Preparation Of Monoclonal Antibodies Specific For West Nile Virus Nonstructural Protein 1 And Development Of Immunoassay In Early Diagnosis Of West Nile Virus Infection

West Nile virus (WNV), a member of the Japanese encephalitis serocomplex in the Flavivirus of the family Flaviviridae, is transmitted through bited of mosquito-bird-mosquito and causes a range of illnesses in humans including West Nile fever and West Nile encephalitis. WNV was first isolated in the West Nile province of Uganda in 1937 from the blood of a woman suffering from a mild febrile illness, then spread to be a global threat to human and animal health across the United States and Canada as well as in temperate regions such as Europe and Africa. Since 1999, WNV has spread rapidly throughout North America causing epidemics of WNV encephalitis, meningitis, and acute flaccid paralysis in naive populations. There were 43822 cases of WNV infection in the United States from 1999 to 2015, and 1184 deaths have been reported (http://www.cdc.gov/ncidod/dvbid/westnile/index.htm).In recent years, since West Nile virus has continued spreading across the world, there also many people died of West Nile encephalitis in Canada, Russia and Israel. Though it has no West Nile virus epidemic in China, there are at least 11 mosquito species which can transmiss West Nile virus in China . As the significant increase in international travel and trade, transportation convenience, West Nile virus spreads in China would be possiblely and conditioned. Liang provided evidence of WNV human infections comfirmed by IgM ELISA and seroconversion by 90% plaque reduction neutralization tests of paired serum samples obtained from a part of patients with febrile illness and viral encephalitis in Xin jiang, China in 2004 and 2011, which suggests that infections with WNV might be greatly underestimated or neglected in China. It should be very important to make prevention in advance and provide technical reserves for diagnosis in case of appearance of WNV in china.Nowadays, a specific therapy or vaccine has not been approved for WNV infection in humans, although passive transfer of specific antibodies and immunoglobulins has been used as a therapeutic attempt , therefore, an early, specific, accurate and early diagnosis of WNV infection might be more important and necessary, particularly in regions where multiple flaviviruses co-circulate. The current diagnostic methods for WNV infection include serological tests, viral isolation, virus nucleic acid and infected cell detection. Viral isolation is a gold standard for the diagnosis WNV infections, but this method must be carried out in a biosafety level 3 (BSL-3) facility as viable WN viruses are used in this assay, and could not provide diagnostic result rapidly. RT-PCR requires specialised laboratory equipment and experienced technicians and could result in a false negative because of the short duration of viraemia and low virus titres during human WNV infection . Serological tests, including detection of antibodies and virus protein, are considered to be specific and convenient, however, there are also problems to be resolved. The currently available commercial kits based on IgM antibody detection could be used to identify WNV infection. However, because of the 3～4 day window phase, the kits might be limited for early diagnosis as a result of the delay between initial infection and seroconversion. Cross-reactivity with other antiflavivirus antibodies, prior exposure to heterologous flavivirus or vaccinations could interfere with and limit the usefulness of serological diagnostic tests . Therefore, a method for the specific detection of viral antigens for early diagnosis of acute WNV infection may be necessary and notable.The two commercial immunochromatographic antigen test kits have recently become available for WNV detection in birds:the VecTest WNV antigen assay (Medical Analysis Systems, Inc., Camarillo, CA, USA) and the Rapid Analyte Measurement Platform (RAMP) WNV test (Response Biomedical Corp., Burnaby, BC, Canada). The sensitivities of these two assay systems are only 105.17 PFU/ml and 103.17 PFU/ml for viral culture supernatants or 94% and 65% for WNV-positive mosquito pools , suggesting that the kits are less useful for screening for WNV infection because of their low sensitivities . Based on the presence of NS1 (nonstructural protein 1, NS1) in the acute-phase sera of DENV and WNV-infected patients and hamsters, more attention has been focused on the development of antigen-capture assays for the detection of the NS1 protein of flavivirus. NS1 is a highly conserved glycoprotein that is secreted at high levels and can be detected in the serum of flavivirus-infected patients. The NS1 antigen-capture assay used in WNVinfected hamsters and mice appears to set up an early detection of infection even prior to the appearance of IgM . The detection limit of these two assays are more than 0.5 ng/ml, and the assays appear not to be sensitive enough to enable detection at 3 days after infection, implying that these assays might not be useful for early diagnosis in clinical applications. In addition, the NS1 capture ELISA reported by Macdonald is unable to distinguish between WNV and other flavivirus infections because the antibody used in this assay is directed against cross-reactive antigenic determinants shared by WNV and other flaviviruses. Saxena et al. used a WNV-NS1 polyclonal antibody as the capture antibody for the NS1 capture ELISA, which can distinguish between WNV and other flavivirus infections, but the inherent batch-to-batch variation ensures that this assay is not suitable for standardisation . In the present study, our aim is to develop a rapid, accurate early diagnostic assay that should be easy to perform, and can be carried out in less-well-equipped laboratory settings, and can be used to study the function of NS1 protein and anti-NS1 antibodies in the pathogenesis of WNV-infection.There are four parts in our work as follows.Ⅰ. Optimization of the expression and refolding conditions and antigenic characterization of recombinant WNV nonstructural protein 1The full-length NS1 gene of WNV (NY-99) was cloned into the prokaryotic expression vector pGEX-5X-3 in frame and upstream of the GST coding sequence as our previous study . By optimization forthe induction of protein expression, purification and refolding conditions, we obtained the high purify and soluble form of WNV-NS1 protein. To determine the antigenicity of the NS1 protein, anti-DENV1-NS1 antibodies and anti-GST mAb were used to test the reaction with the recombinant NS1 fusion proteins by ELISA and Western Blot, based on the cross-reaction among the Flavivirus. The immunoreactive protein bands of the GST-NS1 fusion protein were shown corresponding to their expected molecular mass was 66 kDa. These results indicated that the recombinant NS1 fusion protein with good antigenicity could react to specific antibodies of Flavivirus nonstructural protein 1. This NS1 protein could be used as an antigen marker for WNV-infection diagnosis after identification of distinguishing NS1 form WNV and Dengue virus.Ⅱ. Preparation and characterization of monoclonal antibodies against NSl protein of West Nile virusIn this study,10 Balb/c mice were immunizede by alternately injection into Intraperitoneal and subcutaneous with recombinant NSl protein emulsified with complete Freund’s adjuvant for the first injection and Freund’s incomplete Freund’s adjuvant for the following three booster injections. Then the splenocytes of the immunized mice were fused with myeloma cells to generate hybridoma cell lines that could secrete anti-WNV NS1 protein antibodies. Western Blot (WB) analysis, immnofluorescence assay (IFA) and enzyme-linked immunosorbent assay (ELISA) were applied to identify the specificity of antibodies. Ultimately,65 hybridoma cell lines steadily secreting antibodies against NS1 protein were obtained. Eight mAbs were of IgG2a isotype, nine were IgG2b, two were IgG3, and the other forty-six were all IgGl. Thirty-nine out of the sixty-five mAbs were specific to WNV without cross-reactivity with the four serotypes of dengue virus, Japanese encephalitis virus and yellow fever virus. Competitive inhibition assay showed that the thirty-nine WNV-NS1 specific mAbs recognized at least thirteen distinct antigen epitopes of NS1 and the fourteen cross-reactivity mAbs were recognized at least eight distinct antigen epitopes of WNV-NS1 protein. These results provide a potential value for early diagnosis and vaccine research of West Nile virus infection.Ⅲ. Development and application of the antigen capture ELISA based on mAbs against the NS1 protein of WNVTo select the best combination of capture and detection mAbs for a NS1 antigen-capture ELISA,39 mAbs specific to WNV-NS1 were paired according to their abilities to recognise distinct epitopes of NS1. Finally the mAb pair of immobilised M6 and HRP-M4 was demonstrated to be the most effective to detect WNV. The detection limit of the rWNV-NSl protein was approximately 15-30pg/ml, and the detection linear range is approximately 15.62-125ng/ml. The specificity and sensitivity of the WNV-NS1 capture ELISA was further assessed by testing for the presence of NS1 in the culture supernatants of WNV, four serotypes of DENV, JEV, YFV, and TBEV. Using this assay, the native NS1 was reliably detected starting from day 1 after the WNV infection in mice, with peak NS1 concentration at day 4, whereas the NS1 protein began to decline on day 6, this finding is consistent with the results of Chung[24]. The positive detection rates of WNV-NS1 increased from 85.7% at 1-4 days to 100% at 5-7 days post infection inthe WNV-infected mice serum samples, which is greater sensitivity than that of real-time RT-PCR(P= 0.035). Additionally, by this NS1 antigen-capture ELISA, there was no positive in 107 serum specimens with DENV1-NS1 antigen-positive that were comfirmed by an immunoassay developed by our team. Therefore, these results indicated that the NS1 antigen-capture assay is specific for the detection of WNV, and can be used for early diagnosis of WNV infection.Ⅳ. A primary study for cross reaction of anti-NSl mAbs with human plateletSince the great homogenesis of NS1 protein from WNV and Dengue virus (DENV), and DENV NS1 and anti-NS1 antibody were proved to be associated with reduced platelets in hemorrhage fever patients, and serious hemorrhage and platelets decrease in 2 WNV patients were reported this year, we would like to know whether the anti-WNV-NS1 monoclonal antibodies can react with human platelets. In this part, indirect ELISA, flow cytometry and Western blot assay were used to analyze the binding of 51 anti-NS1 mAbs to human platelet. The results showed that there were 16 mAbs bind with human platelet by indirect ELISA, and 19 mAbs combined with human platelet by flow cytometry. Among them,12 mAbs were simultaneously cross reactive with human platelet by indirect ELISA and flow cytometry. Four of the twelve mAbs showed positive response bands by western blot, speculating that these 8 mAbs only recognize conformational but not linear epitopes of human platelet proteins. This result may provide a potential and primary evidence for further studying the activities of WNV-NS1 protein and anti-NS1 antibodies.In conclusion, the WNV-NS1 protein with good antigenicity was successfully expressed, purified and refolded, and 39 mAbs specific to the WNV-NS1 and 26 mAbs cross-reactivity to the NS1 of other flavivirus were obtained. With these 39WNV-NS1 specific mAbs, a mAbs-based antigen-capture ELISA for detection NS1 protein was successfully established. It is specific to detect WNV-NS1 protein, without cross-reaction with the other closely related members of the flavivirus, such as four serotypes of DENY, YFV, JEV and TBEV, and also great sensitive for detectionof circulating NS1 protein from early infection with West Nile virus in mice.This characteristics will be helpful for rapid early diagnosis of West Nile virus infections. Additionally, Western blot analysis (WB), immnofluorescence assay (IFA) and enzyme-linked immunosorbent assay (ELISA) showed there were eight mAbs specific binding with human platelet. This may provide a potential and primary evidentce for further studying the activities of NS1 and anti-NS1 antibodies, which would be helpful to explore the pathogenesis of West Nile virus infection and vaccine design.