Varicella-zoster virus (VZV) glycoprotein E is a serological antigen for detection of intrathecal antibodies to VZV in central nervous system infections, without cross-reaction to herpes simplex virus 1

Herpes simplex virus 1 (HSV-1) and varicella-zoster virus (VZV) cause serious central nervous system (CNS) diseases that are diagnosed with PCR using samples of cerebrospinal fluid (CSF) and, during later stages of such infections, with assays of intrathecal IgG antibody production. However, serological diagnoses have been hampered by cross-reactions between HSV-1 and VZV IgG antibodies and are commonly reported in patients with herpes simplex encephalitis (HSE). In this study we have evaluated VZV glycoprotein E (gE) as a new antigen for serological diagnosis of VZV-induced CNS infections. Paired samples of CSF and serum from 29 patients with clinical diagnosis of VZV CNS infection (n = 15) or HSE (n = 14), all confirmed by PCR, were analyzed. VZV gE and whole VZV were compared as antigens in enzyme-linked immunosorbent assays (ELISAs) for serological assays in which the CSF/serum sample pairs were diluted to identical IgG concentrations. With the gE antigen, none of the HSE patients showed intrathecal IgG antibodies against VZV, compared to those shown by 11/14 patients using whole-VZV antigen (P < 0.001). In the patients with VZV infections, significantly higher CSF/serum optical density (OD) ratios were found in the VZV patients using the VZV gE antigen compared to those found using the whole-VZV antigen (P = 0.001). These results show that gE is a sensitive antigen for serological diagnosis of VZV infections in the CNS and that this antigen was devoid of cross-reactivity to HSV-1 IgG in patients with HSE. We therefore propose that VZV gE can be used for serological discrimination of CNS infections caused by VZV and HSV-1.     

The insulin degrading enzyme binding domain of varicella-zoster virus (VZV) glycoprotein E is important for cell-to-cell spread and VZV infectivity, while a glycoprotein I binding domain is essential for infection

Varicella-zoster virus (VZV) glycoprotein E (gE) interacts with glycoprotein I and with insulin degrading enzyme (IDE), which is a receptor for the virus. We found that a VZV gE deletion mutant could only be grown in cells expressing gE. Expression of VZV gE on the surface of cells did not interfere with VZV infection. HSV deleted for gE is impaired for cell-to-cell spread; VZV gE could not complement this activity in an HSV gE null mutant. VZV lacking the IDE binding domain of gE grew to peak titers nearly equivalent to parental virus; however, it was impaired for cell-to-cell spread and for infectivity with cell-free virus. VZV deleted for a region of gE that binds glycoprotein I could not replicate in cell culture unless grown in cells expressing gE. We conclude that the IDE binding domain is important for efficient cell-to-cell spread and infectivity of cell-free virus.     

Recombinant glycoprotein E produced in mammalian cells in large-scale as an antigen for varicella-zoster-virus serology

A recombinant glycoprotein E (gE) from varicella-zoster virus (VZV) was generated and produced in Chinese Hamster Ovary (CHO) cells, in the development of a specific antigen for analysis of IgG antibodies to VZV. Several stable gE-secreting clones were established and one clone was adapted to growth in serum-free suspension culture. When the cells were cultured in a perfusion bioreactor, gE was secreted into the medium, from where it could be easily purified. The recombinant gE was then evaluated as a serological antigen in ELISA. When compared to a conventional whole virus antigen, the VZV gE showed similar results in ELISA-based seroprevalence studies of 854 samples derived from blood donors, students, ischemic stroke patients and their controls, including samples with border-line results in previous analyses. Eight samples (0.9%) were discordant, all being IgG-negative by the VZV gE ELISA and positive by the whole virus ELISA. The sensitivity and specificity of the VZV gE ELISA were 99.9% and 100%, respectively, compared to 100% and 88.9% for the VZV whole virus ELISA. The elderly subjects showed similar reactivities to both antigens, while VZV gE gave lower signals in the younger cohorts, suggesting that antibodies to gE may increase with age. It was concluded that the recombinant VZV gE from CHO cells was suitable as a serological antigen for the detection of IgG antibodies specific for VZV.     

Ability of yeast Ty-VLPs (virus-like particles) containing varicella-zoster virus (VZV)gE and assembly protein fragments to induce in vitro proliferation of human lymphocytes from VZV immune patients.

Yeast Ty virus-like particles (VLPs) containing viral protein inserts have previously been shown to be potent immunogens, inducing both humoral and cell mediated immunity (CMI). The antigenicity of hybrid VLPs containing fragments of the varicella-zoster virus (VZV) gE protein or the assembly protein (AP) was assessed by lymphocyte proliferation. Peripheral blood mononuclear cells (PBMCs) from patients with a recent natural VZV infection were stimulated in vitro with VZV-VLPs together with control antigens. PBMC samples from both varicella (85%) and zoster (75%) patients proliferated in responses to at least one of the gE VZV-VLPs. As reported for the first time, VZV specific lymphocyte responses were also identified towards the VZV AP in two varicella and two zoster patient samples. The results demonstrate specific CMI recognition of the VZV gE fragments tested and the VZV AP delivered in the form of recombinant Ty-VLPs, and highlights their potential use as a recombinant antigen delivery system for vaccination.     

Membrane fusion mediated by herpesvirus glycoproteins: the paradigm of varicella-zoster virus

Varicella-zoster virus (VZV) is well known for its propensity to cause polykaryons (syncytia) in the vesicles within infected skin. Similarly in cultured cells, VZV induces extensive syncytial formation by virus-mediated cell-to-cell fusion. Statistical analyses of fusion parameters demonstrated three-way interactive effects among all three tested variables (incubation temperature, cell type and virus strain). For example, fusion was greatly enhanced at 33 degrees C vs 37 degrees C; also fusion was pronounced in epidermal cells but negligible in fibroblast cells. As with all herpesviruses, VZV gH was a major fusogen. VZV cell fusion was inhibited by antibody to gH, but surprisingly was enhanced by antibody to gE. Other evidence implicating a role for VZV gE in the fusion process was provided by two mutant viruses, in which gE cell surface expression was enhanced. Under transfection conditions, VZV fusion formation occurred after expression of the gH/gL complex; in contrast, pseudorabies virus requires expression of gH, gL and gB, while the herpes simplex virus (HSV) types 1 and 2 require the quartet of gH, gL, gB and gD. VZV has no gD gene and no apparent gD functional homologue. On the other hand, VZV gE exerts a greater effect than HSV gE on membrane fusion. Taken together, the data in this review suggest that VZV has evolved viral glycoprotein machinery more geared toward cell-to-cell fusion (fusion-from-within) than toward virus-to-cell fusion (entry/fusion-from-without), as a means for syncytium formation within the human epidermis.     

Expression of the simian varicella virus glycoprotein E

Simian varicella virus (SVV) is closely related to human varicella-zoster virus (VZV) and induces a varicella-like disease in nonhuman primates. The SVV genome encodes a glycoprotein E (gE) which is homologous to the gE of VZV and other alphaherpesviruses. The SVV gE was expressed in Escherichia coli and rabbits were immunized with the recombinant gE fusion proteins to generate polyclonal gE antiserum. Immunofluorescence and immunoprecipitation analyses demonstrated that the SVV gE is expressed on the surface and within SVV-infected cells. The gE is also expressed on SVV virions as indicated by serum neutralization assay. The mature SVV gE is glycosylated and is similar in size ( approximately 100 kd) to the mature VZV gE. Immunohistochemical analysis detected gE within skin vesicles and lung tissue of SVV-infected monkeys. Analysis of the humoral immune response to gE in an SVV-infected monkey determined that anti-gE antibody is induced as early as day 9 postinfection and persists at high titer for longer than 4 months. The simian varicella model offers an opportunity to investigate the role of gE in viral pathogenesis and immunity and to evaluate its potential as a varicella vaccine.     

Varicella zoster virus glycoprotein E-specific CD4+ T cells show evidence of recent activation and effector differentiation, consistent with frequent exposure to replicative cycle antigens in healthy immune donors

Varicella zoster viru (VZV)-specific T cell responses are believed to be vital in recovery from primary VZV infection and also in the prevention of viral reactivation. While glycoprotein E (gE) is the most abundant and one of the most immunogenic proteins of the virus, there are no data addressing potential T cell epitopes within gE, nor the phenotype of specific T cells. Using interferon gamma enzyme-linked immunospot assays and intracellular cytokine assays, we identified gE-specific immune responses in 20 adult healthy immune donors which were found to be dominated by the CD4+ subset of T cells. We characterized three immune dominant epitopes within gE restricted through DRB1*1501, DRB1*07 and DRB4*01, and used DRB1*1501 class II tetrameric complexes to determine the ex vivo frequency and phenotype of specific T cells. In healthy immune donors, the cells were largely positive for CCR7, CD28 and CD27, but expressed variable CD62L and low levels of cutaneous lymphocyte associated antigen with evidence of recent activation. In summary, we show that circulating gE-specific CD4+ T cells are detected at a relatively high frequency in healthy immune donors and show evidence of recent activation and mixed central and effector memory phenotype. These data would be compatible with frequent exposure to replicative cycle antigens in healthy donors and are consistent with a role for gE-specific CD4+ T cells in the control of viral replication.     

Varicella-zoster virus gpI and herpes simplex virus gE: phosphorylation and Fc binding

gpI, the predominant varicella-zoster virus (VZV) envelope glycoprotein, was shown to be phosphorylated exclusively on serine and threonine residues, and phosphorylated gpI was detected in isolated virions. In cells infected with herpes simplex virus type 1 (HSV-1), a related neurotropic alpha-herpesvirus, HSV gE, the homolog to VZV gpI, and HSV gB, the homolog to VZV gpII, were also phosphorylated. The phosphate on gB and gE was alkali labile and resistant to endo H, suggesting linkage to serine and/or threonine. Although VZV gpI and HSV gE share sequence homology and similar post-translational modifications, no Fc-binding activity similar to that associated with gE was detected for gpI or any of the VZV glycoproteins.     

Purification, characterization and immunogenicity of recombinant varicella-zoster virus glycoprotein gE secreted by Chinese hamster ovary cells

The gene of the varicella-zoster virus (VZV) glycoprotein gE, engineered to code for a truncated molecule lacking the anchor and carboxy-terminal tail domains, was transfected into Chinese hamster ovary (CHO) cells via the pEE14 mammalian expression vector. One recombinant cell line, CHO-gE-2–9, secreted high levels of truncated gE into the culture medium. The product was purified to near homogeneity by a combination of anion exchange, hydrophobic and metal-chelate chromatographies. Purified recombinant gE showed the expected amino-terminal sequence and its glycosylation pattern proved similar to that of the natural product. When injected into mice, using either Freund's or alum as adjuvant, the native truncated gE induced complement-dependent neutralizing antibodies. In contrast, when the molecule was first denatured, it lost immunogenicity with alum. These data show that the recombinant gE, although truncated, could potentially be included in a subunit vaccine against VZV infection.     

Varicella-zoster virus infection facilitates VZV glycoprotein E trafficking to the membrane surface of melanoma cells

Varicella-zoster virus glycoprotein E (gE) is the most abundant VZV glycoprotein on the surface of virus-infected cells. VZV gE has targeting sequences for the trans-Golgi network (TGN) and is transported from the ER to the TGN in infected and gE-transfected cells. In this study, VZV gE expressing melanoma cell lines were generated. gE is expressed under the control of the reverse Tet repressor (Tet-On). gE induced by Tet-On is retained at the ER as well as in the cis Golgi by immunofluorescence confocal microscopy. To test whether other viral protein(s) may facilitate gE trafficking and surface localization, MSPgE-vOka virus that contains MSPgE in place of wt gE was made. MAb 3B3 anti-gE does not bind to MSPgE. This MAb was used to track the localization of gE in Met-gE cells post MSPgE-vOka infection. gE became detectable mostly at the TGN and on the cell surface after viral infection. These data indicate that viral proteins facilitate the trafficking and cell surface expression of gE.     

Are false negative direct immnufluorescence assays caused by varicella zoster virus gE mutant strains?

Strains of Varicella zoster virus (VZV) have been described recently in which a single base mutation in the gE epitope abrogates binding of the 3B3 monoclonal antibody, which is widely used for virus detection in diagnostic laboratories. These strains, named VZV-MSP, are associated with a distinct phenotype in both in vitro culture and in SCID-hu mice. We investigated the possibility that negative direct immunofluorescence results, using the 3B3 antibody, where the presence of virus was confirmed by polymerase chain reaction (PCR) or tissue culture are due in some cases to the MSP strain of VZV. A total of 249 vesicle fluid specimens from people with suspected shingles were examined using direct immunofluorescence, tissue culture and a nested multiplex PCR for VZV, herpes simplex virus type 1 (HSV-1) and 2 (HSV-2). VZV was detected in 218 of 249 (87.6%) cases. Forty-five confirmed VZV specimens, but with negative (30) or indeterminate (15) immunofluorescence results, were analysed further. PCR was used to amplify a fragment in ORF 68 that encodes the VZV gE ectodmain recognised by 3B3 antibody. The fragments were sequenced and analysed for the single base change G448A (D150N), which is present in VZV-MSP as compared with the reference Dumas strain. No VZV gE mutant (MSP/MSP-like) was detected. Overall, PCR was found to be the most sensitive method of confirming VZV infection. False negative VZV immunofluorescence results are unlikely to be due to virus variants.     

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.     

Influence of Frequent Infectious Exposures on General and Varicella-Zoster Virus-Specific Immune Responses in Pediatricians

Reexposure to viruses is assumed to strengthen humoral and cellular immunity via the secondary immune response. We studied the effects of frequent exposure to viral infectious challenges on immunity. Furthermore, we assessed whether repetitive exposures to varicella-zoster virus (VZV) elicited persistently high immune responses. Blood samples from 11 pediatricians and matched controls were assessed at 3 time points and 1 time point, respectively. Besides the assessment of general immunity by means of measuring T-cell subset percentages, antibody titers and gamma interferon (IFN-γ)/interleukin 2 (IL-2)-producing T-cell percentages against adenovirus type 5 (AdV-5), cytomegalovirus (CMV), tetanus toxin (TT), and VZV were determined. Pediatricians had lower levels of circulating CD4⁺-naive T cells and showed boosting of CD8⁺ effector memory T cells. Although no effect on humoral immunity was seen, repetitive exposures to VZV induced persistently higher percentages of IFN-γ-positive T cells against all VZV antigens tested (VZV glycoprotein E [gE], VZV intermediate-early protein 62 [IE62], and VZV IE63) than in controls. T cells directed against latency-associated VZV IE63 benefitted the most from natural exogenous boosting. Although no differences in cellular or humoral immunity were found between the pediatricians and controls for AdV-5 or TT, we did find larger immune responses against CMV antigens in pediatricians. Despite the high infectious burden, we detected a robust and diverse immune system in pediatricians. Repetitive exposures to VZV have been shown to induce a stable increased level of VZV-specific cellular but not humoral immunity. Based on our observations, VZV IE63 can be considered a candidate for a zoster vaccine.     

Stability of a varicella-zoster virus glycoprotein E epitope

Summary.  The epitope stability of a varicella-zoster virus (VZV) glycoprotein E (gE) was analyzed with monoclonal antibodies (mAbs) in cells infected with different passages of various VZV strains and isolates. The gE-specific mAbs recognized same antigenic sites (epitopes) in VZV isolates with various passage history. All VZV strains and virus-isolates reacted with an anti-gE monoclonal antibody by immunoprecipitation, or indirect fluorescent antibody staining test. Sera from VZV seropositive individuals reacted with a truncated VZV gE glycoprotein, designated TgpI-511. Also, human mononuclear cells (MNCs) stimulated with TgpI-511 glycoprotein were shown to produce VZV-specific antibodies in vitro. The results demonstrated the stability of these gE epitopes tested in this study in TgpI-511 and among the VZV-isolates obtained from different passages. These results also suggest that VZV glycoproteins as well as live attenuated or killed varicella vaccines containing these epitopes could be used as therapeutic booster vaccines in adults and the elderly to prevent zoster. Received May 12, 1999/Accepted August 20, 1999     

Neutralizing antibody responses induced by varicella-zoster virus gE and gB glycoproteins following infection,reactivation or immunization

The purpose of this study was to compare the antibody responses to varicella-zoster virus (VZV) gE and gB after natural VZV infection and after vaccination with live attenuated OKA vaccine in order to determine the relative importance of these proteins as components of a subunit vaccine. Anti-VZV antibody titers determined by IFA were of the same order of magnitude in sera from individuals with a history of varicella and in vaccinated children but higher in individuals given booster vaccination. The titers of anti-gE and anti-gB antibodies were measured by ELISA using recombinant gE or gB as capture antigen. From these experiments, it appears that the ratio of anti-gE to anti-gB antibody is highly variable from one individual to another but relatively stable over a long period of time for a particular individual, even after a zoster episode. Neutralizing antibodies directed against gE or gB were also measured by subtracting the neutralization titers obtained before and after depletion of the specific antibodies on immobilized recombinant gE, gB, or both. This showed that, with respect to neutralization, anti-gE and anti-gB are equally prevalent in vaccinated children and that anti-gE is generally, but not always, predominant over anti-gB in VZV-infected individuals. Finally, antibodies to these two glycoproteins appear to be predominant among the neutralizing antibodies directed to other VZV antigens. J. Med. Virol. 53:63–68, 1997. © 1997 Wiley-Liss, Inc.     

Targets of the humoral autoimmune response in multiple sclerosis

Multiple sclerosis (MS) is an inflammatory disorder of the central nervous system (CNS) with heterogeneous clinical, genetic and pathophysiological characteristics. The establishment of reliable biomarkers for diagnosis, prognosis and treatment of MS has therefore proven to be very difficult. During the last decades, mounting evidence has been collected for the involvement of B cells and antibodies in MS pathogenesis. A wide variety of autoantibodies has been described in MS and these autoantibodies could be useful biomarkers for MS.     

Increased prevalence of varicella zoster virus DNA in cerebrospinal fluid from patients with multiple sclerosis

In order to investigate the possible involvement of viruses in Multiple Sclerosis (MS), the study evaluated the presence of viral genomic sequences in cerebrospinal fluid (CSF), as markers of viral replication within the central nervous system (CNS). A total of 85 CSF samples were collected from 38 MS patients, 28 patients with other neurological diseases and 19 subjects without neurological diseases. Using nested-PCR, the investigation focused on the presence of human herpes virus DNA, including herpes simplex virus 1 (HSV-1) and 2 (HSV-2), the Epstein-Barr virus (EBV), varicella zoster virus (VZV), human cytomegalovirus (HCMV), human herpes virus 6 (HHV-6) and JC virus (JCV). All the CSF samples from the individuals without neurological diseases were negative for viral DNA. Genomic sequences of HSV-1, HCMV, EBV, HHV6, and JCV were found in patients with MS and other neurological diseases without significant differences between the two groups. VZV DNA was detected more frequently (P < 0.05) in the MS group (31.6%), particularly among the relapsing-remitting MS patients (43.5%), compared with patients with other neurological diseases (10.7%). In addition, the results indicated that JCV and HHV-6 were replicating actively in the CNS of a small, but significant number of patients with MS and other neurological diseases. Most importantly, the study revealed a high frequency of VZV DNA in the CSF of patients with MS, suggesting a possible role of this virus in the pathogenesis of MS.     

A novel varicella-zoster virus gE mutation discovered in two Swedish isolates.

BACKGROUND: Two VZV glycoprotein E (gE D150N) mutant strains were collected in North America in 1995 and in 1999. We now report a novel VZV gE mutant virus discovered in Europe in two VZV strains collected in Stockholm, Sweden, in 1990 and 1999. OBJECTIVES: To characterize the two isolates identified among a total of 634 VZV isolates collected over a 15-year period at the Karolinska University Hospital. STUDY DESIGN: VZV genes were amplified by polymerase chain reaction and their sequences were compared to the genomic sequence of VZV-Dumas prototype strain. RESULTS: A mutation within the gE gene in an epitope recognized by the 3B3 monoclonal antibody was identified in both isolates. A different residue was changed (R152S) compared to the North American strains (D150N). CONCLUSIONS: The occurrence of a VZV gE mutant virus is unusual, but probably a recurring event in Europe and North America. It is unknown whether widespread varicella vaccination programs will alter the rate at which these mutant viruses are isolated.     

The requirement of varicella zoster virus glycoprotein E (gE) for viral replication and effects of glycoprotein I on gE in melanoma cells

The glycoprotein E (gE) of varicella zoster virus (VZV), encoded by ORF68, is the most abundant viral glycoprotein. In the current experiments, we demonstrated that ORF68 deletion was incompatible with recovery of infectious virus from VZV cosmids. Replacing ORF68 at a nonnative AvrII site in the genome restored infectivity. Further, we found that VZV gE could be expressed under the control of the Tet-On promoter in stably transfected melanoma cell lines (Met-gE cells) without evidence of toxicity. In these Met-gE cells, gE colocalized with gamma-adaptin, a trans Golgi network marker, in perinuclear sites, but did not reach plasma membranes. In order to investigate how infection altered gE localization, we made a recombinant virus, vOka-MSPgE, with ORF68 from the VZV MSP strain. VZV MSP encodes a mutant gE protein (D150N) that lacks the mAb epitope, 3B3 (Santos et al., Virology 275, 306-317, 2000), whereas Met-gE protein binds mAb 3B3. Within 48 h after Met-gE cells were infected with vOka-MSPgE, the steady-state distribution of Met-gE protein extended beyond the perinuclear areas to other cytoplasmic sites and to plasma membranes. A second recombinant, vOka-MSPgE without gI (vOka-MSPgEdeltagI), was constructed to investigate Met-gE protein distribution in the absence of gI. The redistribution of Met-gE protein which was observed by 48 h after vOka-MSPgE infection did not occur until 5 days (140 h) within vOka-MSPgEdeltagI infected cells. After vOka-MSPgE infection of Met-gE cells, most Met-gE protein was in the final 94K mature form by 72 h. However, progression to predominance of mature gE was delayed in Met-gE cells infected with vOka-MSPgEdeltagI. These observations confirm our hypothesis that VZV gE is essential, based upon the demonstration of restored infectivity after replacing ORF68 in a nonnative site in the genome, and provide further evidence of the role of gI in facilitating the maturation and intracellular distribution of this critical VZV glycoprotein.     

Identification and functional characterization of the Varicella zoster virus ORF11 gene product

The deletion of ORF11 severely impaired VZV infection of human skin xenografts. Here, we investigate the characteristics and functions of the ORF11 gene product. ORF11 is expressed as a 118 kDa polypeptide in VZV-infected cells; the protein is present in the nucleus and cytoplasm and is incorporated into VZ virions. Although ORF11 had little effect in transactivating VZV gene promoters in transfection assays, deleting ORF11 from the virus was associated with reduced expression of immediate early proteins IE4, IE62 and IE63, and the major glycoprotein, gE. ORF11 was identified as an RNA binding protein and its RNA binding domain was defined. However, disrupting the ORF11 RNA binding domain did not affect skin infection, indicating that RNA binding capacity, conserved among the alphahepesviruses homologues, is not essential while the contribution of ORF11 to the expression of the IE proteins and gE may be required for VZV pathogenesis in skin in vivo.