Construction of bovine herpesvirus-1 (BHV-1) recombinants which express pseudorabies virus (PRV) glycoproteins gB,gC, gD,and gE

Summary We have improved the method for constructing recombinants of bovine herpesvirus type-1 (BHV-1). Using this method, we constructed three recombinants in which the pseudorabies virus (PRV) thymidine kinase (tk) gene was inserted at three different sites in the unique short region of BHV-1. These three sites are located in the open reading frame of gE, gG and gI genes. Previously, two sites (tk and gC) had been used to insert foreign DNA fragments to BHV-1 genome. Therefore we now have 5 sites in BHV-1 where DNA can be inserted. The gB, gC, gD, gE and gI genes of PRV were successfully inserted at the tk or the gC gene of BHV-1 genome and Western blot analyses confirmed that the recombinants express PRV gB, gC, gD and gE. Anti-PRV gB and gC antibodies as well as anti-PRV polyclonal serum neutralized BHV-1 recombinants which express PRV gB and gC. The latter was neutralized more strongly. However, anti-gD monoclonal antibody and anti-PRV polyclonal serum failed to neutralize gD-expressing recombinants. This suggests that PRV gC and some gB are integrated into the viral envelope of the recombinants, but very little gD is present in the viral envelope.     

Characterization of B virus glycoprotein antibodies induced by DNA immunization

Genes encoding glycoproteins gB, gC, gD, gE, and gG of herpes B virus (species Cercopithecine herpesvirus 1) were cloned into mammalian expression vector pcDNA3.1/V5-His. Abilities of the plasmid constructs to express recombinant glycoproteins were confirmed by Western blot analysis of transfected CHO-K1 and COS-7 cells. Antibody production was induced in rabbits by intramuscular injections with the expression constructs at four-weekly intervals. Antibodies to gB were detected after the second DNA inoculation, while it took an additional plasmid injection to induce responses to gC, gD and gE. The gG plasmid failed to stimulate antibody production. Antisera ELISA titers varied greatly depending on the gene, with gB inducing highest (21,000) and gE inducing lowest (60) antibody titer. The induced antibodies were predominantly conformation-dependent. The gB, gC, and gD antisera contained HSV cross-neutralizing antibodies, but only gB antisera contained B virus neutralizing antibodies. The gB antisera cross-reacted with HSV antigens in Western blot, ELISA, dot-blot, plaque immunostaining and immunoprecipitation assays, whereas gD and gC antisera were mostly B virus-specific. Thus, polyclonal antibodies to B virus glycoproteins can be generated by DNA immunization and used as diagnostic and research reagents.     

Role of anti-gB and -gD antibodies in antibody-induced endocytosis of viral and cellular cell surface glycoproteins expressed on pseudorabies virus-infected monocytes

The addition of porcine pseudorabies virus (PrV)-specific polyclonal IgG antibodies to PrV-infected monocytes induces internalization of plasma membrane-anchored viral glycoproteins and major histocompatibility complex (MHC) class I. Using PrV deletion strains, it was shown that gB and gD are essential for the process to occur. The purpose of the current study was to evaluate whether antibodies directed against single viral glycoproteins are able to induce endocytosis. It was shown that monoclonal antibodies directed against viral glycoprotein gB and gD, but not against gC and gE, are able to induce internalization of their respective ligand. Adding a combination of monoclonal antibodies against gB and gD resulted in endocytosis levels, comparable to the endocytosis levels observed when adding porcine PrV-specific polyclonal antibodies. The addition of genistein and tyrphostin 25, two inhibitors of tyrosine kinase activity, abolished endocytosis induced by monoclonal anti-gB and -gD antibodies in a concentration-dependent manner. The addition of similar concentrations of tyrphostin 1, an inactive tyrphostin, had no effect on endocytosis. It was also shown that a mixture of polyclonal, but not monoclonal, antibodies against gB and gD is able to induce cointernalization of MHC class I. This indicates that MHC class I cointernalization results from a passive catching of the molecules rather than from a specific interaction of the MHC class I molecules with one or more viral glycoproteins. In conclusion, it can be stated that antibody-induced crosslinking of gB and gD induces the activation of a tyrosine phosphorylation-dependent signal transduction pathway, leading to their endocytosis. Cointernalization of other viral glycoproteins and MHC class I is most likely caused by a passive catching of these molecules in the gB and gD aggregates.     

Comparative studies of HSV-1 antigens solubilised from infected cells by using non-ionic or zwitterionic detergents

HSV-1 antigen preparations solubilised from Vero cells by using either the non-ionic detergent Nonidet P40 or the zwitterionic detergent Empigen BB, and purified on sucrose density gradients or over a sucrose cushion, were tested by ELISA with anti-HSV-1 glycoprotein monoclonal antibodies and by radioimmunoprecipitation (RIP) with polyclonal HSV-1 antiserum. Amongst several proteins detected in these preparations, the four major HSV-1 glycoproteins, gB, gC, gD, and gE, were found to be present. Differences between NP40 or Empigen-solubilised HSV-1 antigen preparations with respect to two of these glycoproteins, gB and gE, were detected by using a small panel of monoclonal antibodies. Comparative studies in mice showed the Empigen-solubilised HSV-1 antigen preparations elicited greater antibody responses and greater protection against lethal HSV-1 challenge infection than the NP40-solubilised preparation.     

Studies on herpes simplex virus type 1 glycoproteins using monoclonal antibodies

Monoclonal antibodies against herpes simplex virus type 1 glycoproteins were isolated and utilized to study the synthesis and processing of glycoproteins B, C, and D (gB, gC, gD, respectively). Monoclonal antibodies against both gB and gD had higher virus-neutralizing activity when compared to that of gC. Differences among these glycoproteins were observed in their time of appearance in the virus-infected cells. The presence of gD was detected at a very early stage of infection when compared to gB and gC. The localization of these glycoproteins during their synthesis and processing was studied.     

Analysis of canine herpesvirus gB, gC and gD expressed by a recombinant vaccinia virus

Summary.  The genes encoding the canine herpesvirus (CHV) glycoprotein B (gB), gC and gD homologues have been reported already. However, products of these genes have not been identified yet. Previously, we have identified three CHV glycoproteins, gp145/112, gp80 and gp47 using a panel of monoclonal antibodies (MAbs). To determine which CHV glycoprotein corresponds to gB, gC or gD, the putative genes of gB, gC, and gD of CHV were inserted into the thymidine kinase gene of vaccinia virus LC16mO strain under the control of the early-late promoter for the vaccinia virus 7.5-kilodalton polypeptide. We demonstrated here that gp145/112, gp80 and gp47 were the translation products of the CHV gB, gC and gD genes, respectively. The antigenic authenticity of recombinant gB, gC and gD were confirmed by a panel of MAbs specific for each glycoprotein produced in CHV-infected cells. Immunization of mice with these recombinants produced high titers of neutralizing antibodies against CHV. These results suggest that recombinant vaccinia viruses expressing CHV gB, gC and gD may be useful to develop a vaccine to control CHV infection. Accepted November 20, 1996 Received October 10, 1996     

DNA priming and killed virus boosting vaccination strategy can augment the immune response to pseudorabies virus

The immune efficacy of DNA vaccines containing three plasmids encoding gB, gC, and gD glycoproteins (Mix DNA) of Pseudorabies virus (PRV) or the plasmid for gC only (gC DNA), killed virus (KV) vaccine or combination of gC DNA, Mix DNA and KV vaccines was evaluated in mice using primeboost strategy. The mice vaccinated twice with Mix DNA, and once with KV generated higher levels of gCspecific and virus neutralization (VN) antibodies and a stronger cellular immune response than the mice vaccinated three times with the Mix DNA vaccine only. The highest level of VN antibodies were detected in mice vaccinated twice with KV vaccines alone or with combination of DNA and KV vaccines. The challenge of vaccinated mice with the lethal dose of PRV showed that the complete protection against PRV was achieved in the group of mice immunized with the DNA and KV vaccines combined. The results suggested that DNA priming followed by KV vaccine boosting could enhance the antibody response and cellular immunity against PRV infection in mice.     

Characterisation and physical mapping of an HSV-1 glycoprotein of approximately 115 X 10(3) molecular weight

A type-specific monoclonal antibody that efficiently neutralises HSV-1 immunoprecipitated a glycoprotein of slightly greater electrophoretic mobility than gB from HSV-1 infected cells. Pulse and pulse chase experiments indicate that this glycoprotein is distinct from HSV-1 glycoproteins gB, gC, gD, and gE. This was confirmed by the reactions of LP11 with a series of intertypic recombinants the results of which indicate that the LP11 target gene is located close to the HSV-1 thymidine kinase gene between map positions 0.28 and 0.31. In accordance with the presently agreed convention this glycoprotein should be designated gH-1, and it may correspond to the 110K glycoprotein described by S. D. Showalter, M. Zweig, and B. Hampar (1981), Infect. Immun. 34, 684-692. Antibody LP11 inhibits plaque formation when added to cell monolayers after infection suggesting that gH-1 may play a role in cell-to-cell spread of infectious virus.     

Antibody activity to herpes simplex virus in mouse Ig classes and IgG subclasses

Summary Recent studies indicate that Ig class and IgG subclass induction varies for different proteins and further that some Ig subclasses, like IgG2a, are more efficient in important biologic processes such as antibody-dependant cell-mediated cytotoxicity (ADCC). Many proteins of herpes simplex virus type 1 (HSV-1) are immunogenic and induce immunoglobulin responses. To determine the distribution of immunoglobulins induced by HSV-1 proteins, we studied immune mouse serum using an Ig isotype specific Elisa assay for antiviral activity. We found by endpoint analysis that the antiviral titer was 1:12,903 for IgG1, 1:5141 for IgG2a, 1:2140 for IgG2b and 1:229 for IgG3. To identify which isotypes were induced by individual glycoproteins and other viral proteins, Western blots containing HSV-1 proteins were probed with immune serum and isotype specific second antibodies. gB, gC, gD and the 42/44KDa nucleocapsid complex induced strong IgG1, IgG2a, IgG2b responses. IgG3 reactivity with viral proteins appeared weaker. Among the IgG3 reactivities detected on immunoblots, gB and gC were the most intense. Other proteins which elicited IgG1, IgG2a and IgG2b responses were 170KDa, 154KDa and gE. IgA responses were induced by 154KDa, gC, gB, gE and gD. Prominent IgM responses included gB, gC, gD and the 42KDa protein. These results indicate that HSV-1 glycoproteins induce prominent responses in all IgG isotypes except IgG3. The biologic implications of the data are discussed.     

Requirements for transport of HSV-1 glycoproteins to the cell surface membrane of human fibroblasts and Vero cells

The intracellular transport of the HSV-1 glycoproteins gA/gB, gC and gD has been followed by the indirect immunofluorescence technique (IIF). Infected tissue culture cells were stained with monoclonal antibodies made to the individual glycoproteins and with fluorochrome-coupled wheat germ agglutinin reacting specifically with Golgi apparatus of the cells. Staining of either infected, human fibroblasts or of VERO cells at 9 hours p.i. with antibodies to gA/gB showed a prominent ring-like nuclear fluorescence and distinct staining of the Golgi apparatus in the cells. Antibodies to gC and gD stained mainly the Golgi apparatus and areas close to or at the surface of the cells. By immunocytolysis of HSV-1-infected VERO cells the viral glycoproteins were demonstrable at the surface of cells but growth of infected cells in the presence of either TM or monensin inhibited the expression of most of the viral glycoproteins at the cell surface. Blocking of the glycosylation of the viral glycoproteins with tunicamycin (TM) was followed by accumulation of the core of the glycoproteins gA/gB and gD in granular structures close to the nucleus as seen by immunofluorescence microscopy. Antibodies to gC did also stain granules close to the nucleus but in addition the periphery of the cells were stained. Inhibition of intracellular transport from the Golgi apparatus by the carboxylic ionophore monensin was followed by accumulation of all the HSV-1 glycoproteins in vesicles derived from the Golgi apparatus in both human fibroblasts and VERO cells. Our data thus support the hypothesis that the HSV-1 glycoproteins are processed in the Golgi apparatus before the transport to and incorporation into the cell surface membrane of infected cells and into virion envelopes.     

A rapid procedure for the enrichment of undenaturated, antigenically active herpes simplex virus glycoproteins

The usefulness of lentil lectin affinity chromatography for the rapid enrichment of HSV glycoproteins in an undenatured state for both research and clinical purposes was investigated. In order to compare the lentil lectin-binding characteristics and immunologic specificities of undenatured HSV-1 and HSV-2 glycoproteins, [35S]methionine-labelled extracts of virus-infected HEp-2 cells were subjected to lentil lectin affinity chromatography. Individual HSV-1 and HSV-2 glycoproteins in bound and unbound fractions were identified using monoclonal antibodies. With the exception of a portion of pgD and gD, all major viral glycoprotein species (gA, gB, gC, gD, gE and gF) and their glycosylated processive intermediates bound to lentil lectin indicating that all possess predominantly mannosyl and/or glucosyl carbohydrate moieties. Although the unbound pgD and gD species were glycosylated, no gD and only a portion of pgD bound to lentil lectin when reapplied to the column indicating that these subspecies possess alterations in factors required for efficient lectin binding. Immunoprecipitation of undenatured lectin-bound glycoproteins from infected cells using HSV-1 and HSV-2-specific rabbit and human antisera confirmed previous findings that the predominant type-specific glycoproteins of HSV-1 and HSV-2 are gC and gE/gF, respectively.     

Recovery of an unusual fusogenic herpes simplex virus type 2 strain from a clinical specimen

A highly unusual herpes simplex virus type 2 strain, strain Burr, was isolated from a female genital tract clinical specimen. This virus induced remarkably rapid and extensive syncytium formation in Vero cells involving hundreds of cells but was less fusion active in HEp-2 cells, MRC-5 cells, and mink lung cells. Virus-infected cells produced the glycoproteins gB, gC, gD and gE.     

Monoclonal antibodies to herpes simplex virus type 1 proteins, including the immediate-early protein ICP 4.

Monoclonal antibodies were prepared against herpes simplex virus type 1 (strain 14012) by two immunization procedures. Procedure A utilized infectious virus propagated in mouse cells, and procedure B utilized mouse cells infected with herpes simplex virus in the presence of cycloheximide and harvested 1 h after removal of the inhibitor. A total of 52 monoclonal antibodies were obtained against 10 herpes simplex virus proteins, including four glycosylated proteins (a 110,000-molecular-weight protein, gB, gC, and gD) and six nonglycosylated proteins (a 68,000-molecular-weight protein, ICP 9, ICP 8, ICP 6, ICP 5, and the immediate-early ICP 4). The antibodies were assayed against herpes simplex virus types 1 and 2 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioimmunoprecipitates, immunofluorescence, and neutralization. Using the reagents prepared, we concluded that the 110,000-molecular-weight protein, gD, ICP 9, ICP 9, ICP 6, and the 68,000-molecular-weight protein express both type-specific and cross-reactive antigenic determinants. In contrast, nine antibodies against gB all cross-reacted with herpes simplex virus type 2, whereas eight antibodies to gC all reacted type specifically.     

Differential effect of systemic acyclovir treatment of genital HSV-2 infections on antibody responses to individual HSV-2 proteins

Western blot and reflectance densitometry were used to evaluate the antibody response from patients treated with systemic acyclovir during their primary episodes of genital HSV-2 infection. Of 39 patients studied, 10 received oral acyclovir, 10 received intravenous acyclovir, and 19 received placebo. Total antibody levels as well as levels of antibodies to individual HSV-2 proteins (gB, gG, gC/gE, VP16, gD, and p45) were determined in convalescent phase sera. The median number of HSV-2 proteins recognized and the total amount of HSV-2 antibody were significantly lower in acyclovir than placebo treated patients (P less than or equal to 0.01). Levels of antibodies to individual proteins were also lower in sera from acyclovir versus placebo treated patients: gB (P = 0.013), gC/gE (P = 0.017), VP16 (P = 0.001), gD (P = 0.009), and p45 (P = 0.015). Antibody response to gG-92 and to a newly described gG species, gG-70, was not significantly different among treatment groups. A low number of proteins recognized by convalescent serum antibodies were associated with a higher number of lesions at first recurrence (P = 0.02) and with a longer duration of the first recurrent episode (P = 0.02). Low levels of total antibody were associated with shorter times to first recurrence (P = 0.05). Low levels of antibody to VP16 (P = 0.05) and gD (P = 0.01) were associated with longer duration of the first recurrence.     

Production of herpes B virus recombinant glycoproteins and evaluation of their diagnostic potential

B virus (cercopithecine herpesvirus 1) is the only deadly alphaherpesvirus that is zoonotically transmissible from macaques to humans. The detection of humoral immune responses is the method of choice for the rapid identification of B virus-infected animals. We evaluated the diagnostic potential of recombinant B virus glycoproteins for the detection of immunoglobulin G (IgG) antibodies in monkey and human sera. Glycoproteins B, C, and E and secreted (sgG) and membrane-associated (mgG) segments of glycoprotein G (gG) were expressed in the baculovirus expression system, while gD was expressed in CHO cells. We developed recombinant protein-based IgG enzyme-linked immunosorbent assays (ELISAs) and compared their diagnostic efficacies by using B virus antibody-negative (n = 40) and -positive (n = 75) macaque sera identified by a whole antigen-based ELISA and Western blotting. The diagnostic sensitivities of the gB-, gC-, gD-, and mgG-ELISAs were 100, 97.3, 88.0, and 80.0%, respectively. The specificities of the gB-, gC-, and gD-ELISAs and of the mgG-ELISA were 100 and 97.5%, respectively. In contrast, the sensitivities and specificities of sgG- and gE-ELISAs were low, suggesting that sgG and gE are less effective diagnostic antigens. Sera from nonmacaque monkeys cross-reacted with gB, gC, and gD, and only baboon sera reacted weakly with mgG. Human herpes simplex virus type 1 (HSV-1)- and HSV-2-positive sera pools reacted with gB and gD, whereas sera from B virus-infected individuals reacted with all four antigens. These data indicate that gB, gC, gD, and mgG have a high diagnostic potential for B virus serodiagnosis in macaques, whereas mgG may be a valuable antigen for discrimination between antibodies induced by B virus and those induced by other, closely related alphaherpesviruses, including HSV-1 and -2.     

Humoral immune response to HSV-1 and HSV-2 viral proteins in patients with primary genital herpes

The humoral immune response to HSV-1 and HSV-2 proteins was examined in patients with primary first-episode genital herpes. Ten patients had culture-proven HSV-1 infections, 37 had HSV-2 infections, and all were seronegative to HSV proteins before developing their infections. Development of serum antibodies to individual HSV proteins and glycoproteins was determined by immunoprecipitation of radiolabeled HSV-1- and HSV-2-infected cell proteins and subsequent gel electrophoresis. In HSV-1 patients, a sequential development of antibodies to HSV-1 proteins was observed with early appearance of antibodies to the nucleocapsid protein p148 and to glycoproteins gB and gC. Seroconversion to gD and to a polypeptide of 88,000 molecular weight (p88) occurred next, and, finally, seroconversion to gE and to a nonglycosylated 66,000 dalton protein p66. In HSV-2 patients, antibodies to HSV-2 proteins p148, gB, and p88 appeared within 1 week of onset of symptoms. Seroconversion to p66, gD, and to a complex of glycoproteins gC and gE ("g80") occurred later, at a mean time of approximately 3 weeks. Seroconversion to HSV-1 gB, p88, and p66 occurred significantly later than seroconversion to the homologous counterparts. Seroconversion within 21 days of onset to HSV-2 gD, g80, and p66 was associated with a longer time to the first recurrence in HSV-2 patients, suggesting a possible role of these antibodies, alone or in combination, in the maintenance of HSV-2 latency in humans.     

Role of glycoprotein gD in the adhesion of pseudorabies virus infected cells and subsequent cell-associated virus spread

Summary Pseudorabies virus (PrV) infected cells in suspension are able to adhere to a monolayer of uninfected cells by means of PrV glycoproteins expressed at the outer cell membrane, with gB and gC playing a major role as ligands and a heparinlike substance as receptor. In order to investigate the role of gD in this process and subsequent transmission of infectivity to contact cells, experiments with a gD deletion mutant, heparin and a monoclonal antibody (Mab) against gD were performed. The first indication that gD is active during cell adhesion was found by the observation that the binding of gD^– PrV infected cells was five times weaker than that of wild type (WT) PrV infected cells. Further evidence was given by the use of a Mab against gD. Preincubation of WT PrV infected cells with this Mab led to a reduction of the percentage adhering cells from 69% to 49%. The same Mab inhibited the heparin independent and heparin resistant binding of WT PrV infected cells indicating that gD is important during both processes. Furthermore, it was demonstrated in a plaque assay that, after contact with a monolayer, gD^– PrV infected cells in suspension were able to induce plaques with an efficiency of 1%. In conclusion, we can state that beside the interaction of the ligands gB and gC with a heparinlike receptor also the interaction of gD with a receptor which differs from a heparinlike substance mediates the binding of WT PrV infected cells to uninfected cells and that gD is not essential for the subsequent cell-to-cell spread of the virus.     

Intratypic variation of herpes simplex virus type 2 isolates detected by monoclonal antibodies against viral glycoproteins

Monoclonal antibodies (MAbs) to herpes simplex virus (HSV) glycoproteins gD, gG, gB, and gE were used to analyze antigenic variations of 128 genital HSV-2 isolates by an indirect enzyme-linked immunosorbent assay (ELISA). Isolates were considered significantly different from the standard HSV-2 strain 186 when their optical density (OD) in ELISA was less than half that of strain 186. This criterion gave 30 patterns of reactivity among the genital HSV-2 isolates. The MAbs to gB, gG, and 2 of the gD antibodies reacted with more than 90% of the isolates, suggesting that these MAbs recognized highly conserved epitopes. However, the gE MAb reacted with only 47% of the isolates, and one of the gD antibodies with only 39%. Thus, HSV-2 can readily tolerate modifications in some parts of the gD and gE molecules while remaining infectious.     

Zwitterionic detergent solubilisation of HSV-1 surface antigens

Summary A preparation was obtained from herpes simplex virus type 1 (HSV-1)-infected cells using a zwitterionic detergent, Empigen BB. The preparation was partially-purified either by ultracentrifugation over a cushion of 20% sucrose or on a sucrose density gradient. Partial characterisation of these materials by ELISA, using both polyclonal and monoclonal antibodies showed them to contain at least four major HSV glycoproteins, gB, gC, gD and gE. Comparison of Empigen-extracted HSV-1 antigen preparations with preparations obtained using the non-ionic detergents Nonidet P40 or Triton-X-100 indicate that, using conventional procedures, separation of glycoproteins, B, C, D, and E from unwanted proteins may be facilitated using the former detergent.Immunization of mice with Empigen-extracted, partially-purified or gradient-purified antigen preparations elicited good levels of antibody detectable by ELISA and a high degree of protection against both HSV-1 and HSV-2 challenge infection. Such protection could be achieved using aqueous antigen preparations, but was augmented using aluminium hydroxide gel as an adjuvant. In general, Empigen-extracted HSV-1 antigen preparations elicited higher ELISA antibody levels and more complete protection against HSV challenge infection than NP40 or Triton-X-100-extracted preparations.The value and usefulness of the detergent Empigen for obtaining HSV surface antigen preparations and the role of these as potential vaccines against HSV infections, is discussed.