Characterization of neutralizing domains on varicella-zoster virus glycoprotein E defined by monoclonal antibodies

Summary. The genome of varicella-zoster virus (VZV), encodes at least six glycoproteins and they elicit the formation of complement-independent, complement-dependent, and non-neutralizing antibody responses. We have used our library of MAbs to VZV glycoprotein E (gE) to determine the neutralizing epitopes of gE, and shown that gE has 3 distinct neutralizing domains. In this report we have used the baculovirus expression system to identify the antigenic domains of gE. We have generated 3 recombinant baculoviruses, expressing the full-length gE and two overlapping truncated forms (the amino-terminal and the carboxy-terminal) of gE. By immuno-fluorescence and immunoblotting we have explored the physical interactions of Mabs to gE on these constructs. Our panel of MAbs revealed 3 district antigenic domains on gE. All MAbs reacted with the full-length gE; MAbs with high titered complement-dependent neutralizing activities reacted with the N-terminal truncated gE; MAbs with low titered or non-neutralizing activities reacted with the C-terminal truncated gE; MAbs with complement-enhanced neutralizing activities reacted with both truncated constructs. However, although the antibody binding in immunofluorescence and immunoblotting was carried out under denatured conditions, whereas the neutralization is under non-denatured conditions, still the antigenic mapping was similar in both conditions. Received April 24, 1996 Accepted July 29, 1996     

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.     

Novel varicella-zoster virus glycoprotein E gene mutations associated with genotypes A and D

Here, we describe the association of certain varicella-zoster virus (VZV) genotypes with unique glycoprotein E (gE) gene mutations. Within 45 analyzed VZV wild-type strains of genotypes A and D, five novel gE mutations were discovered. A statistically significant (P < 0.0001) association of certain gE mutations with VZV genotype D was found.     

E1 glycoprotein of rubella virus carries an epitope that binds a neutralizing antibody

We identified by immunoprecipitation and Western blot analysis, using a monoclonal antibody that neutralizes rubella virus, that E1 glycoprotein carries an epitope linked with neutralization. Glycosidase treatment of virus does not prevent blotting of this monoclonal antibody with the E1 glycoprotein, dissociating this epitope from the hemagglutination epitope which is linked with the oligosaccharide side chains. We also investigated by Western blot analysis human serum reactivity toward E1 glycoprotein and the two other structural proteins of rubella virus, E2 and C: all positive sera detected E1 and C, irrespective of their titers, indicating the importance of glycoprotein E1 in immunity. Frequent lack of reactivity against E2 might suggest that this glycoprotein is either less exposed or less immunogenic.     

Intracellular transport and stability of varicella-zoster virus glycoprotein K

VZV gK, an essential glycoprotein that is conserved among the alphaherpesviruses, is believed to participate in membrane fusion and cytoplasmic virion morphogenesis based on analogy to its HSV-1 homolog. However, the production of VZV gK-specific antibodies has proven difficult presumably due to its highly hydrophobic nature and, therefore, VZV gK has received limited study. To overcome this obstacle, we inserted a FLAG epitope into gK near its amino terminus and produced VZV recombinants expressing epitope-tagged gK (VZV gK-F). These recombinants grew indistinguishably from native VZV, and FLAG-tagged gK could be readily detected in VZV gK-F-infected cells. FACS analysis established that gK is transported to the plasma membrane of infected cells, while indirect immunofluorescence demonstrated that gK accumulates predominately in the Golgi. Using VZV gK-F-infected cells we demonstrated that VZV gK, like several other herpesvirus glycoproteins, is efficiently endocytosed from the plasma membrane. However, pulse-labeling experiments revealed that the half-life of gK is considerably shorter than that of other VZV glycoproteins including gB, gE and gH. This finding suggests that gK may be required in lower abundance than other viral glycoproteins during virion morphogenesis or viral entry.     

Expression of varicella-zoster virus glycoprotein I in cells infected with a vaccinia virus recombinant

BSC-1 cells infected with a vaccinia virus recombinant containing the coding sequences for varicella-zoster virus (VZV) glycoprotein I (gpI) were analyzed by indirect immunofluorescence and immunoprecipitation for the expression and processing of gpI. The processing of gpI in cells infected with recombinant virus was the same as that observed during VZV infection. Immunofluorescence revealed localization of gpI to the membranes of recombinant virus-infected cells.     

Stability of varicella-zoster virus open reading frame 63

The stability of varicella-zoster virus (VZV) open reading frame (ORF) 63 was analyzed by sequential passage of a virus strain in cell culture. VZV was propagated in culture for 1,206 passages. ORF63 from six passages (18, 220, 516, 730, 1060, and 1,206) was selected and sequenced. Among the six passages, only passage 1,206 showed point mutations at three locations: 551, 618 and 661. In addition, western blot analysis with anti-ORF63 monoclonal antibodies showed no discernable difference in the size of the ORF63 gene product from passage 18 and that from passage 1,206. These results indicate the stability of VZV ORF63 gene in culture over 1,206 passages. Disclaimer: The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the funding agency.     

Characterization of antibody responses against a neutralizing epitope on the glycoprotein E2 of classical swine fever virus

The sequence TAVSPTTLR is a conserved and linear neutralizing epitope on the glycoprotein E2 of classical swine fever virus. In this study, TAVSPTTLR-directed antibodies, induced either by virions or by an epitope-focused immunogen, were characterized. The results revealed that despite the same epitope specificity, the antibodies induced by different immunogens varied significantly both in the neutralizing test and in binding inhibition assays. This suggests that the protective immunity induced by this epitope is due to more than simply the epitope specificity and that this epitope might need essential contributions from its flanking context to induce functional epitope-specific antibodies.     

Neutralizing monoclonal antibody to the E1 glycoprotein epitope of rubella virus mediates virus arrest in VERO cells

The best-known mechanism of action of antibody-mediated virus neutralization is to impede the entrance of viruses to host cells, as determined by neutralization assays. Antibodies may also inhibit the exit of rubella virus (RV) from infected host cells; in this case, the interaction of the antibodies with their domains must occur on the plasma membrane, because antibodies cannot enter the cells. In the present study, we were able to block temporally the exit of virions from RV-infected cells by the binding of monoclonal antibody (mAb) H3 to their surface. The objective was accomplished in three steps: first, we determined the duration of the viral replication cycle; then we established the kinetics of the presence of the domains defined by our mAbs in the cytoplasm of RV-infected VERO cells; and, finally, we assessed the release of viral particles to the supernatant of infected VERO cells in the presence or absence of mAbs or positive and negative mice sera. RV-specific mice sera and mAb H3, which binds to the amino acid sequence 208-239 of the RV-E1 glycoprotein, were able to delay for 24 hours the release of virions from infected cultures, suggesting that the reaction of mAb H3 with its epitope may arrest any change necessary for the assembly and/or release of virions. In conclusion, the neutralizing domain recognized by mAb induces antibodies that can block the viral replication by several mechanisms of action, such as the obstruction of virus entry into cells and the delay of viral release. All of these mechanisms are intimately involved in the critical virus-host cell interactions that allow self-limitation of the infection.     

Identification of an Epstein-Barr virus glycoprotein which is antigenically homologous to the varicella-zoster virus glycoprotein II and the herpes simplex virus glycoprotein B

The Epstein-Barr virus (EBV) antigenic homologue of the varicella-zoster virus glycoprotein II and the herpes simplex virus (HSV) glycoprotein B (gB) was identified through cross-reactivity with anti-glycoprotein II and anti-glycoprotein B peptide sera. The homologue is the previously characterized EBV glycoprotein, with an apparent molecular weight of 125,000 Da, which is synthesized late during productive EBV infection and appears to be encoded by the BamHI A EBV fragment. This glycoprotein, but not other EBV proteins, reacted with the antisera in immunoprecipitation experiments and by ELISA. In addition, absorption of the sera with the purified EBV 125-kDa glycoprotein removed the cross-reacting antibody. Whether the EBV gB homologue has the same biological functions associated with HSV gB has yet to be determined.     

Sequence analysis of the glycoprotein E gene of varicella-zoster virus strains of clades 1, 3 and 5

Eighty-six varicella-zoster virus (VZV) strains of clades 1, 3 and 5, isolated from varicella and zoster patients in Germany, were analyzed by sequencing the glycoprotein E gene. Four novel non-synonymous and 10 novel synonymous mutations were detected. Of these, two synonymous (C513T, C885T) and two non-synonymous mutations (T485G, C524T) were located within the coding regions of e1 and c1. The profile of single-nucleotide polymorphisms was found to be significantly associated with the VZV clades 1, 3 and 5.     

Identification and sequence of the gene encoding gpIII, a major glycoprotein of varicella-zoster virus

The genome of varicella-zoster virus (VZV) encodes three major glycoproteins, two (gpI and gpII) having been mapped and sequenced, which carry epitopes capable of eliciting neutralizing antibodies. The product of the third major glycoprotein gene (gpIII) was purified, and seven consecutive amino acids at its N-terminus were identified. A degenerate pool of oligonucleotides based upon this sequence was used as a probe to localize the gpIII gene to the HindIII B fragment of the VZV genome. An analysis of the DNA sequence from this region revealed an open reading frame (ORF) encoding 841 amino acids. Rabbit antisera against three synthetic peptides derived from the putative gpIII gene recognized a protein which comigrated with gpIII in Western blots and immunoprecipitation analysis. Preclearing with a monoclonal antibody to gpIII specifically abolished immunoprecipitation of this protein. Also a polypeptide translated from mRNA selected by the putative gpIII gene could be immunoprecipitated by the anti-peptide sera. Therefore, we conclude that gpIII is encoded by the identified ORF in HindIII B. In addition, gpIII is implicated as essential for the cell-to-cell spread of VZV.     

Identification and structure of the gene encoding gpII, a major glycoprotein of varicella-zoster virus

The genome of varicella-zoster virus (VZV) encodes three major families of glycoproteins (gpI, gpII, and gpIII). mRNA from VZV-infected cells was hybrid selected using a library of VZV recombinant plasmids and translated in vitro; polypeptide products were immunoprecipitated by polyclonal monospecific guinea pig antibodies to gpII. The mRNA encoding a 100-kD polypeptide precipitable by anti-gpII antibodies mapped to the HindIII D fragment near the center of the UL region. DNA sequence analysis of this region of the VZV genome revealed a 2.6-kbp open reading frame (ORF) potentially encoding a 98-kDa polypeptide possessing the characteristics of a glycoprotein. The 100-kDa polypeptide was specified by mRNA isolated by hybrid selection using a plasmid containing part of the 2.6-kbp ORF, and immunoprecipitation of this protein by anti-gpII antibodies and by convalescent zoster serum was blocked specifically by purified gpII. We conclude that the 2.6-kbp ORF encodes gpII. The imputed primary amino acid sequence of gpII shows a high degree of homology to that of herpes simplex virus type 1 (HSV-1) gB, a result consistent with the equivalent map locations of the respective genes in the HSV and VZV genomes and with the recently reported serological cross-reactivity of HSV-1 gB and VZV gpII. Unlike the mature gene products of gB, those of gpII have been described as a pair of glycoproteins with approximate molecular weights of 60 kDa in reducing gels, products of a single glycoprotein species with approximate mol mass of 125-140 kDa in nonreducing gels. Amino-terminal sequences of purified gpII were determined and compared to the imputed amino acid sequence. This comparison implies that the primary translational product is cleaved approximately into halves in vivo and suggests that mature gpII is a disulfide-linked heterodimer.     

The protective immune response induced by B cell epitope of classical swine fever virus glycoprotein E2

Classical swine fever virus (CSFV) envelope glycoprotein E2 is a major protective immunogen responsible for eliciting neutralizing antibodies and conferring protective immunity against the virus. Based on the core sequence (TAVSPTTLR, 829-837 aa) of the B cell linear epitope of the CSFV E2 protein identified by Lin et al., two oligonucleotides MF and MR were synthesized and used to construct by PCR a gene cassette encoding a 15 amino acid polypeptide M (CTAVSPTTLRTEVVK), which spans 828-842 amino acids of E2. The gene cassette was fused in-frame to 3' terminal of glutathione S transferase gene (GST) of the prokaryotic expression vector pGEX-6p-1, resulting in the recombinant plasmid pGEX-M. After transformation into Escherichia coli BL21 a soluble fusion protein GST-M with expected size of 28 kDa was expressed after inducing with isopropyl-beta-d-thiogalactoside (IPTG). Enzyme-linked immunosorbent assay (ELISA) and Western blot analysis showed that the purified GST-M had good reactivity with swine anti-CSFV serum and rabbit anti-CSFV E2 serum. Further vaccination trials showed that the fusion protein GST-M could elicit effectively immune response protecting rabbits and pigs from virulent challenge. This study showed a possibility for developing epitope-based vaccines against CSFV.     

戊型肝炎病毒线性抗原表位克隆重及表达的初步 …

目的 探讨戊型肝炎病毒线性抗原表位的克隆重表达策略。方法 合成ＨＥＶ ＯＲＦ３羧基端１９个氨基酸编码ＤＮＡ序列，在序列的５’端加入了两个甘氨酸序列，通过基因扩增载体中的５’ＧＧＧＧ／ＣＣＣＣ非对称粘性末端，实现线性表位序列的首尾串联排列。结果 获得了２ｍｅｒ、４ｍｅｒ、６ｍｅｒ、８ｍｅｒ４个首尾串联的重复序列；分别克隆重到表达载体ｐＱＥ３０中后，在ＩＰＴＧ诱导下主要以包涵体形式表达，其中４ｍｅｒ蛋     

Classical swine fever virus: Antigenic architecture of the E2 glycoprotein elucidated by epitope mapping using synthetic peptides

Detailed epitope characterization of the major envelope glycoprotein E2 of the classical swine fever virus (CSFV) is important for our understanding of interactions between the virus and the host immune system, as well as for the development of CSFV-specific diagnostic assays and epitope- or peptide-based marker vaccines. As was shown previously by competitive binding assay, monoclonal antibodies obtained by our group recognize eight epitopes on the E2 protein. Here, we report mapping of five linear, nonoverlapping B-cell epitopes that use a set of synthetic peptides, which encompass the full sequence of the CSFV E2 protein (Shimen strain). Two of the epitopes are located in the antigenic domain A of the E2, while another three belong to a highly structured region of this glycoprotein. The alignment of the identified gene sequences was performed for 12 CSFV strains, three strains of bovine viral diarrhea virus (BVDV), and two strains of the border disease virus (BDV). The data obtained could be used to improve CSFV diagnostic assays, as well as to investigate the effects of aminoacid substitutions in E2 on its antigenic properties.     

Identification by monoclonal antibodies of a new epitope in the glycoprotein complex of Sindbis virus

Monoclonal antibodies against a deletion mutant of Sindbis virus were produced and characterized in order to determine the fine mapping and functional activities of single viral epitopes. All monoclonal antibodies so far tested showed a certain degree of reciprocal competition and were directed against an antigenic determinant which was present only on the undissociated complex of the E1 and E2 glycoproteins. A biological assay measuring viral haemagglutination showed no decrease in the titre of viral samples preincubated with monoclonal antibodies. Conversely, a reduction in viral infectivity was demonstrated, particularly with two of these antibodies. The results suggest that the antibodies which we characterized seem to recognize a new epitope which is represented on both glycoproteins on the surface of this mutant of Sindbis virus.     

戊型肝炎病毒新潜在中和性表位的发现

目的 探讨戊型肝炎病毒(HEV)衣壳蛋白是否存在除主要免疫优势中和表位aa459～606以外的其他中和表位.方法 通过对几株单克隆抗体及其表位的性质进行分析,对比位于主要免疫优势表位区aM59～606和位于该表位N端序列aa394～458区域的数个表位的中和活性.结果 发现位于aa423～437的表位对应的单抗具有潜在中和活性,不同于已知的HEV中和性表位(aa459～606),该表位是一个线性非免疫优势表位.结论 HEV ORF2 aa423～437为新的潜在的线性非免疫优势中和表位,该发现丰富了对HEV衣壳结构的认识,为HEV预防与治疗提供了新的针对靶点.     

Immunization of monkeys with varicella-zoster virus glycoprotein antigens and their response to challenge with simian varicella virus

African green monkeys (Cercopithecus aethiops) were immunized with three intramuscular injections of gpI, gpII, or gpIII glycoprotein antigens of varicella-zoster virus (VZV). Antibody responses to VZV were determined by enzyme-linked immunosorbent assay (ELISA) and to simian varicella virus (SVV) by immunofluorescence and by serum neutralization assays. Two weeks following the third immunization with VZV glycoproteins, the monkeys were challenged by inoculation of SVV. Antibodies to gpII or gpIII partially prevented infection by SVV, while the presence of antibodies to gpI was ineffective in preventing disease induced by SVV challenge. Factors affecting the immunogenicity of these antigens in this model are discussed.