Monoclonal antibody raised against envelope glycoprotein peptide neutralizes Japanese encephalitis virus

Summary.  Epitopes on envelope glycoprotein of Indian strain of Japanese encephalitis virus were delineated by prediction methods. Monoclonal antibodies (MAb) raised against a putative B cell epitope peptide, reacted with the virion in ELISA and immunofluorescence assays. One MAb was also able to neutralize the virus. The reactivity of this MAb against a Sri Lankan strain was checked, since this strain had a substitution within the B cell epitope at position Egp 153 (G → W). The MAb was able to bind to, but was not able to neutralize the Sri Lankan isolate. The data indicated that the predicted B cell epitope is a neutralizing epitope and may be included in a peptide-based vaccine against the virus. Received July 20, 2000 Accepted September 28, 2000     

Expression of envelope glycoprotein (E) of Japanese encephalitis virus by recombinant vaccinia virus

Vaccinia virus recombinants inserted with cDNA clones of Japanese encephalitis (JE) virus envelope glycoprotein (E) gene were constructed. The E gene product was detected in the recombinant virus-infected BHK21 cells by immunofluorescence (IF) and Western blotting. The intensity of IF observed was higher by the recombinant of the TK promoter--P7.5 promoter--inserted cDNA construct than by the P7.5 promoter--TK promoter--inserted cDNA construct. The E gene product was hardly detected by the recombinant carrying the TK promoter only upstream to the inserted cDNA, although the glycoprotein E mRNA had been transcribed.     

Immunogenicity of peptides cleaved by cyanogen bromide from Japanese encephalitis virus envelope glycoprotein E

Envelope glycoprotein (E) prepared from purified Japanese encephalitis (JE) virus was cleaved with cyanogen bromide (CNBr) followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Mice were immunized with 36 kD, 27 kD, and 8 kD bands from CNBr-cleaved and 54 kD band from control specimens. Neutralization test was positive in one out of two anti-54 kD, in none of the anti-36 kD, and all of anti-27 kD and anti-8 kD sera at 1:10 dilution. Geometrical mean ELISA titre was the highest for anti-54 kD followed by anti-36 kD, anti-27 kD, and anti-8 kD sera. Reactivity of these sera to CNBr-cleaved fragments in Western blotting indicated that the 8 kD fragment was a part of the 27 kD but was not included in the 36 kD fragment, while the 27 kD and 36 kD fragments shared an overlapping part. These fragments were located on the E protein by N-terminal amino acid sequencing of each fragment purified by reversed-phase high-performance liquid chromatography and by comparison with the nucleotide sequence of the E protein gene. The 36 kD fragment was located between the third and the ninth methionine and covered most of the N-terminal side of the E protein. In contrast, the 27 kD fragment was located between the fourth and the tenth methionine and included the 8 kD fragment which was situated between the ninth and the tenth methionine near to the C-terminus of the E protein. Denaturation-resistant neutralizing epitope(s) appeared to be present on the 8 kD fragment, but not on the 36 kD fragment.     

Recognition of helper T cell epitopes in envelope (E) glycoprotein of Japanese encephalitis, west Nile and Dengue viruses.

Helper T (Th) cell antigenic sites were predicted from the primary amino acid sequence (approximately 500 in length) of the envelope (E) glycoprotein (gp) of Japanese encephalitis (JE), West Nile (WN) and Dengue (DEN) I-IV flaviviruses. Prediction of Th epitopes was done by analyzing the occurrence of amphipathic segments, Rothbard-Taylor tetra/pentamer motifs and presence of alpha helix-preferring amino acids. The simultaneous occurrence of all these parameters in segments of E gp were used as criteria for prediction as Th epitopes. Only one cross reactive epitope was predicted in the C-terminal region of the E gp predicted segments of all flaviviruses analyzed. This region is one of the longest amphipathic stretch (approximately from 420 to 455) and also has a fairly large amphipathic score. Based on the predicted findings three selected peptides were synthesized and analyzed for their ability to induce in vitro T cell proliferative response in different inbred strains of mice (Balb/c, C57BL6, C3H/HeJ). Synthetic peptide I and II prepared from C-terminal region gave a cross reactive response to JE, WN and Den-II in Balb/c and C3H/HeJ mice. Synthetic peptide III prepared from N-terminal region gave a proliferative response to DEN-II in Balb/c strain only, indicating differential antigen presentation.     

Structural and molecular analyses of functional epitopes and escape mutants in Japanese encephalitis virus envelope protein domain III

Immunologic Research - The Japanese encephalitis virus (JEV) is one of the vector borne causes of encephalitis found in southeastern Asia. This positive single-stranded RNA virus is a member of the...     

Effect of tunicamycin on expression of epitopes on Japanese encephalitis virus glycoprotein E in porcine kidney cells

The effect of tunicamycin (Tm), a glycosylation inhibitor, on the epitopes expressed on Japanese encephalitis virus (JEV) glycoprotein E (gpE) in porcine kidney stable (PS) cells was studied. At Tm concentration of 2 micrograms/ml, the virus-infected cells showed markedly reduced or no reactivity with any of the monoclonal antibodies (MAbs) directed against JEV gpE except NHs-2 and also with polyclonal antibodies (PAbs) directed against JEV. With the increase in Tm concentration to 3 micrograms/ml, a complete loss of the conventionally detected reactivity of the MAbs except NHs-2 was recorded, while the Pabs showed no decrease in their reactivity. However, the MAb NHs-2 and PAbs lost their reactivity when the cells treated with 3 micrograms/ml Tm were stained for epitopes expressed on their surface indicating that glycosylation plays a role in this phenomenon. Tissue culture fluid (TCF) displayed a low virus content in the presence of 3 micrograms/ml Tm, indicating probably a down-regulation of virus maturation inside the cells. Since preM and NS-1 proteins possess besides gpE conserved N-glycosylation sites and play a role in the maturation of JEV, their expression in nascent, i.e. non-glycosylated form might be responsible for the observed low virus content of TCF. Thus, the glycosylation of JEV gpE seems essential for the acquisition of native conformation of its epitopes and their expression in cells.     

猪瘟病毒E2糖蛋白抗原表位的预测、多肽合成及特性研究

目的：研究CSFV E2蛋白免疫原性肽的生物学特性。方法：应用计算机分析软件对猪瘟病毒＆糖蛋白的抗原表位进行了预测，结合＆基因的变异分析，人工合成了两条多肽(Pep1和Pep2)序列，与抗mE2蛋白的兔血清和抗mE2的8株单抗进行反应，并将两条多肽分别与载体蛋白(BSA)进行偶联和免疫家兔。结果：两条多肽均与兔的抗血清及单抗A11反应；多肽Pep2与单抗D5和D8反应。多肽Pep1与BSA载体蛋白偶联(Pep1-BSA)，免疫家兔后能够产生针对多肽Pep1的抗体。结论：两条多肽Bep1和Pep2中，只有多肽Pep1具有很好的反应原性和免疫原性。     

Identification of immunodominant epitopes in envelope glycoprotein of human T lymphotropic virus type II.

A series of synthetic peptides derived from the envelope glycoprotein of human T lymphotropic virus type II (HTLV-II) was used in an enzyme immunoassay to determine the immunodominant epitopes of envelope glycoprotein. Of the 11 synthetic peptides spanning the external glycoprotein of HTLV-II (gp52) and the 3 from the transmembrane protein (gp21), 3 peptides from gp52 (termed Env-20(85-102), Env-202(173-209), and Env-203(219-256] reacted with most of the polymerase chain reaction-confirmed HTLV-II specimens (83, 95, and 76%, respectively); all other peptides reacted minimally with these specimens. Env-202(173-209) reacted with a greater percentage (91 to 100%) of specimens from different risk groups, including intravenous drug users (n = 30), North American Indians (n = 13), Guaymi Indians from Panama (n = 22), and routine U.S. blood donors (n = 34), when compared with Env-20(85-102) (73 to 100%) or Env-203(219-256) (68 to 83%). Furthermore, Env-20(85-102) and Env-202(173-209) had some reactivity (8-25%) with sera from HTLV-I-infected individuals, whereas Env-203(219-256) reacted with 58% of HTLV-I specimens. We conclude that peptides Env-20(85-102) and Env-202(173-209) represent the type-specific immunodominant epitopes of HTLV-II external glycoprotein.     

Expression of Japanese encephalitis virus envelope protein in transgenic tobacco plants

The virus envelope (E) protein of Japanese encephalitis virus induces virus-neutralizing antibodies and is therefore a potential vaccine antigen. In a mammalian system, co-expression of another viral structural protein prM is necessary for proper expression and folding of E protein. Transgenic tobacco plants were produced carrying JEV cDNA encoding prM and E proteins under the control of the CaMV 35S promoter. E protein, however, was not detectable in these plants. In vitro translation studies showed that the presence of the prM sequence inhibited transgene expression in the plant system. Accordingly, JEV E protein could be expressed in transgenic tobacco plants only without the prM protein.     

Characterization of Japanese encephalitis virus envelope protein expressed by recombinant baculoviruses

Recombinant baculoviruses containing the coding sequences of the viral structural proteins, i.e., the capsid (C) protein, the precursor to premembrane (preM) protein, and the envelope (E) protein, as well as a nonstructural protein, NS1, of Japanese encephalitis virus (JEV) were constructed. Infection of Spodoptera frugiperda cells with these recombinant viruses produced PreM and E proteins. The E proteins synthesized by the recombinants were shown to be glycosylated and similar in size to the authentic E protein. The E protein was found on the surface of infected cells. The antigenic properties of recombinant E proteins were evaluated using a panel of monoclonal antibodies produced against JEV E protein. It was demonstrated that all of the epitopes detectable on the authentic JEV E protein were present on the recombinant E protein expressed by a recombinant baculovirus containing the coding sequence for a part of C, PreM, E, and a part of NS1 proteins. However, for E protein expressed by a recombinant baculovirus having the coding sequence of only a part of PreM, but all of E and a part of NS1, one of the flavivirus cross-reactive epitopes was not detected. Mice immunized with cells infected with the recombinant baculoviruses developed neutralization antibodies.     

Fragment of Japanese encephalitis virus envelope protein produced in Escherichia coli protects mice from virus challenge.

A fragment from the N-terminal part (E(A)) and a fragment from the C-terminal part (E(B)) of the envelope (E) protein of Japanese encephalitis virus (JEV) was synthesized in Escherichia coli. These two fragments were overlapping with each other by nine amino acids, however, they were not cross-reacting with each other at the antisera level. Both E(A)and E(B)are antigenic by themselves when injected into mice, but when tested against sera from mice, rabbit, swine and human that had been immunized or naturally infected with JEV, E(B)acted as a better antigen than E(A)by ELISA assays. E(B)also proved to be a better immunogen in protection against lethal JEV infection than E(A). The protection appears to be correlated with the neutralizing titres of the anti-JEV sera. The response elicited by E(B)is a Th1 response and the antibody produced contained higher neutralizing titre than E(A)fragment. The major difference between E(A)and E(B)fragments is the solubility during expression in E. coli, while E(B)fragment is soluble, E(A)was isolated from the insoluble inclusion bodies. Therefore the antigenicity and immunogenicity expressed by the E(B)fragment may probably be due to its proper folding to assume a correctly assembled form during expression in E. coli, a quality that is important for a protein to qualify as a good vaccine candidate.     

Serologically defined linear epitopes in the E2 envelope glycoprotein of Semliki Forest virus

Summary A set of 41 overlapping peptides, representing the complete sequence of SFV-E2 protein were synthesized and analyzed in the ELISA test against murine anti-SFV sera. No single peptide was recognized by all antisera. Eight peptides were found to be highly reactive with hyperimmune anti-SFV sera. Six out of the eight peptide sequences coincide with the most hydrophilic regions of SFV-E2. Out of these, four peptides (amino acid positions 16–35, 61–80, 166–185, 286–305) that contain the least number of alphavirus conserved residues were selected. This panel constitutes the minimal number of peptides necessary and sufficient for specific recognition of hyperimmune mouse anti-SFV sera.     

Japanese encephalitis and Japanese encephalitis virus in mainland China

Japanese encephalitis (JE), caused by Japanese encephalitis virus (JEV) infection, is the most important viral encephalitis in the world. Approximately 35,000–50,000 people suffer from JE every year, with a mortality rate of 10,000–15,000 people per year. Although the safety and efficacy of JE vaccines (inactivated and attenuated) have been demonstrated, China still accounts for 50% of the reported JE cases worldwide. In this review, we provide information about the burden of JE in mainland China and the corresponding epidemiology from 1949 to 2010, including the morbidity and mortality of JE; the age, gender, and vocational distribution of JE cases; its regional and seasonal distribution; and JE immunization. In addition, we discuss the relationships among vectors, hosts, and JEV isolates from mainland China; the dominant vector species for JEV transmission; the variety of JEV genotypes and the different biological characteristics of the different JEV genotypes; and the molecular evolution of JEV. Copyright © 2012 John Wiley & Sons, Ltd.     

Neutralizing peptide ligands selected from phage-displayed libraries mimic the conformational epitope on domain III of the Japanese encephalitis virus envelope protein

The envelope (E) protein of Japanese encephalitis virus (JEV) contains 500 amino acids with six "conserved" disulfide bonds to maintain its conformational structure. Neutralizing epitopes located on the E protein are mostly conformational dependent. In this study, we used phage-displayed 12-residue combinatorial peptide libraries to select high-affinity peptide ligands bound to monoclonal antibody E3.3. The specific peptide ligands presented on ten high-affinity phage clones displayed six different amino acid sequences, all showing a novel cis-proline turn structure. After being superimposed onto the best fit of the three-dimensional structure of JEV E protein, these peptide structures were mapped to a conformational region constituted by three continuous polypeptide segments (E307-E309, E327-E333, E386-E390) in domain III. Synthetic peptide ligands based on one peptide sequence (E18) were further investigated using alanine scanning within the cis-proline turn structure to demonstrate its unique molecular characteristics. Our results showed that three residues forming the novel cis-proline turn structure were all important in eliciting JEV-specific neutralizing antibodies in mice.     

Synthetic peptides approach to identification of epitopes on bovine leukemia virus envelope glycoprotein gp51

Peptides corresponding to residues 21-28, 39-48, 57-67, 59-69, 78-92, 144-155, 144-157, 195-205, 255-268, and 260-268 of envelope glycoprotein gp51 of bovine leukemia virus (BLV) were chemically synthesized and coupled to keyhole limpet hemocyanin or tetanus toxoid. All peptides were immunogenic in rabbits and induced production of antipeptide antibodies. Enzyme-linked immunosorbent assays using Tween 80-purified gp51 or BLV particles showed that antibodies against peptides 78-92, 255-268, and to a lesser extent 39-48 and 144-157 were able to react with the parent glycoprotein, purified or as an integer part of BLV particles. Antisera against peptides 39-48, 78-92, and 144-157 neutralized VSV (BLV) pseudotypes in vitro, the highest neutralization titer being obtained with the 78-92 cyclized peptide. These observations confirm that the NH2 moiety of gp51 carries epitopes involved in virus infectivity.     

Biochemical and biological characteristics of epitopes on the E1 glycoprotein of western equine encephalitis virus

Antigenic determinants identified by monoclonal antibodies (Mabs) on the E1 glycoprotein of western equine encephalitis (WEE) virus have been characterized by their serological activity, requirements for secondary structure, expression on the mature virion, and their role in protecting animals from WEE virus challenge. On the basis of a cross-reactivity enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition assay, eight antigenic determinants (epitopes) on the E1 glycoprotein have been identified, ranging in reactivity from WEE-specific to alphavirus group reactive. No neutralization of virus infectivity was demonstrable with any of the Mabs. An alphavirus group-reactive hemagglutination (HA) site, a WEE complex-reactive HA site, and a WEE virus-specific HA site were identified. Spatial arrangement of these epitopes was determined by a competitive binding ELISA. Four competition groups defining three distinct antigenic domains were identified. Antibodies directed against four E1 epitopes were capable of precipitating the E1/E2 heterodimer from infected cells or purified virus disrupted with nonionic detergents. These same antibodies precipitated only E1 in the presence of 0.1% SDS. That E1 conformation was important was shown by the inability of antibodies specific for seven of the epitopes to bind to virus denatured in 0.5% SDS. As determined by equilibrium gradient analysis of virus-antibody mixtures, four epitopes were found to be fully accessible on the mature virion, three epitopes were inaccessible, and one epitope was partially accessible to antibody binding. Antibodies specific for three epitopes were able to passively protect mice from WEE virus challenge.     

Design and characterization of polytope construct with multiple B and TH epitopes of Japanese encephalitis virus

Japanese encephalitis (JE) remains a major public health threat with vaccination as the only measure for its prevention. Epitope-based vaccination is a promising approach for achieving protective immunity and avoid immunopathology in Japanese encephalitis virus (JEV) infection due to flavivirus cross-reactivity. We have mapped B-cell epitopes from JEV envelope protein, responsible for elicitation of neutralizing antibodies. Incorporation of T helper (T(H)) epitopes, along with these, imparted protective immunity to the host. In the present study, based on in silico epitope selection we optimized and proposed a polytope DNA construct (P-JEV) consisting B-cell and T(H) epitopes from the JEV envelope (E) protein as well as non-structural protein-1 (NS1). The immunogenicity and protective efficacy of P-JEV was assessed by in vitro and in vivo experiments. The expressed P-JEV showed reactivity in in vitro assays with JEV monoclonal antibodies. Protective efficacy of P-JEV was assessed in BALB/c mice. Our findings indicate that P-JEV may be a candidate vaccine for the prevention of JEV infection.     

B-cell epitopes of varicella-zoster virus glycoprotein II

Summary B-cell epitopes of varicella-zoster virus glycoprotein II were mapped by means of solid phase ELISA, synthetic oligopeptides (constructed according to the Davison-Scott sequencing of the varicella-zoster virus genome) and sera from varicellae and herpes zoster patients. The individual pattern of antibody peptide binding varied considerably but at least 9 more reactive sites seemed discernible. A 31-mer-peptide corresponding to a hydrophilic segment of the glycoprotein (aa 417–447) was constructed. This peptide reacted with 2 out of 4 varicellae and 5 out of 9 zoster sera, respectively.     

Administration of noninternal image monoclonal anti-idiotypic antibodies induces idiotype-restricted responses specific for human immunodeficiency virus envelope glycoprotein epitopes.

A mouse monoclonal anti-idiotypic antibody (anti-Id), designated MC1, was generated against chimpanzee antibodies specific for a synthetic peptide corresponding to a native epitope associated with gp41 of human immunodeficiency virus (HIV). This anti-Id recognized a shared idiotope/idiotype (Id) on a second chimpanzee anti-gp41 peptide preparation but failed to detect this Id on rabbit and mouse anti-gp41 peptide antibodies induced by immunization with the gp41 synthetic peptide. The chimpanzee Id-MC1 reaction was not inhibited by either synthetic peptide or recombinant gp160 suggesting that MC1 exhibits noninternal image, Ab-2 alpha-like characteristics. Immunization of syngeneic Balb/c mice with MC1 induced an antigen-positive (Ag+) response capable of binding the synthetic peptide, recombinant gp160, and gp41, whereas MC1-immunized rabbits did not produce any detectable anti-peptide and/or anti-HIV envelope glycoprotein antibody response. The MC1-induced anti-Id response (Ab-3) in both mice and rabbits expressed a similar Id with the Ab-1, which is not normally expressed in the anti-gp41 peptide antibody response induced by the nominal antigen in Balb/c mice and in rabbits. Together, these studies indicate that a mouse monoclonal anti-Id of the Ab-2 alpha class can induce an anti-HIV response specific for a gp41 epitope defined by a synthetic peptide, which does not cross species barriers.