Charles Grose

Varicella-zoster virus-specific gp140: a highly immunogenic and disulfide-linked structural glycoprotein.

A 140,000-dalton disulfide-linked glycoprotein (gp140) specified by varicella-zoster virus (VZV) in infected cultured cells was identified and precipitated by two murine monoclonal antibodies (VZ-151 and VZ-158). When analyzed under reducing conditions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gp140 was cleaved predominantly into a 66,000 lower-molecular-weight product (gp66). This protein was classified as a viral structural component, since it was observed in the polypeptide profile of metrizamide gradient-purified enveloped virions. By immunofluorescence analyses with a monoclonal antibody probe, gp140 expression was documented to be highly conserved both within cultured cells inoculated with homologous (VZV-Oka) and heterologous (VZV-32) strains and in infected human tissues from chicken pox and zoster patients. That the glycoprotein was highly immunogenic was confirmed by the presence of high-titer anti-gp140 antibody in the sera of both hyperimmunized laboratory animals and naturally infected humans. Temporally, the humoral response to gp140 following primary VZV infection preceded that against the other viral glycoproteins. These studies describe, therefore, an immunogenic, disulfide-linked viral structural glycoprotein, which must be included among the other five previously described VZV-specific fucosylated species--gp118, gp98, gp62, and gp45.

The synthesis of glycoproteins in human melanoma cells infected with varicella-zoster virus.

Abstract The synthesis of glycoproteins has been investigated in cultured human melanoma cells before and after infection with varicella-zoster virus (VZV). The glycosylated polypeptides were labeled with tritiated precursors of the sugar moiety and were analyzed by polyacrylamide gel electrophoresis. When the electrophoretic profiles of uninfected and infected cell lysates were compared, a marked reduction in host cell glycoprotein synthesis was evident during the late phase of viral infection. In addition, at least three infected-cell specific (ICS) glycosylated polypeptides were detected. After immune precipitation of [ 3 H]glucosamine-labeled VZV cell extracts with human and rabbit VZV antisera, a total of five ICS glycoproteins were identified and designated gp 45, gp 62, gp 88, gp 98, and gp 118, according to their apparent molecular weights (× 10 3 ). Four of the five polypeptides (but not gp 88) were visualized after immune precipitation of infected cell extracts labeled with [ 3 H]fucose. The ICS glycopeptides corresponded in molecular weight to [ 35 S]methionine-labeled polypeptides detected in a virus-enriched fraction obtained by centrifugation of sonically disrupted infected cells in a combination density-viscosity gradient. A prominent ICS high-molecular weight (∼150,000) nonglycosylated polypeptide was identified also in the latter fraction.

Immunoprecipitable polypeptides specified by varicella-zoster virus.

Abstract Polypeptides encoded by varicella-zoster virus (VZV) in infected cell cultures have been identified by radioimmune precipitation techniques. Detergent-solubilized extracts of VZV-infected cells were reacted with highly specific VZV antisera raised in strain-2 guinea pigs immunized with sonicates of syngeneic virus-infected cells. Fractionation of the immunoprecipitates in acrylamide slab gels demonstrated an average of 16 polypeptides, which ranged in molecular weight from 32,000 to a200,000. These included the three major immunogenic glycoproteins (gp 62, gp 98, and gp 118) and a prominent higher molecular weight nonglycosylated polypeptide at 155,000. One of the [ 35 S]methionine-labeled polypeptides comigrated with purified actin. Not all polypeptides were visible in any one particular fluorogram, but comparative analysis of polypeptide profiles derived from electrophoreses performed with different gel concentrations and different crosslinkers (methylene-bisacrylamide and N,N -diallyltartardiamide) clearly established a consistent and reproducible pattern of radioactive bands. A low background of radio-activity was nonspecifically precipitated by the antigen-antibody-protein A complexes; however, with the exception of a common band comigrating with actin, the electrophoretic profiles representing virus-specific and nonspecific immunoprecipitates were easily distinguished.

Cryopreservation of varicella-zoster virions without loss of structural integrity or infectivity.

Varicella-zosterer virions present in infected cells or in a cell-free state were freeze-dried without loss of structural integrity of infectivity. Generally, yields of greater than 5 log10 foci/ml (infected cells) or greater than 4 log10 PFU/ml (cell-free virus) were recovered from varicella-zoster virus-infected human melanoma cells both before and after lyophilization in phosphate-buffered media containing 0.1-1.0 M sucrose. Virus frozen in solutions lacking sugar had little or no residual infectivity after vacuum sublimation was completed. Visualization by electron microscopy demonstrated large numbers of enveloped virions in the virus preparations lyophilized in media containing sucrose; in marked contrast, virus subjected to freeze-drying in buffered solutions without sugar consisted mainly of naked nucleocapsids. Water analyses by Karl Fischer titration suggested that residual moisture retained by sugar prevented disenvelopment of the varicella-zost virion.

Complete spectrum of the varicella congenital defects syndrome in 5-year-old child.

Chickenpox in the pregnant woman occasionally leads to infection and teratogenesis of the early to midgestational embryo. We describe the developmental assessment and clinical management of a 5-year-old girl with classic varicella congenital defects syndrome. This patient is unique with respect to (1) a generally favorable outcome in spite of the severity of her anomalies involving the eyes, limbs and skin, (2) a completely normal evaluation of her immunologic status and (3) a serologically documented herpes zoster infection at age 41 months.

Varicella-Zoster Virus Infection of Diploid and Chemically Transformed Guinea-pig Embryo Cells: Factors Influencing Virus Replication

Factors influencing the replication of varicella-zoster virus (VZV) in guinea-pig embryo cells were evaluated using both diploid cells (GPEC) and a chemically transformed cell line (GPT). Wild-type and vaccine strains of VZV were successfully isolated and serially propagated in GPEC prepared from early gestation whole embryos (less than 2 cm in length). Low passage GPEC (less than or equal to 5 subcultivations) were more susceptible to VZV infection than high passage GPEC (greater than 5 subcultivations), and guinea-pig cells were consistently less permissive than human diploid cells. Cell-free virus was produced from VZV-infected GPEC cultures by sonication and peak yields of 10(3) p.f.u./ml were obtained. In addition, we report the isolation and propagation of VZV, as well as production of cell-free virus, in GPT. Both GPEC and GPT cells were less susceptible to VZV infection than human cells. However, viral replication was enhanced by incubation of VZV-infected GPT cultures at 32 degree C rather than 36 degree C.

1105 HOW VARICELLA VACCINE WORKS: AN APPRAISAL WITH MONOCLONAL ANTIBODIES

Studies with the live attenuated strain (VZV-Oka) of varicella zoster virus vaccine demonstrate a high degree of efficacy in protecting children with leukemia from contracting wild-type chickenpox. The purpose of this study was to determine which were the most important immunogenic proteins of VZV-Oka and to identify neutralization epitopic sites. Prior investigations of the immune response to wild type chickenpox indicate that the predominant antibody response is directed against the major viral glycoproteins. In order to evaluate VZV-Oka, murine monoclonal antibodies were produced against its viral glycoproteins. Specificities of antibodies were determined by techniques of immunoprecipitation and immunoblotting. By these studies we divided the glycoproteins into three groups: (1) gp118 (2) gp98/gp62/gp45 and (3) gp140/gp66. Biologic activity of each antibody was assayed by the technique of plaque reduction. A panel of antibodies against VZV gp118 were shown to possess marked neutralization activity (with titers to 1:40,000) against homologous and heterologous VZV strains. A titer of this magnitude indicates that glycoprotein gp118 of VZV-Oka harbors a major neutralization epitope. Glycoproteins gp140/gp66 also contained a complement-independent neutralization epitope but none was detected on the gp98 complex. These analyses with monoclonal antibodies define biologically important antigenic sites of VZV-Oka and suggest a role for the same glycoproteins in any future subunit vaccine.

1020 THE ANTIGENIC DETERMINANTS OF VARICELLAZOSTER VIRUS

Antigenicity and immunogenicity are important considerations in the laboratory evaluation of candidate varicella-zoster virus (VZV) vaccines. Therefore, high titered antisera were prepared in guinea pigs and rabbits against two VZV strains: VZV-32 (a laboratory strain) and VZV-Oka (a vaccine strain). Radiolabeled proteins and glycoproteins were precipitated from VZV-infected cells with the VZV immune sera, as well as human zoster sera, and analyzed by polyacrylamide gel electrophoresis. Three major glycopeptides of approximate mol. wt. 62,000, 98,000, and 118,000 were identified; two of the three glycoproteins have been found in the membranes of VZV-infected cells and in culture medium overlying VZV-infected cells. At least seven nonglycosylated antigens, which ranged in mol. wt. from ∼30,000 to 150,000, also were present in the precipitates. The prominent high mol. wt. antigen probably corresponds to the major capsid polypeptide found in herpesviruses. In summary, at least 10 polypeptides were consistently precipitated by VZV anti-sera obtained from hyperimmunized animals. Since both strains elicited the same spectra of VZV antibodies, they could not be distinguished from one another on the basis of their electrophoretic profiles.

774 HUMORAL IMMUNE RESPONSE FOLLOWING VARICELLA-ZOSTER VIRUS INFECTION

Previous investigations of the humoral immune response to varicella-zoster virus (VZV) infection were hampered by the insensitivity of the complement-fixing antibody test. Development of the indirect fluorescent method for detecting antibody to VZV-membrane antigen provided a means of separating immune from susceptible individuals and confirmed the ability of zoster immune globulin (ZIG) to attenuate disease if given to susceptible children shortly after exposure. Because of the important role of antibody in modifying clinical disease, we have evaluated the neutralizing antibody response in neonates and older children with VZV infection. Utilizing a newly developed ‘semi-micro’ plaque reduction assay we found (i) that the titer of neutralizing antibody was enhanced 2-4 fold by the addition of complement and (ii) that complement-dependent neutralizing antibody occasionally was detectable when anti-membrane antibody was negative (<1:2). These results suggest that neutralizing antibody titers may be required to fully assess the VZV immune status of exposed newborn and immuno-suppressed children. In addition, this test may define the role of humoral immunity in modulating VZV reactivation.