Nucleotide sequence analysis of varicella-zoster virus glycoprotein E epitope coding regions.

Varicella-zoster virus (VZV) glycoprotein E [gE] contains 623 amino acid residues. Fifty percent of the gE gene, codons 39 to 344 that encompasses two epitope coding regions e1 and c1, was sequenced and analyzed for variation among the 30 VZV isolates. A total of eleven isolates showed variance when compared with Dumas VZV strain sequence through base substitutions, with two isolates showing an amino acid change of tryptophan to arginine outside the coding regions of the epitopes e1 and c1 that are recognized by monoclonal antibodies 4F9 and c1, respectively. The results suggest that these epitopes were stable in the various VZV isolates. Thus, VZV glycoproteins with conserved epitopes are suitable candidates for both primary and booster vaccines.

Zoster in patients infected with HIV: a review.

Varicella-zoster virus (VZV), a member of the human herpesvirus family, causes childhood chickenpox (varicella), becomes latent in sensory ganglia, and reactivates years later in immunocompromised and elderly persons to produce shingles (herpes zoster). Early in the AIDS epidemic, zoster was noted in adults and children infected with HIV. Severe and debilitating zoster-associated dermatological, ophthalmic, and neurological complications may occur in patients infected with HIV. Antiviral therapy can modify the duration of zoster and alleviate its attendant complications. Varicella vaccine may boost the immunity and prevent virus reactivation. VZV immune globulin (VZIG) prevents or modifies clinical illness in persons who have been exposed to varicella or zoster.

Stability of a varicella-zoster virus glycoprotein E epitope.

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.

Boosting immune response with a candidate varicella-zoster virus glycoprotein subunit vaccine.

A varicella-zoster virus (VZV) seropositive individual was immunized with 100 micrograms of purified VZV TgpI-511 glycoprotein subunit antigen formulated with monophosphoryl lipid A. Serum samples were obtained during a 40-day period post-immunization (PI) and analysed by immunoprecipitation and virus neutralization tests. The results from immunoprecipitation studies revealed an increase in VZV anti-gpI antibody titer as early as 6 days PI which continued to rise during 40 days PI. In addition, virus neutralization tests showed a 21.0% VZV neutralization 6 days PI with an increase to a 96.7% VZV neutralization 40 days PI. These results suggested that the candidate VZV glycoprotein subunit vaccine (TgpI-511) was capable of boosting the production of neutralizing antibodies in the immunized VZV seropositive human subject.

Antibody-binding sites on truncated forms of varicella-zoster virus gpI(gE) glycoprotein.

Varicella-zoster virus (VZV)-seropositive human sera were shown to be reactive with the truncated VZV gpI(gE) candidate subunit vaccine (TgpI-511). To identify the location of antibody-binding sites (epitopes) on TgpI-511, three truncated forms of TgpI-511 glycoprotein (TgpI-124, TgpI-160, TgpI-316) DNA encoding the N-terminal region of this glycoprotein with amino acid residues of 124, 160 and 360, respectively, were inserted into the vaccinia virus genome. Infection of cells with recombinant vaccinia viruses resulted in the secretion of all three truncated gpI(gE) as well as TgpI-511 from the infected cells. Immunoprecipitation of these truncated glycoproteins with VZV-seropositive human sera and gpI(gE)-specific monoclonal antibodies identified the location of four new antibody-binding sites on the VZV TgpI-511 glycoprotein. In addition, tunicamycin treatment and O-glycanase digestion revealed the presence of both N-linked and O-linked oligosaccharides on TgpI-511. These results revealed the location of new epitopes on VZV TgpI-511 and demonstrated that the epitopes on TgpI-511 were recognized by human sera from VZV-seropositive individuals.

Antigenicity of a candidate varicella-zoster virus glycoprotein subunit vaccine.

A 1642 bp DNA fragment, encoding the N-terminal region and 511 amino acid residues of varicella-zoster virus (VZV) glI gene, was inserted into vaccinia virus genome. The expression of recombinant vaccinia virus (designated VVTgpI-511) yielded the synthesis of a 60 kDa protein species which was processed to a secretory 76 kDa polypeptide (designated TgpI-511). The antigenicity of this protein was examined by subcutaneous inoculation of one rabbit with 100 micrograms purified TgpI-511 in the Ribi adjuvant system. The animal was boosted 3 weeks after the initial inoculation and antisera were tested 7 days after the last injection by immunoprecipitation and neutralization tests. The results showed that rabbit antibodies to TgpI-511 (RAnti-TgpI-511) were reactive with purified TgpI-511 as well as native gpI in VZV-infected cells. In addition, TgpI-511 was capable of eliciting complement-dependent VZV neutralizing antibodies. These results suggested that the purified preparation of TgpI-511 may have the potential to be used as a candidate VZV subunit vaccine.

Latency in vitro of varicella-zoster virus in cells derived from human fetal dorsal root ganglia.

A potential in vitro model of varicella-zoster virus (VZV) latency was developed. Dissociated human dorsal root ganglion cultures were infected with VZV and maintained for 1 wk in the presence of bromovinyl arabinosyl uracil, a potent inhibitor of VZV. Seven to 21 d after removing the inhibitor (> or = 14 d after infection), the cells were trypsinized, passed to monolayers of human embryonic lung fibroblasts, and observed for VZV reactivation as indicated by typical cytopathic effects and the appearance of VZV antigens. VZV reactivated from 56% of the cultures containing both neurons and satellite cells but not from cultures specifically enriched for either neurons, satellite cells, or ganglion-derived fibroblasts. The failure to isolate VZV from cell suspensions that were sonicated before cocultivation with fibroblasts indicated that infectious VZV was not present before reactivation. Moreover, immunohistochemical and immunoprecipitation studies revealed no VZV-specific antigens in any cultures before the reactivation stimulus. VZV antigens were detected after trypsinization and cocultivation. These findings suggest that cultures containing both neurons and satellite cells provide a model system for VZV persistence that possesses many properties of a latent infection.

Specific lysis of targets expressing varicella-zoster virus gpI or gpIV by CD4+ human T-cell clones.

Varicella-zoster virus (VZV)-specific CD4-positive T cells are known to lyse targets expressing VZV antigen, but little is known of the glycoprotein specificity or phenotype of these cells. To test the ability of T cells to distinguish between gpI and gpIV (which share an antibody-defined epitope), we prepared clones from blood from four healthy individuals by limiting dilution. Among 68 T-cell clones from four donors which were VZV specific in tests of proliferation, 30 lysed autologous Epstein-Barr virus-transformed lymphoblasts which had been superinfected with a recombinant vaccinia virus which included the whole VZV gpI sequence. These clones were characterized as major histocompatibility complex class II restricted by inhibition of their cytotoxicity with HLA-DR and CD4 monoclonal antibodies. Twenty-one clones lysed targets expressing gpIV. Fifteen of these clones lysed targets expressing gpI and gpIV. Four clones with gpI-gpIV specificity were examined in detail, and their dual specificity was confirmed by cold target inhibition. These four clones failed to kill target cells infected with a mutant gpIV recombinant vaccinia virus from which amino acid residues 212 to 354 had been deleted. This region includes one of the two gpIV decapeptides which have 50% homology with amino acids 111 to 121 of gpI. Our data confirm that T-cell-receptor-associated structures are required for specific lysis of VZV targets and indicate that (i) gpI-specific CD4 cytotoxic T cells outnumber gpIV-specific T cells in blood and (ii) 50% of gpI-specific T-cell clones also lyse gpIV-expressing targets.

Neutralizing antibodies induced by recombinant vaccinia virus expressing varicella-zoster virus gpIV.

Monoclonal antibodies generated against varicella-zoster virus (VZV) glycoprotein I (gpI) also recognize VZV gpIV (A. Vafai, Z. Wroblewska, R. Mahalingam, G. Cabirac, M. Wellish, M. Cisco, and D. Gilden, J. Virol. 62:2544-2551, 1988). To determine whether the virus-neutralizing activity of these antibodies belongs to gpI, gpIV, or both, the open reading frame encoding gpIV was inserted into the vaccinia virus genome. Immunoprecipitation of recombinant vaccinia virus-infected cells with anti-gpIV monoclonal antibody yielded synthesis and processing of gpIV similar to those expressed in VZV-infected cells. Antibodies raised against VVgpIV in a rabbit recognized both native gpI and gpIV and neutralized VZV infectivity. In addition, antibodies raised against recombinant vaccinia virus carrying VZV gpI neutralized VZV infection. These results indicate a structural relationship between VZV gpI and gpIV and show that gpI and gpIV each induce virus-neutralizing antibody.

In vitro transcription and translation of proteins encoded by the BamHI-B genomic fragment of herpes simplex virus-1.

The BamHI-B DNA fragment of herpes simplex virus type-1 (HSV-1) is associated with intraperitoneal pathogenicity. Among the recently mapped RNA transcripts from this fragment (15), one was reported to be associated with latency. To relate the RNA transcripts to virus pathogenicity, the in vitro-transcribed RNAs from BamHI-B fragments of three HSV-1 strains--F (pathogenic), R19, and HFEM (apathogenic), were studied by in vitro translation. When the BamHI-HpaI (0.738-0.755 map units) DNA fragment from HSV-1 strain F was transcribed rightward and translated, three proteins of 70, 63, and 51 kD were detected. The 63 kD protein resembles in size and orientation the protein encoded by the ICP-27 (IE-2) gene (0.740-0.749 mu). The 51 kD polypeptide is assumed to be a prematurely terminated form of this protein. No proteins were obtained from RNA transcribed in the opposite direction. The SalI-NcoI (0.746-0.761 mu) fragment of the three HSV-1 strains yielded two proteins of 25 and approximately 15 kD when transcribed rightward and a 35 kD polypeptide from RNA transcribed in the opposite direction. As a result of the genomic deletion in HFEM, it was possible to obtain the 35 kD protein from the SalI-SalI DNA fragment (0.746-0.761 mu) as well. In vitro transcription and translation of the PstI-SalI (0.778-0.790 mu) DNA fragment (the right-hand side of HpaI-P) did not result in protein synthesis. The possibility that the UL56 gene is connected with the intraperitoneal pathogenicity of HSV-1 is discussed.

Latent varicella-zoster viral DNA in human trigeminal and thoracic ganglia.

BACKGROUND: Some human herpesviruses become latent in dorsal-root ganglia. Primary infection with the varicella-zoster virus causes chickenpox, followed by latency, and subsequent reactivation leading to shingles (zoster), but the frequency and distribution of latent virus have not been established. METHODS: Using the polymerase chain reaction, we performed postmortem examinations of trigeminal and thoracic ganglia of 23 subjects 33 to 88 years old who had not recently had chickenpox or shingles to identify the presence of latent varicella-zoster viral DNA. Oligonucleotide primers representing the origin of replication of the varicella-zoster virus and varicella-zoster virus gene 29 were used for amplification. RESULTS: Among the 22 subjects seropositive for the antibody to the virus, both the viral origin-of-replication and gene-29 sequences were detected in 13 of 15 subjects (87 percent) in whom trigeminal ganglia were examined and in 9 of 17 (53 percent) in whom thoracic ganglia were examined. Viral DNA was not detected in brain or mononuclear cells from the seropositive subjects. None of three thoracic ganglia from the one seronegative subject contained varicella-zoster viral DNA. CONCLUSIONS: These findings indicate that after primary infection with varicella-zoster virus (varicella), the virus becomes latent in many ganglia--more often in the trigeminal ganglia than in any thoracic ganglion--and that more than one region of the viral genome is present during latency.

In-vitro synthesis of functional varicella zoster and herpes simplex viral thymidine kinase.

The varicella zoster virus (VZV) and herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) genes were cloned into the transcription vector pGEM4. In-vitro translation (ivt) of RNA transcribed from these genes showed prominent expression of functional TK proteins with the expected molecular weights of 36 kD for VZV and 43, 39, and 38 kD for HSV-1. The TK proteins were recognized by rabbit anti-VZV and anti-HSV-1 antibodies, respectively. Analysis of the ivt products by thin-layer chromatography revealed the conversion of thymidine to its phosphorylated forms (TMP, TDP, and TTP) by both the VZV and HSV-1 TK genes. The estimated specific activities of the in-vitro translated VZV and HSV-1 TKs were comparable. VZV TK templates were linearized at internal restriction sites and RNAs transcribed from these templates directed the synthesis of polypeptides with sizes consistent with the colinearity of the VZV TK gene. Deletion of 107 amino acids at the carboxy terminus of the VZV TK gene abolished the in-vitro TK activity. In addition, immunoprecipitation of truncated proteins synthesized in vitro suggested the possible involvement of the region between amino acid residues 101 and 168 from the amino terminus of the VZV TK molecule in the formation of structures necessary for antigenicity.

Monoclonal antibody to immediate early protein encoded by varicella-zoster virus gene 62.

Monoclonal antibodies (mAbs) were prepared against varicella-zoster virus (VZV)-infected cell proteins, and 10 mAbs which reacted with nuclear antigens were selected. These mAbs recognized a major 175-180 kDa and three minor VZV-specific phosphoprotein species. Immunofluorescence staining of VZV-infected cells showed that the 175-180 kDa protein was synthesized within 6 h after infection. The synthesis of this protein was inhibited by cycloheximide (CH); however, reversal of CH treatment and addition of actinomycin D (ActD) resulted in the synthesis of the 175-180 kDa protein. To determine whether the 175-180 kDa protein seen in the infected cells is encoded by VZV immediate early (IE) gene 62, the predicted open reading frames of VZV genes 61 and 62 were cloned into pGEM transcription vectors. RNA was transcribed from each gene, translated in vitro and immunoprecipitated with a mAb which recognizes a major 175-180 kDa and three minor proteins. The reactivity of the in vitro translation products encoded by gene 62 with this mAb suggested that the 175-180 kDa protein is encoded by VZV IE gene 62.

Antigenic cross-reaction between a varicella-zoster virus nucleocapsid protein encoded by gene 40 and a herpes simplex virus nucleocapsid protein.

Human sera from varicella-zoster virus (VZV) and herpes simplex virus type 1 (HSV-1) seropositive individuals contain antibody to a 155-kilodalton (155 kDa) viral protein. In this study, we show that monoclonal antibodies (mAb10.1 and mAb1A1.4) prepared against VZV and HSV-1 proteins, respectively, reacted with nuclear antigens and recognized a 155 kDa protein in the infected cells. Immunoprecipitation of whole virions and viral nucleocapsids with these mAbs showed that the 155 kDa protein is located in VZV and HSV-1 nucleocapsids. In addition, immunofluorescence and cross-reaction experiments revealed the antigenic cross-reactivity between the VZV and HSV-1 155 kDa nucleocapsid proteins. To map the coding region of the VZV 155 kDa protein, a truncated DNA fragment from the predicted open reading frame 40 was cloned into an in vitro transcription vector (pGEM). The RNA transcribed from the inserted DNA was translated in vitro and immunoprecipitated with mAb10.1. The reactivity of the in vitro translation products with mAb10.1 indicated that the 155 kDa nucleocapsid protein is encoded by VZV gene 40. These findings demonstrated that the VZV 155 kDa nucleocapsid protein encoded by gene 40 induces humoral response which cross-reacts with both VZV and HSV.

Persistence of varicella-zoster virus DNA in blood mononuclear cells of patients with varicella or zoster.

Varicella-zoster virus (VZV) DNA was detectable by in-situ hybridization in blood mononuclear cells (MNCs) of patients with varicella or zoster for 2-56 days after the onset of a rash. VZV DNA was present in many MNCs from one acute varicella patient 2 days after the onset of the rash and was rarely found in MNCs during acute zoster, convalescent zoster, and convalescent varicella. The morphology of MNCs containing VZV was heterogenous, although most viral-DNA-containing MNCs were large monocytoid cells. Serial examination of blood MNCs from one adult with varicella revealed VZV DNA up until 8 weeks, but not 16 weeks, after the appearance of the rash; parallel studies in four zoster patients showed VZV DNA up until 3 weeks, but not later than 7 weeks after the appearance of the rash. These results indicate that MNCs become infected with VZV during the primary encounter with VZV (varicella) and during reactivation (zoster) and that infection continues for weeks after the onset of the skin rash. Furthermore, the detection of VZV DNA in blood MNCs of uncomplicated zoster patients coincides with the period during which these patients experience pain.

Existence of similar antigenic-sites on varicella-zoster virus gpI and gpIV.

We previously identified the antibody-binding site of a monoclonal antibody (mAb 79.0) on varicella-zoster virus (VZV) glycoprotein I (gpI) and showed that this monoclonal antibody binds to both VZV gpI and gpIV (Vafai et al., J. Virol. 62, 2544, 1988). In this study, a synthetic peptide comprising the mAb 79.0 binding site (designated el) was prepared and anti-peptide antibodies (RAnti-el) were raised in rabbit. RAnti-el recognized the primary translation products encoded by VZV genes 67 (gpIV) and 68 (gpI). To further localize the binding site of RAnti-el on VZV gpIV, the gpIV gene cloned in pGEM transcription vector was cleaved at different locations to generate four truncated DNA fragments. RNA was transcribed from each truncated gpIV fragment, translated in vitro and immunoprecipitated with RAnti-el. The results indicated that RAnti-el binds an antigenic determinant within the first 153 amino acid residues on the primary translation product of VZV gpIV. In addition, RAnti-el recognized the high-mannose intermediate but not the mature from of gpI in the infected cells or the translation products of gpIV glycosylated in vitro in the presence of canine microsomal membrane. These results: (a) confirmed the existence of a shared antigenic determinant on both VZV gpI and gpIV; and (b) indicated that the addition of terminal sugar modification may influence the conformation of gpI and gpIV with respect to the antigenic determinant recognized by RAnti-el.

Human newborn natural killer cell responses to activation by monoclonal antibodies. Effect of culture with herpes simplex virus.

Deficient responses by NK cells may contribute to the susceptibility of human newborns to severe HSV infections. Furthermore, HSV disseminates widely during neonatal infection and may also infect and interfere with the function of blood mononuclear cells (MNC). To determine whether the function of newborn NK cells would be affected by contact with HSV, and whether newborn NK cells might be permissive for HSV replication, newborn MNC were cultured with HSV in vitro for 3 days. Before culture, the intracellular calcium mobilization in newborn NK cells induced by mAb to CD2 and CD16 did not differ from that of adult NK cells. This result argues against immaturity of an early NK cell activation event in human newborns. After culture with HSV the Ca2+ flux response was unaffected by lysis of K562 targets by newborn NK cells and NK-dependent suppression of HSV replication in fibroblasts were preserved or increased. Incorporation of thymidine by NKH-1 cells following stimulation with PHA and IL-2 was not suppressed. NK cells recovered from these cultures did not contain infectious HSV and synthesis of HSV-specific proteins was not detected by immunoprecipitation although HSV genome was detected by DNA hybridization. Our results extend the in vitro model stimulation systems in which newborn NK cells respond positively to include triggering through the CD2 Ag, cross-linking of cell surface CD 16 Ag and response to a pathogen, HSV.

Non-productive infection of human newborn blood mononuclear cells with herpes simplex virus: effect on T cell activation, IL-2 production and proliferation.

Proliferative responses by T cells from newborn cord blood stimulated with PHA or CD3 were reduced following infection with live (but not killed) herpes simplex virus in vitro although activation (measured by calcium flux) and IL-2 production were unaffected. The impairment of proliferation was not reversed by exogenous IL-2, phosphonoacetic acid, indomethacin or anti-alpha or anti-gamma interferon antibodies. HSV DNA was detected by hybridization in DNA extracted from unseparated MNC and from subsets sorted for CD3+ and for HLA DR+ expression. HSV DNA replication was not detected by thymidine uptake and only small amounts of virus were recovered in an infectious centre assay, suggesting that infection was non-permissive. Nevertheless, in-vitro synthesis of a limited range of HSV proteins including ICP4 was detected by metabolic labelling.

Recognition of similar epitopes on varicella-zoster virus gpI and gpIV by monoclonal antibodies.

Two monoclonal antibodies, MAb43.2 and MAb79.0, prepared against varicella-zoster virus (VZV) proteins were selected to analyze VZV gpIV and gpI, respectively. MAb43.2 reacted only with cytoplasmic antigens, whereas MAb79.0 recognized both cytoplasmic and membrane antigens in VZV-infected cells. Immunoprecipitation of in vitro translation products with MAb43.2 revealed only proteins encoded by the gpIV gene, whereas MAb79.0 precipitated proteins encoded by the gpIV and gpI genes. Pulse-chase analysis followed by immunoprecipitation of VZV-infected cells indicated reactivity of MAb43.2 with three phosphorylated precursor species of gpIV and reactivity of MAb79.0 with the precursor and mature forms of gpI and gpIV. These results indicated that (i) MAb43.2 and MAb79.0 recognize different epitopes on VZV gpIV, (ii) glycosylation of gpIV ablates recognition by MAb43.2, and (iii) gpIV is phosphorylated. To map the binding site of MAb79.0 on gpI, the pGEM transcription vector, containing the coding region of the gpI gene, was linearized, and three truncated gpI DNA fragments were generated. RNA was transcribed from each truncated fragment by using SP6 RNA polymerase, translated in vitro in a rabbit reticulocyte lysate, and immunoprecipitated with MAb79.0 and human sera. The results revealed the existence of an antibody-binding site within 14 amino acid residues located between residues 109 to 123 on the predicted amino acid sequences of gpI. From the predicted amino acid sequences, 14 residues on gpI (residues 107 to 121) displayed a degree of similarity (36%) to two regions (residues 55 to 69 and 245 to 259) of gp IV. Such similarities may account for the binding of MAb79.0 to both VZV gpI and gpIV.

Chronic progressive varicella-zoster virus encephalitis in an AIDS patient.

A patient with AIDS developed chronic, progressive encephalitis. Pathologic changes indicated that the encephalitis was produced primarily by a human herpesvirus. Hybridization of radiolabeled RNA probes transcribed from cloned DNA fragments of varicella-zoster virus (VZV), herpes simplex virus, cytomegalovirus, and the human immunodeficiency virus to DNA extracted from the patient's brain identified VZV as the causative agent. The results suggest that VZV should be considered in the differential diagnosis of chronic encephalitis of unknown etiology, particularly in immunosuppressed patients.