152 and Phalloidin-TRITC together. So mAb B152 may recognize both the DV2 antigen and the antigen of actin cystoskeleton.3. PRNT verified three mAb all have the ability of the neutralization. Confluent VERO cells were grown on the 24-well plate. 9×105 PFU/ml of DV2 wasdiluted into about 90 PFU/0.1 ml. mAb groups and normal mouse sera group were prepared to the plaque reduction test, and every group were diluted into 1:10, 1:40, 1:100, 1:160 and 1:640. The admixture of 0.5 ml diluted DV2 and 0.5 ml diluted mAb (or diluted normal mouse sera) was incubated at 37 ℃ for 1 h, and then infected the cell in the 24-well plate. At days 7-8 post-infection, the cells were stained by crystal violet and virus titer was calculated. Results are as follows: (1) the average PFU of positive control is 90; The PFU of negative control is zero; (2) The PFU of mAb groups decreased at different level, and it showed mAb titer-dependent manner. The normal mouse sera can decrease the PFU only in the 1:10 dilution and can not decrease the PFU in the other dilution. That indicated that three mAb all have the ability of neutralization. 50 % plaque inhibition concentration (IC50) of the three mAb are: mAb Ii is about 1:160, mAb I9 is about 1:100 and mAb B152 is about 1:40.4. mAb all recognize the epitope of DV2 envelope protein.DV2 sample and C6/36 cells sample were used as detection group and control group respectively. All the samples were electrophoresed in a 10% polyacrylamide gel, and then the gelatum was transferred onto nitrocellulose membrane. The mAb was used as the first antibody, and mouse-anti-DV2 serum is the positive control. Goat-anti-mouse IgG-HRP is the second antibody. The results are: (1) There is no stain in two groups when did not add first antibody; (2) There are several stained bands in detection group and no staining in control group when mouse-anti-DV2 sera was used as the first antibody; (3) There is only one stained band in detection group and no stain in control group when mAb was used asthe first antibody. The location of three mAb stained band is the same, but the thickness of three band is different that show the different affinity of three mAb or the different titer of mAb. The molecular weight ratio (Mr) of mAb stained band is above 43 000 and under 66 200. So we confirm that is the envelop protein of DV2 because the Mr of E protein is 55 000~60 000. E protein is the most possible structure protein that can induce neutralized antibody. Taken together, we suggested that the three mAb all recognize the epitope of DV2 envelop protein and have the ability of neutralization.