Production and characterization of monoclonal antibodies directed against pseudorabies virus

Hybridoma cell lines producing monoclonal antibodies to pseudorabies virus (PRV) were established. The monoclonal antibodies were characterized with respect to their antigenic specifications and biological activities. Two monoclonal antibodies immunoprecipitated the 50 kDa PRV glycoprotein (gp50) and two immunoprecipitated the 82 kDa glycoprotein (gp82). The monoclonal antibodies were used to analyze the biological roles of these two glycoproteins. One monoclonal antibody directed against each glycoprotein did not require complement for in vitro viral neutralization while the other monoclonal antibody directed against the glycoprotein required complement for neutralization. The monoclonal antibodies against gp50 were shown to be directed against different epitopes within the glycoprotein. In contrast, the monoclonal antibodies against gp82 were shown to be directed against the same antigenic site on the glycoprotein. In vivo passive immunity studies in mice showed that monoclonal antibodies directed against either gp50 or gp82 could be protective.     

Identification of the pseudorabies virus glycoprotein gp50 as a major target of neutralizing antibodies

Summary A total of 108 monoclonal antibodies specific for pseudorabies virus (PRV) were isolated in a cellular fusion, using spleen cells from mice which had been immunized with a live strain (Kojnok strain). Twelve of them neutralized the Kojnok strain in vitro in the absence of complement, as well as 28 virulent strains of various geographical origin and isolated from various animal species. All of the 12 clones were specific for glycoprotein gp50. Eighteen other clones with no neutralizing activity were studied: 11 reacted with glycoprotein GIII, 3 with glycoprotein GII, 3 with the glycoprotein gp63 and 1 with the glycoprotein GI. Transfer to mice of ascitic fluids corresponding to clones reacting with gp50 and GIII showed that some of them provided the mice with the ability of resisting to virulent challenge. Thus it appears that glycoproteins gp50 and GIII are major immunogens of the virion.     

The scavenger receptor,cysteine-rich domain-containing molecule gp-340 is differentially regulated in epithelial cell lines by phorbol ester

Gp-340 is a glycoprotein belonging to the scavenger receptor cysteine rich (SRCR) group B family. It binds to host immune components such as lung surfactant protein D (SP-D). Recent studies found that gp-340 interacts directly with pathogenic microorganisms and induces their aggregation, suggesting its involvement in innate immunity. In order to investigate further its potential immune functions in the appropriate cell lines, the expression of gp-340 in four conventional immune cell lines (U937, HL60, Jurkat, Raji), and two innate immune-related epithelial cell lines (A549 derived from lung and AGS from stomach), was examined by RT-PCR and immunohistochemistry. The resting immune cell lines showed weak or no gp-340 mRNA expression; while the two epithelial cell lines expressed gp-340 at much higher level, which was differentially regulated by phorbol myristate acetate (PMA) treatment. In the A549 cells, gp-340 was up-regulated along with the PMA-induced proinflammatory expression of both IL-6 and IL-8. In AGS cells, PMA down-regulation of gp-340 was seen in parallel with an up-regulation of the two mature gastric epithelial specific proteins TFF1 (trefoil factor 1) and TFF2, which are implicated as markers of terminal differentiation. Analysis of the distribution of gp-340, together with the TFFs and SP-D in normal lung and gastric mucosa, supported further our in vitro data. We conclude that the differential regulation of gp-340 in the two epithelial cell lines by PMA indicates that gp-340 s involvement in mucosal defence and growth of epithelial cells may vary at different body locations and during different stages of epithelial differentiation.     

DIVA diagnostic of Aujeszky's disease using an insect-derived virus glycoprotein E

Commercial vaccines against Aujeszky's disease are mainly formulated using deleted versions of attenuated or inactivated Pseudorabies virus (PRV) particles lacking of the structural glycoprotein E (gE). Complementary diagnostic assays used to differentiate infected from vaccinated animals (DIVAs), are based on the detection of serum antibodies against gE. A recombinant version of the PRV gE protein was expressed in a baculovirus vector system in Trichoplusia ni insect larvae in order to obtain this diagnostic reagent for large scale diagnosis at reduced costs. A recombinant gE gene (gEr), lacking of signal peptide and transmembrane domains, was cloned into a modified baculovirus vector to allow glycosylation of the protein and its subsequent exportation to the extracellular space. Analysis by SDS-PAGE, Western-blotting and glycoprotein staining revealed that a glycosylated protein of the expected electrophoretic mobility was obtained in infected larvae. Time course experiments revealed that maximum expression levels were reached 72h post-infection using 10(4)pfu of the recombinant baculovirus (BACgEr) per inoculated larva. An indirect PRV gE-ELISA was developed using gEr as a coating antigen. A comparison between larvae-derived PRV gE-ELISA and two commercially available PRV diagnostic kits showed good correlation between assays and better sensitivity when testing certain sera pig samples using the gEr ELISA. More than 30,000 ELISA determinations could be performed from crude extracts obtained from a single larva infected with the recombinant baculovirus, indicating the feasibility of this strategy for inexpensive production of glycosylated antigens for PRV diagnosis.     

Characterization of the pseudorabies virus-specific immunoglobulin M response and evaluation of its diagnostic use in pigs with preexisting immunity to the virus.

Despite preexisting immunity to pseudorabies virus (PRV), pigs may become infected and may or may not show clinical signs of disease. To investigate whether detection of immunoglobulin M (IgM) antibodies to PRV is suitable for diagnosis of recent infection in pigs with (or without) preexisting immunity, the IgM responses of pigs were examined after both experimental and natural infections. Upon inoculation of seronegative pigs with a low dose of a mildly virulent strain of PRV, IgM was first detectable at day 7 postinoculation (p.i.), reached a maximum at day 14 p.i., and became undetectable again at about days 32 to 36 p.i. In inoculated pigs with maternal antibodies against PRV, the IgM response began later and ended sooner, and peak titers were also lower. In immune pigs with maternally derived antibodies, there was apparently no correlation between the virulence of the inoculated strain and the IgM response. The suitability of the IgM enzyme-linked immunosorbent assay (ELISA) for detection of recent infection in the field was compared with that of the virus neutralization (VN) assay and with an ELISA which specifically detects antibodies directed to glycoprotein I (gI) of PRV. Paired sera were obtained from pigs suspected of PRV infection in an area endemic for PRV infection in which vaccination against PRV is often applied. Practically all pigs had antibodies to PRV in the acute phase of the disease. Compared with the VN assay, the specificity of the IgM ELISA was high but its sensitivity was low. However, all three serotests apparently failed to detect some PRV infections. The IgM ELISA appeared to be especially useful as a diagnostic aid for detection of recent infections in pigs with high levels of neutralizing and gI antibodies, probably maternally derived, in the acute phase of the disease. Such pigs may fail to develop a significant rise in VN antibody titer. The IgM ELISA may be the only serotest for monitoring infections in such pigs.     

Translation of papaya ringspot virus RNA in vitro: detection of a possible polyprotein that is processed for capsid protein, cylindrical-inclusion protein, and amorphous-inclusion protein

The genomic RNA of papaya ringspot virus (PRV), a member of the potyvirus group, was translated in a rabbit reticulocyte cell-free system as an approach to determining the translation strategy of the virus. The RNA directed synthesis of more than 20 distinct polypeptides ranging from apparent molecular weight of 26,000 (26K) to 220K. Antiserum to PRV capsid protein (CP) reacted with a subset of these polypeptides, including a 36K protein that comigrated with PRV CP during electrophoresis. Immunoprecipitation with antiserum to PRV cylindrical-inclusion protein (CIP) defined another set of polypeptides including 70K, 108K, 205K, and 220K proteins as major precipitates. The 70K protein comigrated with authentic CIP, and the 205K and 220K proteins were related to both CP and CIP. Immunoprecipitation with antiserum to PRV amorphous-inclusion protein (AIP) defined a unique set of polypeptides which contained a 112K protein as the major precipitate and 51K, 65K, and 86K proteins as minor precipitates. The 51K protein comigrated with authentic AIR A major product of 330K was observed when translation was done without the reducing agent, dithiothreitol. Immunological analyses and kinetic studies indicated that the 330K protein zone was related to the presumed CP, CIP, and AIP zones and 330K possibly is the common precursor for these viral proteins. The presence of a polyprotein of Mr corresponding to the entire coding capacity of the genomic RNA and its likely precursor relationship to the other polypeptides suggest that proteolytic processing is involved in the translation of PRV RNA.     

A highly specific and sensitive sandwich blocking ELISA based on baculovirus expressed pseudorabies virus glycoprotein B

A direct sandwich blocking enzyme-linked immunosorbent assay (BacgB ELISA) based on the reaction between a monoclonal antibody (MAb) and a recombinant glycoprotein B (gB) of pseudorabies virus (PRV) was developed. This protein was obtained in large quantities from insect cells infected with a PRV gB recombinant baculovirus. Expression of the gB was confirmed by immunoperoxidase monolayer assay (IPMA) with gB specific MAbs. The specificity and sensitivity of the developed BacgB ELISA were evaluated and compared with two commercially available tests by using sets of sera of known PRV infection or vaccination history. For validation, 347 serum samples have been tested. The BacgB ELISA had a high sensitivity and specificity, which were comparable with those of the two commercial tests. In addition, the BacgB ELISA allows detecting anti-gB antibodies in pig serum as early as 7 days following infection. Also maternal antibodies in uninfected pig sera were detected. We conclude that the BacgB ELISA is a useful tool for the detection of as well vaccinated as infected pigs (including derivatives from gE negative vaccine strains), with the added advantage that it uses an antigen that can be produced safely and in large quantities.     

Expression of porcine pseudorabies virus genes by a bovine herpesvirus-1 (infectious bovine rhinotracheitis virus) vector

Summary Recombinant DNA techniques were used to insert foreign genes into bovine herpesvirus-1 [infectious bovine rhinotracheitis virus (IBRV)] vectors which were attenuated by deletion and/or insertion mutations in the IBRV thymidine kinase (tk) gene. In one recombinant, the regulatory and coding sequences of the late pseudorabies virus (PRV) glycoprotein gIII gene, were inserted into the early IBRV tk gene. This recombinant efficiently expressed the PRV gIII gene indicating that immediate early IBRV proteins were competent to transactivate the late PRV gIII gene. IBRV vector viruses were also prepared in which the coding sequences of the early PRV tk gene, the late PRV gIII gene, and theE. coli -galactosidase gene were ligated to the late IBRV gIII promoter. Genotypes and phenotypes of the recombinant viruses were verified by restriction endonuclease and molecular hybridization experiments, thymidine plaque autoradiography, -gal plaque assays, and by immunoprecipitation experiments on extracts from³H-mannose-labelled cells. The recombinant IBRV expressing -gal from the IBRV gIII promoter has been useful as an intermediate in the construction of IBRV vectors harboring foreign DNA sequences. The infectivity of the IBRV recombinant that expressed PRV gIII from the IBRV gIII promoter, was neutralized by polyclonal PRV antisera and by monoclonal antibodies to PRV gIII. The PRV gIII glycoprotein synthesized by the preceding recombinant has been used to coat microtiter test plate wells in a PRV gIII differential diagnostic test kit.     

Induction of protective antibody responses against pseudorabies virus by intranasal vaccination with glycoprotein B in mice

Summary Intranasal vaccination of mice with glycoprotein B (gB) of pseudorabies virus (PRV) induced specific IgA and IgG antibody responses in the secretion of the respiratory tract, resulting in protection of the animals against intranasal challenge with a lethal dose of virulent PRV. The immune response was enhanced by the use of cholera toxin B subunit as an adjuvant. The present results indicate that local vaccination with gB is a promising strategy to confer protective immunity on animals against PRV infection by inducing secretory antibodies on their mucosal surfaces where the primary replication of the virus occurs.     

An indirect double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) using baculovirus-expressed antigen for the detection of antibodies to glycoprotein E of pseudorabies virus and comparison of the method with blocking ELISAs.

Antibodies in porcine sera against glycoprotein E (gE) of pseudorabies virus (PRV) are usually measured in blocking enzyme-linked immunosorbent assays (ELISAs) with one or two murine monoclonal antibodies (MAbs) directed against gE. Our aim was to develop a confirmation assay which is based on another principle and which is able to detect antibodies directed against most potential binding sites on gE with high specificity. Therefore, we developed an indirect double-antibody sandwich assay (IDAS) using recombinant gE expressed by baculovirus (BacgE960). A fragment of the gE gene consisting of nucleotide positions +60 to +1020 of gE, coding for the major antigenic sites of gE but not the transmembrane region, was cloned behind the signal sequence of PRV gG and the p10 promoter in a baculovirus vector. Immunoblot analysis showed that the expressed protein reacted with MAbs directed against five of the six antigenic sites on gE. Although the conformation of some antigenic sites, notably antigenic sites E and C, was not identical to their natural conformation, the expressed protein bound gE-specific antibodies in porcine sera in Western blots (immunoblots) and ELISAs. For the IDAS, a coating MAb directed against the nonimmunodominant antigenic site A on gE was chosen. A major obstacle in binding ELISAs, such as the IDAS, appeared to be the high nonspecific binding activity observed in porcine sera. As a result, sera could be tested only in relatively high dilutions in the BacgE960 IDAS, in contrast to the testing of sera in blocking ELISAs. The sensitivity and specificity of the newly developed BacgE960 IDAS were evaluated and compared with those of five commercially available blocking ELISAs by using several sets of sera of known PRV disease history. The BacgE960 IDAS assay had a high diagnostic specificity and a moderate sensitivity. The five blocking ELISAs differed remarkably in sensitivity and specificity, thereby illustrating the need for standardization and confirmation. We conclude that the BacgE960 IDAS is a useful and specific additional (confirmatory) test for the detection of antibodies to gE.     

Characterization of pseudorabies virus neutralization antigen glycoprotein gIII produced in insect cells by a baculovirus expression vector.

The gene encoding the complete glycoprotein of pseudorabies virus (PRV, Yamagata-S81 strain glycoprotein gIII) has been inserted into the baculovirus transfer vector pAcYM1S derived from the nuclear polyhedrosis virus of Autographa californica (AcNPV). A Spodoptera frugiperda cell line, SF21AE, was efficiently co-transfected with the transfer vector containing the gIII gene and AcNPV DNA by cationic liposomes (Lipofectin). The gene was placed under the control of the AcNPV polyhedrin promoter and expressed to high levels by the derived recombinant virus using SF21AE. Three polypeptides of different molecular weight were expressed. The principal products were glycosylated and transported to the cell surface. The smallest product was not glycosylated. Despite their lower molecular weight, it has been established that the antigenic properties of the peptides were conserved by comparison with those of the authentic glycoprotein gIII of PRV. Immunogenicity of the expressed products was also demonstrated. Intraperitoneal injection of expressed gIII induced neutralizing antibodies in mice. The results have raised the possibility that the protein expressed by baculovirus recombinant may be used to analyze biologically functional sites, develop a subunit vaccine and diagnostic antigens.     

Monoclonal antibodies to bovine coronavirus: characteristics and topographical mapping of neutralizing epitopes on the E2 and E3 glycoproteins

Monoclonal antibodies to the Quebec isolate of bovine coronavirus were produced and characterized. Monoclonal antibodies to both the E2 and the E3 glycoproteins were found to efficiently neutralize virus in vitro. None of the monoclonal antibodies directed against the E1 glycoprotein neutralized virus infectivity. Neutralizing monoclonal antibodies to the E2 glycoprotein were all found to immunoprecipitate gp190, gp100, and their intracellular precursor protein gp170. Neutralizing monoclonal antibodies to the E3 glycoprotein immunoprecipitated gp124 and showed differential reactivity to its precursor proteins gp59 and gp118. These monoclonal antibodies also showed differential reactivity to an apparent degradation product of E3. Neutralizing monoclonal antibodies to E2 bound to two distinct nonoverlapping antigenic domains as defined by competitive binding assays. Neutralizing monoclonal antibodies to the E3 glycoprotein also bound to two distinct antigenic sites as defined by competitive binding assays plus a third site which overlapped these regions. Other results indicated that one domain on the E3 glycoprotein could be further subdivided into two epitopes. Thus four epitopes could be defined by E3-specific monoclonal antibodies.     

Deleting valine-125 and cysteine-126 in glycoprotein gI of pseudorabies virus strain NIA-3 decreases plaque size and reduces virulence in mice

Summary We investigated the function of antigenic domains on gI in virulence and immunogenicity. Three PRV gI mutants were constructed by deleting nucleotides coding for the following amino acids: valine-125 and cysteine-126, located in a discontinuous antigenic domain (M 303); glycine-59 and aspartic acid-60 located in a continuous antigenic domain (M 304); and arginine-67 and alanine-68, located in a discontinuous antigenic domain (M 305). Mismatch primers in the polymerase chain reaction were used to introduce the deletions. Anti-gI monoclonal antibodies were used in an immunoperoxidase monolayer assay to distinguish PRV gI mutants from wild-type PRV. The gI mutant viruses were tested for their growth in vitro and for their virulence in mice. The growth properties of PRV gI mutant virus M 303 were comparable to the growth properties of a PRV gI-negative mutant (M 301): both mutants produced small plaques in various cells, and when grown on swine kidney cells and chicken embryo fibroblasts, their growth was disadvantaged compared to wild-type PRV. However, in embryonal Balb/c mouse cells expressing gI, gI mutant viruses and wild-type PRV produced plaques of the same size, confirming that the mutations in gI are responsible for the small plaque phenotype. The growth properties of PRV gI mutant viruses M 304 and M 305 were comparable to the growth properties of wild-type PRV. When the mean time to death was used as the criterion, the gI mutant viruses M 301 and M 303 were significantly less virulent in mice than wild-type PRV. Four other, independently obtained, PRV mutants all carrying the valine-125 and cysteine-126 deletion (M 308, M 309, M 310 and M 311 respectively) exhibit the same phenotype. Our results show that deleting valine-125 and cysteine-126 in gI decreases plaque size and reduces virulence in mice to the same degree as deleting the gI protein.     

Antibody-mediated polysome precipitation as a method for the size determination of viral mRNA species: viral envelope glycoprotein mRNA of avian sarcoma viruses

A method was developed that allows the in situ isolation of viral mRNA, i.e. from polysomes of infected cells. This was achieved by precipitating intact polysomes via their nascent virus polypeptides using virus-specific antibodies. As a model, antibodies to the major envelope glycoprotein (gp85) of avian sarcoma viruses were employed to precipitate those polysomes from infected cells which synthesized the corresponding p70 virus glycoprotein precursor. Normal immunoglobulin and polysomes from uninfected chicken embryo fibroblasts served as controls. The radioactivity labelled mRNA from antibody-precipitated polysomes could subsequently be extracted and characterized for size. It was found that avian sarcoma virus gp85 envelope glycoprotein is predominantly synthesized by a 22--28 s viral mRNA. In addition, minor amounts of gp85-specific mRNAs of 16--21 s and 28--35 s could be demonstrated. The data indicate the presence in polysomes of viral mRNA species coding for i) gp85 only (16--21 s RNA), ii) gp85 and pp60src protein from the adjacent src-gene (22--28 s RNA), and iii) a large viral precursor protein (28--35 s RNA).     

Expression of melanocyte-associated markers gp-100 and Melan-A/MART-1 in angiomyolipomas

 Angiomyolipomas are tumours of uncertain histogenesis, most often occurring in association with the kidney. A characteristic finding is their reactivity with HMB-45, a monoclonal antibody to the melanocyte-associated antigen gp-100. We tested 18 angiomyolipomas for their reactivity with A103, a monoclonal antibody to Melan-A (MART-1), another melanocyte-associated marker, and compared it with HMB-45. All cases were positive with both antibodies, yet most cases showed a more homogeneous staining pattern with A103. Normal kidney was immunohistochemically negative for both antibodies. We also performed RT-PCR assays for gp-100 and Melan-A in 4 of the 18 angiomyolipoma samples and in three normal kidney samples. All 4 angiomyolipoma specimens revealed mRNA for both melanocyte differentiation markers. gp-100 mRNA was found in the samples of normal kidney, but Melan-A mRNA was not. Our study shows that angiomyolipomas express the melanocyte-associated antigens Melan-A and gp-100 at the protein and at the mRNA level, suggesting a true expression of these antigens rather than cross-reacting epitopes. Based on the mRNA expression pattern, immunohistochemical analysis is the preferred method for the detection of gp-100, while Melan-A can be used at the protein and mRNA levels. Our study demonstrates that A103 is a useful marker for the diagnosis of angiomyolipomas. Received: 3 December 1998 / Accepted: 5 January 1999     

Induction of antibody against in vitro translation products encoded by varicella-zoster virus glycoprotein genes

Antibodies were raised in rabbit against the in vitro translation products encoded by the varicella-zoster virus (VZV) glycoprotein genes gpI and gpIV. The antisera neutralized VZV infectivity and specifically identified two late VZV glycoproteins, gpI and gpIV, in VZV-infected cells and in the envelope of VZ virions. Pulse-chase experiments revealed a 55K precursor protein to gpIV (60K) and a 82K precursor protein to gpI (95K). Immunoprecipitation of 32P-labeled VZV-infected cells showed that the precursor-products of gpI are phosphorylated. These results demonstrate that translation products synthesized in vitro can be used to produce antibodies that recognize native viral proteins and therefore facilitate the identification and analysis of viral gene products in the infected cells.