Screening And Identification Of Linear B Cell Epitopes On Hantaan Virus Structural Proteins

Hantaan virus (HTNV), the prototype of the Hantavirus genus whichbelongs to Bunyaviridae family, together with another member of the Hantavirusgenus, Seoul virus, are the main causative pathogens of hemorrhagic fever withrenal syndrome (HFRS) in China. The annual incidence of HFRS worldwide is60,000~150,000, of which China accounts for about90%. HFRS prevails all overprovinces in China except for one or two provinces, and the motality of thisdisease is about2%~10%.Hantavirus infection could induce strong and long-lasting adaptive immuneresponse, including humoral immune response and cell-mediated immuneresponse. The nucleocapsid protein of hantavirus is the major immunogen forhumoral immune response, and can induce antibody production within severaldays after hantavirus infection. Thus the N protein of hantavirus is often used as adiagnostic agent. The glycoproteins (G1and G2, or Gn and Gc) of hantavirus arebelieved to be the structural proteins which induce neutralizing antibodies, emerging later during the course of HFRS. It has been demonstrated that theinduction of neutralizing antibodies was closely related to the prognosis of HFRSor hemorrhagic fever with pulmonary syndrome (HPS). Antibodies to both the Nprotein and the G proteins in the acute phase increased with time, peaked at latercourse and then sustained for a period as long as decades.Epitopes on both the N protein and the G proteins of Puumala virus andsome New World hantaviruses have been extensively investigated usingmonoclonal antibodies as well as sera from the patients. However, to date, thereare only a few epitopes on HTNV structural proteins reported, which were mainlymapped by using monoclonal antibodies. Here, we report a systemic investigationof epitopes on HTNV structural proteins using the PepScan method with bothHFRS patient sera and neutralizing monoclonal antibodies to HTNV.To map the linear epitopes on the N protein of HTNV, in total,70overlapping peptides covering the entire sequence of N protein were synthesizedand coated on ELISA plates. The sera from35HFRS patients and24normalcontrols were used to screen the peptides recognized by patient serum specificantibodies. The result showed that although HFRS patient serum antibodiesrecognized dozens of synthetic peptides spreading all over the amino acidsequence of the N protein, most of the peptides recognized by specific antibodieswere located at the N-terminal part (amino acid1to amino acid117), followed bythe C-terminal part (amino acid361to amino acid429) and there were only a fewpeptides located sparsely in middle of the amino acid sequence.Competitive ELISA was carried out using several peptides andcorresponding patient sera as a representative to clarify the specificity ofinteractions between synthetic peptides and HFRS patient serum antibodies.Binding of patient serum antibody to coating peptide could be specifically inhibited by the corresponding free peptide, but not by irrelative peptides, in adose-dependent manner, demonstrating that the synthetic peptides displayed asthe linear epitopes recognized by corresponding serum antibodies.Next, we investigated the change of IgG titres specific to identified linearepitopes on the N protein in patient sera collected at different time during thedisease course. Although the total IgG to the N protein increased during thecourse, IgG to specific linear epitopes displayed some discrepancy. During theobserved time, the titres of IgG to some linear epitope increased continually;some increased at first, and then decreased; and some decreased very soon.To identify the neutralizing epitopes on the G proteins of hantaan virus,28115-mer overlapping peptides were synthesized, and grouped into28pools. Eachpool containing mixture of peptides were coated on either ELISA wells ornitrocellulose membranes, and screened with3murine neutralizing monoclonalantibodies3D8,3G1, and8G3, specific to the G2protein of hantaan virus. Thecomponent synthetic peptides from positive reacting pools were coatedindividually and screened with the same antibody again. Our screening resultsdemonstrated that monoclonal antibody3D8specifically recognized a syntheticpeptide corresponding to881-KGFLCPEFPGSFRKK-895in the G proteinsequence. Monoclonal antibody8G3also recognized this peptide as well as othersynthetic peptides. Monoclonal antibody3G1recognized several other syntheticpeptides, indicating that it may recognize a conformational epitope. CompetitiveELISA was carried out and confirmed the specific binding of the neutralizingmonoclonal antibody3D8and8G3to peptide KGFLCPEFPGSFRKK. To finemap the linear epitope recognized by monoclonal antibody3D8, several more15-mer peptides which overlap only one amino acid with each other aroundKGFLCPEFPGSFRKK were synthesized, and screened with the antibody. This led us to realize that the neutralizing monoclonal antibody3D8reacted with asynthetic peptide corresponding to882-GFLCPEFPGSFRKKC-896even morestrongly. To further define amino acid crucial to the epitope, alanine scanningmutagenesis was employed, and the mutated peptides were synthesized andscreened with the monoclonal antibody3D8again. Our results showed thataa885C and aa896C were the most vital amino acids in the epitope, and aa882G,aa883F, aa884L, aa893R, aa894K and aa895K also played an important role inthe recognition of the linear epitope by the neutralizing monoclonal antibody3D8.Our results identified a new neutralizing epitope on the G2protein of hantaanvirus.This thesis report for the first time the systermic mapping of linear B cellepitopes on the Hantaan virus nucleocapsid protein, and the identification a novelneutralizing epitpoe on Hantaan virus G2protein, which may lay the foundationfor further understanding of adaptive immunity induced by HTNV infection,novel vaccine design, and the development of therapeutic monoclonal antibodyfor treatment of HFRS.