Identification Of A Panel Of Murine Monoclonal Antibodies Against P1Protein Of Enterovirus71&Development Of A VP1-ELISA Assay For Rapid Detection Of Neutralizing Antibodies Against Enterovirus71

Hand-foot-and-mouth disease (HFMD) is a common children’s infectious disease characterized by fever and herpes manifesting in hand, foot and mouth. Most cases with HFMD are mild, but a few may suffer severe complications of encephalitis, acute cardiopulmonary failure, multi-organ failure, and even death. HFMD has become a threat to health of children in recent years since the outbreaks of large epidemics that had claimed considerable victims of paralysis or death in Asian-pacific region. It has been reported Enterovirus71(EV71) and Coxsackievirus A16(CoxA16) were the two dominant Enterovirus genotypes responsible for most infections of those HFMD epidemic.EV71succeefully draws most attentions due to its high affinity to central nervous that is associated with neurological complications (e.g. aseptic meningitis, brain stem encephalitis, neurogenic pulmonary edema, et al.) and systemic complication (e.g. multiple organ failure, shock, DIG, et al.) claiming death. EV71, a member of picornaviridae, is composed by RNA core and capsid composed by four proteins (VP1-VP4). Of the four proteins, VP1, VP2and VP3are those locate at outside surface for serving as the main neutralizing epitope of the virus, while the inner locating VP4play the role of maintaining the integrity of capsid. EV71is subgrouped into subtypes of2A, B1-B5and C1-C5basing on the conserved genes of VPl and (or) VP4. It is the C4subtype responsible for all HFMD epidemics in China mainland since1997.Nowadays, there is no effective drug for prevention and treatment to EV71infection. To develop the drugs and vaccine for prevent and treat of patients infectious with EV71is considered as important aim. Immunoglobulin infusion could alleviate the condition of patients in clinical trials. Nevertheless, there is risk of blood infectious diseases and the quality of commercial immunoglobulin in different batches may be inconsistent. Monoclonal antibody (mAb) with high neutralizing activity may avoid the problems mentioned above. And some antivirus monoclonal antibodies (mAbs) have been used to treat virus infectious diseases. As severe HFMD infectious with EV71will lead to sequela to patients, the development of vaccine of EV71is the best strategy to control the epidemic of EV71and reduce the incidence of disability and mortality. The ability of deducing neutralizing antibody is the important index to assess the efficiency of the vaccine. The method based on CPE neutralizing, the classic method in EV71research, which lack of power to provide objective result and timeconsuming forneutralizing process, is not suitable in large scale studies. As a result, it is of great need to find a convenient, fast, objective and high throughput screening method to study EV71.EV71(C4subgroup) and VP4recombination protein were used as antigen to generate mAbs in our previous work by hybridoma technique. To identify their targeted epitopes, recombined EV71VP1, VP2, VP3and VP4Protein were displayed in yeast surface. And classic neutralizing method was used to screen EV71 monoclonal antibody with neutralizing activity. In addition, we tried to develop a convenient, fast, objective and high throughput method of EV71neutralization test. Our work provides the experimental evidence for exploring the structure and function of capsid of EV71, revealing the pathogenic mechanism of EV71and vaccine preparation.There are three parts in our study as follows.Part Ⅰ Four viral capsid protein of EV71displayed on yeast surface.Yeast surface display technology has become a platform for functional protein engineering and got popular concern in recent years for its comparative advantage in protein post-translational modification, secretion, safety, etc.pYDlplasmid EBY100yeast display system were applied to display VP1, VP2, VP3and VP4capsid protein on the surface of EBY100Saccharomyces cerevisiae with the following procedures. We designed four pairs of primer according to the gene sequence of EV71(GZR); too constructed VP1-pYD1, VP2-pYD1, VP3-pYD1and VP4-pYD1recombinant plasmid recombination VP1, VP2, VP3and VP4gene fragments were amplified by PCR, cutted by double enzyme, and cloned into plasmid pYD1.Thenthe recombination plasmids were transformed into EBY100, and the positive yeast clones were culture in2%galactose culture medium, protein would expressed in the term of "Xpress-protein-6×His" fusion protein, on the yeast surface. Expression of target protein, were detected in series time with Xpress antibody or His antibody. Flow cytometry assay showed that expressions of target protein reached peaked at time-point of48hours after induction in about50-60%of yeast. Therefore, recombinant yeast clones were harvested at time-point of48h for identifying monoclonal antibody against EV71viral capsid protein in subsequent experiments. For the yeast display protein was similar to natural proteins, and the whole detection process does not make the protein denaturation, so this method can identify mAbs recognizing both the linear and conformational epitope.Part II Identification of a panel of Murine Monoclonal Antibodies against P1Protein of Enterovirus71Flow cytometry (FCM) combined with yeast display, immunofluorescence (IFA) and Western blot identification were jointly performed to pin down the exact antigens of EV71reacting with the20monoclonal antibodies against EV71that we prepared previously. The20cryopreservated hybridoma cell were revived and inoculated into the peritoneal cavity of mice to produce ascites containing monoclonal antibodies against EV71, then the acquired ascites were added caprylic acid ammonium sulfate to purify targeting antibodies. IFA assay showed that all the20mAbs bind to EV71infected RD cells, but not react with uninfected cells, suggesting that these Mabs were specific EV71antibody by Western blotting, five monoclonal antibodies (M-5, M-8, M-12, M-14, M-18) bind with recombinant VP1protein specifically, three monoclonal antibodies (M-3, M-10, M-13) bind with recombinant VP2protein, monoclonal antibody (M-1) binds with recombinant VP4protein specifically, indicate that these Mabs are recognizing linear epitope on corresponding proteins. By FCM, four Mabs (M-5, M-8, M-14, M-26) bind with VP1recombinant yeast, three (M-3, M-10, M-13) with VP2recombinant yeast, and only one (M-1) binds with recombinant VP4protein specifically, M-12binds both VP1and VP2recombinant yeast, while M18does not binds with either kinds of recombinant yeast. The results by FCM and immunoblotting against7strains of monoclonal antibody identification are consistent, but results of three mAbs are inconsistent.M-26reacted with VP1recombinant yeast specifically in FCM, but not with recombinant VP1protein in Western blotting, implying that the antibody may against natural VP1protein conformational epitope. M-18binds with purified VP1recombinant protein, not with the VP1recombinant displayed on yeast, implying that this antibody may recognize linear epitopes located in internal of the natural protein. Why M-12be capable of binding with the two recombinant proteins, remains to be studied further. We determined the neutralizing activity of mAb against EV71by using classic CPE neutralization test, three neutralization mAb were VP1antibodies. These two strains of monoclonal antibody of neutralizing activity, would be expected to be used as a therapeutic mAbs candidates.Part III Development of a VP1-ELISA Assay for Rapid Detection of Neutralizing Antibodies against Enterovirus71We established a new method of EV71neutralization test, namely EV71ELISA neutralization test (ELISA-MNT) with some adaptions of methods in previous reports. The basic principle of ELISA-MNT is based on VP1protein as a structural protein of EV71virus. In EV71infection, the level of VP1protein was positive correlation with EV71virus proliferation. So we would measure virus infected cells by direct ELISA using anti VP1monoclonal antibody. In this study, we developed a rapid ELISA-MNT, in which RD cells chose as EV71infected target cells, and the amount of virus and detection antibody concentration were optimized. The experimental steps are as follows:serial dilutions of serum or antibody incubated with equal amounts of1:1000diluted virus solution (3.16×104TCID50) for1hours, then the mixture was added to infection of RD cells for24h, infected cells were fixed with acetone and methanol and followed by detection by ELISA using VP1monoclonal antibody (AC17B6-HRP). Under the value A450of antibody or serum test holes less than0.5times of virus control hole’s A450value, and the highest dilution of antibody or serum as antibody or serum neutralization titer, neutralizing titer>10could be considered as neutralization antibody. We used classic CPE neutralization test as the gold standard to evaluate the performance of our ELISA-MNT method. By determining of the neutralizing titers of19serum of human and20EV71mAbs with the two methods, the results showed that the sensitivity of ELISA-MNT was92.9%, the specificity was100%. The results of3neutralizing monoclonal antibodies and10serum assay showed a good correlation within two methods for determination of neutralizing antibody titers (r=0.934, P<0.001), suggesting that the ELISA-MNT method can be applied for the determination of EV71neutralizing antibody. Additionally, ELISA-MNT can be finished within2days. In conclusion, compared to CPE-MNT, ELISA-MNT is time-saving and suitable for high-throughput detection. Conclusion:1. The recombinant yeasts expressing EV71capsid protein VP1, VP2, VP3and VP4were successfully obtained by construction of yeast display system. The recombinant yeasts expressing EV71capsid protein could be used to identify EV71monoclonal antibodies by flow cytometry, which would be a good experimental platform for identification of EV71P1mAb especially those recognizing a conformational epitope on P1.2. By using immunofluorescence, Western blot and the FCM identification,20strains of monoclonal antibodies were confirmed as EV71associated antibodies. In these mAbs,6strains were VP1specific,3were for VP2, and only1strain was VP4associated.3. The results on antibody identification show a good correlation by using Western blot and the FCM.4. Three VP1mAbs, M-5, M-8and M-14, M-5and showed especially high neutralization titers, which would be expected to be used as a therapeutic mAbs candidates.5. A rapid ELISA-MNT assay for measuring neutralizing antibody against Enterovirus71was developed, which shows good sensitivity (92.9%), and specificity (100%), and also a good correlation with CPE-MNT (r=0.934, P<0.001). Compared to CPE-MNT, ELISA-MNT is time-saving, suitable for high-throughput detection.