Protection of mice and swine from pseudorabies virus conferred by vaccinia virus-based recombinants.

Glycoproteins gp50, gII, and gIII of pseudorabies virus (PRV) were expressed either individually or in combination by vaccinia virus recombinants. In vitro analysis by immunoprecipitation and immunofluorescence demonstrated the expression of a gII protein of approximately 120 kDa that was proteolytically processed to the gIIb (67- to 74-kDa) and gIIc (58-kDa) mature protein species similar to those observed in PRV-infected cells. Additionally, the proper expression of the 90-kDa gIII and 50-kDa gp50 was observed. All three of these PRV-derived glycoproteins were detectable on the surface of vaccinia virus-PRV recombinant-infected cells. In vivo, mice were protected against a virulent PRV challenge after immunization with the PRV glycoprotein-expressing vaccinia virus recombinants. The coexpression of gII and gIII by a single vaccinia virus recombinant resulted in a significantly reduced vaccination dose required to protect mice against PRV challenge. Inoculation of piglets with the various vaccinia virus-PRV glycoprotein recombinants also resulted in protection against virulent PRV challenge as measured by weight gain. The simultaneous expression of gII and gp50 in swine resulted in a significantly enhanced level of protection as evaluated by weight evolution following challenge with live PRV.     

Envelope glycoprotein gp50 of pseudorabies virus is essential for virus entry but is not required for viral spread in mice.

Phenotypically complemented pseudorabies virus gp50 null mutants are able to produce plaques on noncomplementing cell lines despite the fact that progeny virions are noninfectious. To determine whether gp50 null mutants and gp50+gp63 null mutants are also able to replicate and spread in animals, mice were infected subcutaneously or intraperitoneally. Surprisingly, both gp50 mutants and gp50+gp63 double mutants proved to be lethal for mice. In comparison with the wild-type virus, gp50 mutants were still highly virulent, whereas the virulence of gp50+gp63 mutants was significantly reduced. Severe signs of neurological disorders, notably pruritus, were apparent in animals infected with the wild-type virus or a gp50 mutant but were much less pronounced in animals infected with a gp50+gp63 or gp63 mutant. Immunohistochemical examination of infected animals showed that all viruses were able to reach, and replicate in, the brain. Examination of visceral organs of intraperitoneally infected animals showed that viral antigen was predominantly present in peripheral nerves, suggesting that the viruses reached the central nervous system by means of retrograde axonal transport. Infectious virus could not be recovered from the brains and organs of animals infected with gp50 or gp50+gp63 mutants, indicating that progeny virions produced in vivo are noninfectious. Virions that lacked gp50 in their envelopes, and a phenotypically complemented pseudorabies virus gII mutant (which is unable to produce plaques in tissue culture cells), proved to be nonvirulent for mice. Together, these results show that gp50 is required for the primary infection but not for subsequent replication and viral spread in vivo. These results furthermore indicate that transsynaptic transport of the virus is independent of gp50. Since progeny virions produced by gp50 mutants are noninfectious, they are unable to spread from one animal to another. Therefore, such mutants may be used for the development of a new generation of safer (carrier) vaccines.     

Glycoprotein gp50-negative pseudorabies virus: a novel approach toward a nonspreading live herpesvirus vaccine.

Essential herpesvirus glycoproteins are involved in membrane fusion processes during infection, e.g., viral penetration and direct cell-to-cell transmission. We previously showed that the gD-homologous glycoprotein gp50 of pseudorabies virus (PrV) is essential for virus entry into target cells but proved to be dispensable for direct viral cell-to-cell spread in cell culture (I. Rauh and T. C. Mettenleiter, J. Virol. 65:5348-5456, 1991). For gp50-negative (gp50-) viruses, after phenotypic complementation necessary for primary infection, the only means of viral spread is by way of direct cell-to-cell transmission. In contrast, virus mutants lacking the essential gB-homologous glycoprotein gII after phenotypic complementation are only able to infect primary target cells and are blocked in further viral spread. To analyze how these in vitro phenotypes translate into virus replication in the animal, mice were infected intranasally with gp50- or gII- PrV mutants after prior phenotypic complementation by propagation on cell lines providing the essential glycoprotein in trans. Our results show that whereas the gII- mutants did not cause disease or any symptoms, gp50- mutants derived from two different PrV strains were fully virulent, with animals exhibiting severe symptoms ultimately leading to death. However, free infectious virus could not be recovered from either gp50- or gII- PrV-infected animals. We conclude that direct cell-to-cell transmission as the only means of viral spread of the gp50- mutants is sufficient for a full virulent phenotype in mice. After infection of pigs with phenotypically complemented gp50- PrV, only mild symptoms were observed, whereas the gII- mutant was totally avirulent. In both cases, shedding of infectious virus did not occur, in contrast to results with animals infected by gX- PrV that showed severe signs of disease and extensive virus shedding. After challenge infection with the highly virulent NIA-3 strain, the previously gII- PrV-infected animals exhibited severe symptoms, whereas the gp50- PrV-infected pigs showed a significant level of protection. In conclusion, vaccination with a PrV mutant lacking glycoprotein gp50, which is unable to spread between animals because of a lack of formation of free infectious virions, can confer on pigs protection against challenge infection. These results provide the basis for the development of new, nonspreading live herpesvirus vaccines based on gp50- PrV mutants.     

DNA sequence of the gene for pseudorabies virus gp50, a glycoprotein without N-linked glycosylation.

The DNA sequence was determined for a region of the pseudorabies virus (PRV) genome to which a mutation defining resistance to a monoclonal antibody has been mapped (M. W. Wathen and L. M. K. Wathen, J. Virol., 51:57-62, 1984). This sequence was found to contain an open reading frame that did not include an amino acid sequence directing N-linked glycosylation. This open reading frame was expressed in uninfected Chinese hamster ovary cells to produce the PRV glycoprotein gp50. When PRV-infected Vero cells were incubated in the presence of tunicamycin, the gp50 that was produced had an identical molecular weight to that produced in the absence of drug. When infected cells were incubated in the presence of monensin, the molecular weight of gp50 was reduced from 60,000 to 45,000, but was not sensitive to endo-beta-N-acetylglucosaminidase H. These observations led to the conclusion that gp50 does not contain N-linked carbohydrate, as predicted from the DNA sequence. A region of the amino acid sequence and the positions of the cysteine residues of PRV gp50 are homologous to glycoprotein D of herpes simplex virus.     

Deletions in vaccine strains of pseudorabies virus and their effect on synthesis of glycoprotein gp63.

The pseudorabies virus vaccine strains Norden and Bartha each have been reported to have deletions in the small unique component of the genome (B. Lomniczi, M. L. Blankenship, and T. Ben-Porat, J. Virol. 49:970-979, 1984). The deletion in Norden was shown to delete the entire coding region for gI but not any of the coding sequences for gp63. However, gp63 in Norden-infected cells was only 36 kilodaltons, and a 44-kilodalton form of gp63 was released into the medium. In Bartha, the deletion removed the coding region for all but 89 amino acids of gp63, and no gp63 was detected in either Bartha-infected cells or medium.     

Reduced yield of infectious pseudorabies virus and herpes simplex virus from cell lines producing viral glycoprotein gp50.

Pseudorabies virus (PRV) glycoprotein gp50 is the homolog of herpes simplex virus (HSV) glycoprotein D. Several cell lines that constitutively synthesize gp50 were constructed. Vero cells, HeLa cells, and pig kidney (MVPK) cells that produce gp50 all gave reduced yields of PRV and HSV progeny viruses when compared with the parent cell line or the same cell line transfected to produce a different protein. The reduction in virus yield was greatest at low multiplicities of infection. The Vero and HeLa cells that produce gp50 showed an even greater reduction in HSV yield than in PRV yield. This phenomenon may be an example in a herpesvirus of the interference observed in retroviruses or cross-protection in plant virus systems.     

Host range restriction of vaccinia virus in Chinese hamster ovary cells: relationship to shutoff of protein synthesis.

Chinese hamster ovary cells were found to be nonpermissive for vaccinia virus. Although early virus-induced events occurred in these cells (RNA and polypeptide synthesis), subsequent events appeared to be prevented by a very rapid and nonselective shutoff of protein synthesis. Within less than 2 h after infection, both host and viral protein syntheses were arrested. At low multiplicities of infection, inhibition of RNA synthesis with cordycepin resulted in failure of the virus to block protein synthesis. Moreover, infection of the cells in the presence of cycloheximide prevented the immediate onset of shutoff after reversal of cycloheximide. Inactivation of virus particles by UV irradiation also impaired the capacity of the virus to inhibit protein synthesis. These results suggested that an early vaccinia virus-coded product was implicated in the shutoff of protein synthesis. Either the nonpermissive Chinese hamster ovary cells were more sensitive to this inhibition than permissive cells, or a regulatory control of the vaccinia shutoff function was defective.     

Culture media optimization for Chinese hamster ovary cell growth and expression of recombinant varicella-zoster virus glycoprotein E

Herpes zoster (HZ) is caused by reactivation of varicella-zoster virus (VZV) latent in the sensory ganglia and causes severe pain, often leading to postherpetic neuralgia (PHN). Two prophylactic vaccines against HZ are currently licensed for human use, a live attenuated vaccine and a subunit vaccine containing recombinant VZV glycoprotein E (gE) as antigen. The latter has superior protective efficacy against HZ and PHN. During HZ subunit vaccine development, we obtained Chinese hamster ovary (CHO) cell clones expressing VZV gE. This study was performed to optimize culture media conditions for CHO cell growth and gE production. Using a high-throughput culture system, three CHO cell clones were cultured in microtiter plates containing 24 different basal media, and three basal media were selected. The clone with the highest gE expression was fed-batch cultured in each of the three basal media in combination with 13 different feed media. A pair of media, BalanCD CHO Growth A and EX-CELL Advanced CHO Feed 1, with the highest productivity was selected for gE production. Scale-up fed-batch cultures of the selected clone cultured in a wave bag bioreactor containing the optimized media yielded 2440 mg gE protein/L culture, a 11.5-fold increase compared to original culture conditions (batch culture in CD OptiCHO medium). The optimized media condition is used to produce VZV gE antigen for an HZ subunit vaccine, which is under phase I clinical trial. This study would provide valuable insights on culture media optimization for CHO cells expressing a recombinant vaccine antigen.

     

Insensitivity of a ricin-resistant mutant of Chinese hamster ovary cells to fusion induced by Newcastle disease virus.

The simian virus 40 (sv40) tumor antigen (T-antigen) and tumor-specific transplantation antigen (TSTA) have been partially purified and studied to clarify their relationship. The T-antigen and the TSTA were partially purified from nuclei of SV AL/N cells, and SV40-transformed mouse embryo fibroblast line, by precipitation with ammonium sulfate and chromatography on DEAE- and DNA-cellulose. The T-antigen was assayed by complement fixation, and the TSTA was assayed by its ability to immunize mice against SV40-containing ascites tumor cells. When T-antigen- and TSTA-containing preparations were sedimented through sucrose gradients, each antigen had a major peak of activity at a sedimentation coefficient of 6.7 and minor peaks in other regions. Antiserum against T-antigen (from tumor-bearing hamsters) immunoprecipitated the TSTA activity. A preparation of T-antigen from human SV80 cells, which exhibited only one protein band after sodium dodecylsulfate-polyacrylamide gel electrophoresis, had TSTA activity when as little as 0.6 microgram of protein per mouse was used for immunization. These experiments demonstrate that the T-antigen, the product of the SV40 early A gene is capable of inducing specific immunity against transplantation of SV40-transformed tumor cells in mice.     

A cowpox virus gene required for multiplication in Chinese hamster ovary cells.

Cowpox virus, in contrast to vaccinia virus, can multiply in Chinese hamster ovary cells. To study the genetic basis for this difference in host range, recombinants between vaccinia and cowpox viruses were isolated and their DNA restriction patterns were examined. The ability to multiply in Chinese hamster ovary cells could be correlated with the conservation of cowpox virus sequences mapping at the left end of the genome. This was further demonstrated by marker rescue of the host range phenotype with restricted cowpox virus DNA. Marker rescue with cloned restriction fragments of decreasing size enabled the fine localization of the host range function to a 2.3-kilobase-pair fragment. Nucleotide sequencing revealed that the fragment encoded a single major polypeptide of approximately 77,000 daltons. It is suggested that the role of the host range gene from cowpox virus is to prevent the early and extensive shutoff of protein synthesis that normally occurs in Chinese hamster ovary cells infected by vaccinia virus.     

Isolation and characterization of a Chinese hamster ovary mutant cell line with altered sensitivity to vaccinia virus killing.

The Chinese hamster ovary (CHO) cell line is nonpermissive for vaccinia virus, and translation of viral intermediate genes was reported to be blocked (A. Ramsey-Ewing and B. Moss, Virology 206:984-993, 1995). However, cells are readily killed by vaccinia virus. A vaccinia virus-resistant CHO mutant, VV5-4, was isolated by retroviral insertional mutagenesis. Parental CHO cells, upon infection with vaccinia virus, die within 2 to 3 days, whereas VV5-4 cells preferentially survive this cytotoxic effect. The survival phenotype of VV5-4 is partial and in inverse correlation with the multiplicity of infection used. In addition, viral infection fails to shut off host protein synthesis in VV5-4. VV5-4 was used to study the relationship of progression of the virus life cycle and cell fate. We found that in parental CHO cells, vaccinia virus proceeds through expression of viral early genes, uncoating, viral DNA replication, and expression of intermediate and late promoters. In contrast, we detect only expression of early genes and uncoating in VV5-4 cells, whereas viral DNA replication appears to be blocked. Consistent with the cascade regulation model of viral gene expression, we detect little intermediate- and late-gene expression in VV5-4 cells. Since vaccinia virus is known to be cytolytic, isolation of this mutant therefore demonstrates a new mode of the cellular microenvironment that affects progression of the virus life cycle, resulting in a different cell fate. This process appears to be mediated by a general mechanism, since VV5-4 is also resistant to Shope fibroma virus and myxoma virus killing. On the other hand, VV5-4 remains sensitive to cowpox virus killing. To examine the mechanism of VV5-4 survival, we investigated whether apoptosis is involved. DNA laddering and staining of apoptotic nuclei with Hoechst 33258 were observed in both CHO and VV5-4 cells infected with vaccinia virus. We concluded that the cellular pathway, which blocks viral DNA replication and allows VV5-4 to survive, is independent of apoptosis. This mutant also provides evidence that an inductive signal for apoptosis upon vaccinia virus infection occurs prior to viral DNA replication.     

Monitoring gene expression in Chinese hamster ovary cells using secreted apoaequorin.

A luminescence method for monitoring gene expression in Chinese hamster ovary cells using apoaequorin as a secreted reporter enzyme is described. In this method, the cell is not disrupted prior to assay as in the earlier aequorin procedure and in the firefly method. The apoaequorin secretion vector is constructed by fusing the DNA fragment of the signal peptide sequence of human follistatin to the apoaequorin gene. Transfection of Chinese hamster ovary cells with the vector causes the apoaequorin to be secreted directly into the culture medium. Assay is carried out by removing a small aliquot of the culture medium, incubating it with coelenterazine, and adding Ca2+ to trigger light emission from the regenerated aequorin. The light intensity is measured with a photomultiplier photometer and is proportional to the amount of apoaequorin present. The method is highly specific and sensitive and can be carried out in a relatively short period of time.     

Protection against lethal simian immunodeficiency virus SIVsmmPBj14 disease by a recombinant Semliki Forest virus gp160 vaccine and by a gp120 subunit vaccine.

Infection of pigtail macaques with SIVsmmPBj14, biological clone 3 (SIV-PBj14-bc13), produces an acute and usually fatal shock-like syndrome 7 to 14 days after infection. We used this simian immunodeficiency virus (SIV) model as a rapid and rigorous challenge to evaluate the efficacy of two SIV Env vaccine strategies. Groups of four pigtail macaques were immunized four times over a 25-week span with either a recombinant Semliki Forest virus expressing the SIV-PBj14 Env gp160 (SFV-SIVgp160) or purified recombinant SIV-PBj14 gp120 (rgp120) in SBN-1 adjuvant. Antibody titers to SIV Env developed in all immunized animals (mean peak titers prior to challenge, 1:1,700 for SFV-SIV gp 160 and 1:10,500 for rgp120), but neither neutralizing antibodies nor SIV-specific T-cell proliferative responses were detectable in any of the vaccinees. All macaques were challenged with a 100% infectious, 75% fatal dose of SIV-PBj14-bc13 at week 26. Three of four control animals died of acute SIV-PBj14 syndrome on days 12 and 13. By contrast, all four SFV-SIVgp160-immunized animals and three of the four rgp120-immunized animals were protected from lethal disease. While all virus-challenged animals became infected, symptoms of the SIV-PBj14 syndrome were more severe in controls than in vaccinees. Mean virus titers in plasma at 13 days postchallenge were approximately 10-fold lower in vaccinated than control animals. However, there was no apparent correlation between survival and levels of peripheral blood mononuclear cell-associated culturable virus, provirus load, or any antiviral immunologic parameter examined. The results indicate that while immunization with SFV-SIVgp160 and rgp120 did not protect against virus infection, these Env vaccines did lower the virus load in plasma and protect against the lethal SIV-PBj14 challenge.     

Amplification and expression of heterologous oncostatin M in Chinese hamster ovary cells.

A stable Chinese hamster ovary (CHO) cell line producing high levels of human oncostatin M (OM) was generated by transfecting a heterologous gene coding for the protein. This novel construct was comprised of the gene for the transforming growth factor-beta 1 (TGF-beta 1) signal peptide fused to the gene for mature human OM. Amplification with methotrexate produced milligram quantities of this recombinant OM, which was processed correctly, glycosylated, and found to have biological functions similar to those of natural OM.     

Relationship between Golgi architecture and glycoprotein biosynthesis and transport in Chinese hamster ovary cells

We have investigated the effect of colcemid-induced disassembly of microtubules, which is accompanied by retraction of the endoplasmic reticulum and fragmentation of the Golgi apparatus, on glycoprotein biosynthesis and transport in Chinese hamster ovary (CHO) cells. CHO cells were metabolically radiolabeled with [6- 3H]galactose or [2- 3H]mannose in the presence of either 0.1% dimethyl sulfoxide or 10 microM colcemid in dimethyl sulfoxide. The fine structure of glycoprotein asparagine-linked oligosaccharide structures synthesized in the presence or absence of colcemid was analyzed by lectin affinity chromatography, ion exchange chromatography, and methylation analysis using radiolabeled glycopeptides prepared by Pronase digestion. The fractionation patterns of [3H]mannose- and [3H]galactose-labeled glycopeptides on immobilized lectins indicated that processing to complex N-linked chains and poly-N-acetyllactosamine modification were similar in control and colcemid-treated cells. In addition, colcemid treatment did not alter the extent of sialylation or the linkage position of sialic acid residues to galactose. Using a trypsin release protocol, it was also found that the transport of newly synthesized glycoproteins to the cell surface was not affected by colcemid. These results demonstrate that the morphologically altered ER and Golgi apparatus in colcemid-treated CHO cells are completely functional with respect to the rate and fidelity of protein asparagine-linked glycosylation. Furthermore, movement of newly synthesized glycoproteins to and through the ER and Golgi apparatus and their transport to the cell surface in nonpolarized cells appears to be microtubule-independent.     

Delay of vaccinia virus-induced apoptosis in nonpermissive Chinese hamster ovary cells by the cowpox virus CHOhr and adenovirus E1B 19K genes.

The infection of vaccinia virus in Chinese hamster ovary (CHO) cells produces a rapid shutdown in protein synthesis, and the infection is abortive (R.R. Drillien, D. Spehner, and A. Kirn, Virology 111:488-499, 1978; D.E. Hruby, D.L. Lynn, R. Condit, and J.R. Kates, J. Gen. Virol. 47:485-488, 1980). Cowpox virus, which can productively infect CHO cells, had previously been shown to contain a host range gene, CHOhr, which confers on vaccinia virus the ability to replicate in CHO cells (D. Spehner, S. Gillard, R. Drillien, and A. Kirn, J. Virol. 62:1297-1304, 1988). We found that CHO cells underwent apoptosis when infected with vaccinia virus. The expression of the CHOhr gene in vaccinia virus allowed for the expression of late virus genes. CHOhr also delayed or prevented vaccinia virus-induced apoptosis in CHO cells such that there was sufficient time for replication of the virus before the cell died. The E1B 19K gene from adenovirus also delayed vaccinia virus-induced apoptosis; however, there was no detectable expression of late virus genes. Furthermore, E1B 19K also delayed cell death in CHO cells which had been productively infected with vaccinia virus. This study identifies a new antiapoptotic gene from cowpox virus, CHOhr, for which the protein contains an ankyrin-like repeat and shows no significant homology to other proteins. This work also indicates that an antiapoptotic gene from one virus family can delay cell death in an infection of a virus from a different family.     

Evidence for allelic exclusion in Chinese hamster ovary cells.

Earlier results suggested that the functional hemizygosity of genes in pseudodiploid Chinese hamster ovary (CHO) cells is due to the silencing of one allele by DNA methylation. From this one could make a strong prediction that we have now been able to confirm by genetic experiments, using thymidine kinase (TK) alleles. TK- mutants induced by ethylmethane sulphonate (EMS) were all revertible to TK+ at high frequency by the demethylating agent 5-azacytidine (5-aza-CR). This revertibility was due to reactivation of a silent nonmutant TK allele. Further mutagenesis by EMS yielded TK- derivatives that were no longer revertible by 5-aza-CR; these are assumed to have mutations in both alleles. TK- cells were also transfected with equine herpes virus TK+ DNA, and the TK+ derivatives were shown to be markedly less stable than cells with the normal TK+ gene. CHO cells lack metallothionein activity (sensitive to cadmium), and also require proline for growth, because genes have become silenced during the establishment of the cell line. In both cases 5-aza-CR reactivates these genes to give the cadmium resistant and proline independent phenotypes. Long-term experiments with reactivants in the absence of selection showed that the genes become silent, presumably as a result of de novo methylation. A strain resistant to cytosine arabinoside (araCR) was also resistant to 5-azadeoxycytidine (5-aza-CdR), but not to 5-aza-CR, which would be expected if the araCR strain lacked deoxycytidine kinase.(ABSTRACT TRUNCATED AT 250 WORDS)