Screening And Analysis Of Epitopes Recognized By Neutralizing Antibodies Against Envelope Protein Domain Ⅲ Of Dengue Virus

Dengue fever (DF) is a insect-borne infectious disease caused by dengue virus (DENV). Diseases caused by dengue virus (DV) infection vary in severity, with symptoms ranging from mild fever to life threatening dengue hemorrhage fever (DHF),dengue shock syndrome (DSS) and even death. A report from WHO in 2014 announced that the geographical range of dengue virus transmission has fastest expanded among insect-borne diseases in the globe, and the number of cases of dengue illness reported worldwide had increased more than 30-fold in the past 50 years, leading to serious health problem and economic burden. Dengue fever got severe pandemic in southern China (mainly Guangdong Province). The number of cases of dengue illness in Guangdong Province reached 45000, which remind the importance of prevention and treatment of dengue fever.Currently, there are no available licensed vaccines or antivirals for dengue. Although a vaccine developed by Sanofi has passed phase III clinical trials, but the result showed partial protection and cannot protect DENV-2 infection. The complex pathogenicity, existence of four viral serotypes and especially the antibody dependent enhance (ADE) for infection by heterotypic serotypes dengue virus have presented significant obstacles to vaccine development.The DENV is a enveloped positive single-stranded RNA viruses, belong to Flaviviridae family, Flavivirus genus that includes DENV serotypes 1-4 (DENV1-4). DENVs genome encodes for 3 structural (C, prM and E) and 7 nonstructural proteins (NS1, NS2A,NS2B, NS3, NS4A, NS4B and NS5). As the existence on virus surface, the E protein is considered as the main antigen that induces production of neutralizing antibody against DENV and the dominate target protein of DENV research. Nonstructural proteins play critical roles in virus proliferation, and most of work related to nonstructural proteins focus on the development of vitro diagnostic reagents. E protein contains three functional domains. The domain I of E protein is located in the middle of the protein,domain Ⅱ is located in the flank of the protein and contains the fusion peptide. The domain Ⅲ(EDⅢ), an immunoglobulin-like domain, is thought to contain the virus receptor-binding sites and contain epitopes recognized by virus-neutralizing monoclonal antibodies. Therefor, EDⅢ attracts critical attention, especially in development of vaccine and exploration of antibody protective mechanism.Previous studies suggested that serotype specific or cross-reactive antibodies against EDⅢ were detected in patients sera after DENV infection. Antibodies against EDⅢ in a large antibody repertoire against DENV exhibit the great potent activity in neutralizing DENV infection of Vero cells. Mouse monoclonal antibodies (mAbs) against recombinant EDⅢ(rEDⅢ) also can effectively protect from DENV infection in vitro or in vivo. Recent studies revealed that antibodies against EDⅢ from human sera after DENV infection only contribute a little on viral neutralization and ADE. It would be attributed to that EDⅢ-specific antibodies are less abundant in the human antibody repertoire and EDⅢ is not a predominante immunogen being capable of provoking effective antibody response. Once antibodies against EDIII reach enough concentration, EDIII-reactive antibodies would play an essential role in neutralizing protection. Accordingly, one of the effective strategies is to resolve EDIII neutralizing epitopes and develop subunit vaccines, and the analyse of EDIII neutralizing epitopes is also the key of the present study.In our previous work, a set of mAbs were generated by immunizing mice with rEDⅢs of DENV1-4. In present work, two mAbs 2B11 A35 and 2D73A7, that were proved as cross-neutralizing protection antibodies against four serotypes DENV by enzyme linked immunospot based on microneutralization test (ELISPOT-MNT). Phage display peptide library technique was used to screen and analyze conservative epitopes which were recognized by these two cross-neutralizing mAbs against four serotypes DENV, which would expected to provide useful information for subunite vaccine design.The present study includes the following three parts as follows:identification of cross-reactive protective monoclonal antibody, screening and analysismimetic peptide sequences and identification onimmunogenicity of peptide mimics.Part I. Identification of cross-neutralizing protective monoclonal antibodies against EDⅢAs the complexity of pathogenic mechanism of the four serotype dengue virus, The ideal vaccine should protect people from four serotype dengue virus, so the epitope recognized by the monoclonal antibodies should be conserved among the EDIII of the four serotype dengue virus. Hence, in this part, we are going to find the monoclonal antibodies that can cross protect and neutralize four serotype dengue virus. In our preliminary work, we have identified 27 strains of cross reactive monoclonal antibodies, with the method of ELISPOT-MNT, we find out two strains of monoclonal antibodies among those antibodies which can cross neutralize four serotype dengue virus. Detail work of this part begin with the ELISPOT-MNT, we use times dilution from 200μg/ml of the monoclonal antibodies, than the monoclonal antibodies incubated with four serotype dengue virus respectively, after incubation, monoclonal antibodies and four serotype dengue virus commonly infected LLC-MK2 cells, the number of cells detected and counted by the method of spot counter. The experimental results showed that the incubation of monoclonal antibodies with dengue virus can significantly decrease the number of infectious dengue virions, the more the concentration of monoclonal antibodies, the fewer the number of infected LLC-MK2 cells, even no LLC-MK2 cells can be infected. Monoclonal antibody 2B11A35 showed a good neutralization activity to DENV-1, DENV-2 and DENV-3, with the half maximal inhibitory concentration (IC50) less than 1.5μg/ml, but have a bad neutralization activity to DENV-4, with IC50 greater than 80μg/ml. Monoclonal antibody 2D73A7 showed a good neutralization activity to DENV-2 and DENV-3, with IC50 less than 3.5μg/ml, and the neutralization activity to DENV-4 taken the second place, with IC50 less than 8μg/ml; but have a bad neutralization activity to DENV-1, with the IC50 above 200μg/ml.The experimental result of this part find out two strains of cross protective monoclonal antibodies, and those monoclonal antibodies showed different neutralization activity to four serotype of dengue virus, but totally showed excellent neutralization activity. This result suggests that those two strains of cross protective monoclonal antibodies might recognise the more conserved epitopes on DENV EDⅢ.Part Ⅱ Biopanning and identification of epitopes recognized by two cross-neutralizing mAbs 2B11A35 and 2D73A7Linear dodecapeptide phage display libraries(Ph.D.-12) and cycleheptapeptide phage display libraries(Ph.D.-C7C) were used to biopanning of epitopes recognized by cross-neutralizing mAb 2B11A35 and 2D73A7, by which the epitope mimics or mimotope to EDIII conservative structure were obtained. After 3 rounds biopanning against Ph.D.-12 using mAb 2B11A35,phages were significantly enriched, and 20 phage clones were picked up from the third round eluted product. After 3 rounds biopanning against Ph.D.-C7C using mAb 2B11A35, phages was significantly enriched, and 20 phage clones were picked up from the third round eluted product. The 3 rounds biopanning for Ph.D.-12 by mAb 2D73A7, displayed an enriched more than 5000 times, and 30 phage clones were picked up from the third round eluted product. The 4 rounds biopanning for Ph.D.-C7C by mAb 2D73A7, phage enriched more than 5000 times and 20 phage clones were picked up. The phage clones picked up from eluted product were identified by phage ELISA in which above two mAbs were used as capture antibodies. The phage clones that bind specifically with the mAbs for biopanning, were as positive clones. All the positive clones were excluded the possibility of sequence binding with plate, nor binding with irrelevant monoclonal antibody.All of positive phage clones were amplified for DNA extracting and sequenced by Shanghai sangon biological engineering co, LTD. In 17 linear 12 mer phage clones bound with binding to 2B11A35, the most repetitive sequence were THNGPGRFTGIL, THYCAWDQKNCL and THQPQPKIPAVT, respectively. In 20 cyclic 7mer phage clones binding to mAb 2B11A35, the most repetitive sequence were WHGHPHH and GHDLHPA. In 26 linear 12mer phage clones binding to mAb 2D73A7, the most repetitive sequence were LHRYSPDGASYA and LHRYDVSNNLPN. In 18 cycle 7merphage clones binding to mAb 2D73A7, the most repetitive sequence were PMYGWDM and WAYGFNM.According to results of ELISA,4 of linear 12mer sequences for MAb 2B11A35 were synthesized and biotinylated in N-terminal. Additionally, the peptide-carrier conjugate was also prepared. Unfortunately, both biotinylated peptides and peptide-carrier conjugates could not bind with mAb, but bind weakly to rabbit antiserum to EDIII, which would be attributed to these peptides mimic to the structural epitope.In this part, the sequences mimics to conservative epitopes on EDIII recognized by neutralizing antibodies were screened from Ph.D.-12 and Ph.D.-C7C phage peptide library using two mAbs 2B11A35 and 2D73A7, which were proved to be conformational epitopes.Part Ⅲ. Antigenicity assessing of the sequences mimics by immunizing mouse with phagesAfer amplification and purification, No.1,9,11,14 phages bio-panned from Ph.D.-12 with mAb 2B11A35 were emulsionized with Freund’s adjuvant and were set to immunize BALB/C mice, respectively. Mice immunized with above phage clones and empty vector phage clones at a fixed titer (1×10 pfu/ml). The binding of antiserum to DENV EDIII was evaluated by ELISA or IFA. The anti-phage antibody titier of the mouse serum is about 800, and the antiserum can also bind to four serotype DENV EDIII. The results suggested that these special sequences displayed on phage recognized by mAb 2B11A35 can induce production of cross-reactive antibodies against four serotype DENV EDIII, which means that the these peptides on the 4 phage clones mimic to cross-reactive antigen epitope.