Replication-defective recombinant Semliki Forest virus encoding GM-CSF as a vector system for rapid and facile generation of autologous human tumor cell vaccines.

This paper describes the production of recombinant Semliki Forest virus encoding murine or human granulocyte-macrophage colony-stimulating factor (GM-CSF) and the capacity of these vectors to transduce murine and human tumor cells ex vivo. High-titer stocks (up to 3 x 10(9) particles/ml) of conditionally infective, replication-defective, recombinant SFV particles were generated using the SFV Helper-2 system. It is shown that the recombinant SFV/GM-CSF virus, as well as recombinant SFV carrying the beta-galactosidase reporter gene, efficiently transduce both murine tumor cell lines as well as primary human renal carcinoma cells. Using ELISA's specific for GM-CSF, levels of GM-CSF production by the cells were determined. Levels of murine GM-CSF (mGM-CSF) produced by SFV/mGM-CSF transduced renal cell cancer cultures were equal to or higher than corresponding levels reported in the literature after transduction of similar renal carcinoma cell cultures using a retroviral vector system. The biological activity of GM-CSF was demonstrated by using cells which are dependent on GM-CSF for growth and by using primary bone marrow cells. All the transduced cell cultures (including the human renal cell carcinoma samples) produced GM-CSF for up to at least 4 days after transduction. The results imply that the recombinant SFV system can be used for rapid and facile preparation of autologous cancer cell vaccines.

Glycoprotein H of herpes simplex virus type 1 requires glycoprotein L for transport to the surfaces of insect cells.

In mammalian cells, formation of heterooligomers consisting of the glycoproteins H and L (gH and gL) of herpes simplex virus type 1 is essential for the cell-to-cell spread of virions and for the penetration of virions into cells. We examined whether formation of gH1/gL1 heterooligomers and cell surface expression of the complex occurs in insect cells. Three recombinant baculoviruses, expressing gL1, gH1, and truncated gH1 (gH1t), which lacks the transmembrane region, were constructed. It was shown that recombinant gH1/gL1 and gH1t/gL1 heterooligomers were produced in insect cells. As in mammalian cells, gH1 and gH1t were not detected on the surfaces of insect cells in the absence of gL1. When coexpressed with gL1, recombinant gH1 was displayed on the surfaces of insect cells. Coexpression of gH1t and gL1 resulted in secretion of the gH1t/gL1 complex into the cell culture medium, indicating that gH1t is also transported to the surfaces of insect cells. Our results indicate that the process of folding and intracellular transport of gH1 and gL1 is comparable in insect cells and mammalian cells and that the baculovirus expression system can be used to examine the complex formation and the intracellular transport of gH1 and gL1. The availability of secreted gH1t/gL1 complex offers the opportunity to further investigate the immunological properties of this complex.

Construction and properties of pseudorabies virus recombinants with altered control of immediate-early gene expression.

To investigate how altered control of expression of the essential immediate-early (IE) gene of pseudorabies virus influences virus replication and virulence, we replaced the IE promoter with the tissue-specific promoters of the bovine cytokeratin IV gene (CKIV), the bovine cytokeratin VIb gene (CKVIb), or the inducible promoter of Drosophila heat shock gene HSP70. We compared expression of the IE gene of the wild-type virus and recombinant viruses in different cell types and at different temperatures and found that IE expression had become cell type or temperature dependent. When a recombinant virus was titrated on nonpermissive cells or was titrated at nonpermissive temperatures in vitro, the plating efficiency was reduced by more than 99%. Mice were inoculated subcutaneously (s.c.), intraperitoneally (i.p.), or intranasally (i.n.) with a dose equal to 100 times the 50% lethal dose of the wild-type virus. After inoculation with temperature-sensitive recombinant N-HSP, two (s.c.), two (i.p.), and four (i.n.) of five mice died. However, at this dose, recombinant N-CKIV, which contains a promoter specific for stratified epithelial tissue of the tongue mucosa, was not lethal when inoculated s.c. or i.p. but killed four mice when inoculated i.n. Recombinant N-CKVIb, which contains a promoter specific for the suprabasal layers of the epidermis, was not lethal after inoculation by any of the three routes. In explant cultures of nasal mucosa of pigs, replication of N-CKIV and N-CKVIb was not markedly reduced in the epithelium. However, in contrast to results obtained with wild-type virus, infection of the stroma was not observed. We conclude that the replicative ability and virulence of pseudorabies virus can be influenced by altering control of expression of the IE gene.

Genetic recombination of pseudorabies virus: evidence that homologous recombination between insert sequences is less frequent than between autologous sequences.

We studied in vivo recombination between a thymidine kinase (TK) negative, glycoprotein E (gE) negative, attenuated strain and a virulent strain of pseudorabies virus (PRV) in pigs. To simplify the detection of recombination we inserted different but overlapping (375 bp) parts of the E1 gene of classical swine fever virus into the gG locus of both virus strains. Recombination between the E1 sequences of these viruses results in reconstitution of the complete E1 coding sequence and expression of the E1 protein. Since E1 is highly immunogenic, we expected to detect in vivo recombination in co-inoculated pigs by the presence of serum antibodies against E1. However, after co-inoculation of pigs with high doses of both virus strains, we were unable to detect antibodies against E1, suggesting that in vivo recombination did not occur or remained below the detection limit. Analysis of individual progeny viruses showed that 13 out of 995 (1.3%) possessed a recombinant TK-negative gE-positive phenotype. In contrast, no E1-positive viruses were detected among 5000 analyzed. This result showed that in vivo recombination between the two virus strains did occur, but was much more frequent between the TK and gE loci than between the E1 sequences. Similar results were obtained in in vitro recombination experiments in which possible growth differences between the various virus strains were excluded. The different recombination frequencies could not be attributed to the difference in distance of the genetic loci since recombination between mutations at a distance of 266 bp in the TK gene occurred as frequent as recombination between the TK and gE genes which are separated by approximately 60 kilobasepairs. These results indicate that some property of the E1 sequence and/or the location of the E1 sequence within the PRV genome affects the frequency of recombination.

In vivo recombination of pseudorabies virus strains in mice.

We studied in vivo recombination of pseudorabies virus (PRV) by inoculating mice with non-lethal mutants that carry a small deletion or insertion in the thymidine kinase (TK) gene or the ribonucleotide reductase (RR) gene. After co-inoculation of mice with two different mutants, homologous recombination between the viral genomes resulted in the generation of wild-type PRV that was highly lethal for mice. Thus, recombination could easily be assessed by monitoring survival of inoculated animals. Our results demonstrated that recombination was only detectable when high doses of virus were used. Intragenic recombination was more efficient between mutations in the TK gene than between mutations in the RR gene. Efficient intragenic recombination in the TK gene occurred between mutations which were separated by as few as 266 nucleotides. When two mutants were inoculated with an interval of 2 h, recombination still occurred. No recombination could be detected when the viruses were inoculated at the same time but in separate parts of the body. When inoculated separately, none of the mutants tested could be isolated from the brains of mice. Virus could be recovered from the brain, however, after co-inoculation. Surprisingly, of these viruses 36-39% possessed the parental mutant genotype. This observation indicates that complementation enables these mutants to replicate in the brain and suggests that complementation may contribute to pathogenicity of PRV.

Vaccine properties of pseudorabies virus strain 783 are not affected by a deletion of 71 base pairs in the promoter/enhancer region of the viral immediate early gene.

Strain 783 of pseudorabies virus (PRV) is a genetically engineered vaccine which contains three deletions. The purpose of this study was to examine the effect of one of the deletions, which until now has not been characterized. The deletion occurs within the inverted repeats. Seventy-one base pairs (bp) were deleted, including one of the repeat sequence elements related to the TAATGARATTC boxes detected within the promoter and enhancer region of the immediate early (IE) genes of herpes simplex virus. The deletion affected neither the transcription of the IE gene nor viral growth in vitro. In our animal experiments, one group of pigs was inoculated with the original strain 783 and another with strain 783 which had had the repeat sequences restored. These two groups were then compared to determine the protective efficacy of the two vaccine strains against PRV infection. The deletion in the inverted repeats does not affect the vaccine properties of PRV strain 783: strain 783, with and without the 71 bp deletion in the repeats, protected pigs equally well.

Virulence and pathogenesis of non-virulent and virulent strains of pseudorabies virus expressing envelope glycoprotein E1 of hog cholera virus.

Pseudorabies virus (PRV) expressing the envelope glycoprotein E1 (E1) of hog cholera virus (HCV) was used as a model to study the potential risks connected with the use of a live herpesvirus vaccine expressing a foreign gene. The gene encoding E1 was inserted into the glycoprotein X (gX) locus of both a virulent PRV strain and a non-virulent PRV strain in which the virulence genes encoding glycoprotein I (gI) and thymidine kinase (TK) had been inactivated. We investigated whether strain M205 (gI-, TK-, gX-, E1+) had a changed cell or host tropism or virulence compared with strain M206 (gI-, TK-, gX-) in pigs, rabbits, hamsters, rats, mice and rhesus monkeys. The insertion of E1 into this non-virulent PRV strain caused no change in cell or host tropism. However, pigs inoculated with M205 shed less virus over a shorter period than pigs inoculated with M206. Theoretically, virulent PRV strains expressing E1 (gX-, E1+) could arise through transfer of the E1 gene of M205 to a virulent PRV strain. Therefore, we inoculated pigs with strain M12 (gX-, E1+) or the control strain M104 (gX-) and compared the virulence and pathogenesis. M12 and M104 were of approximately equal virulence and the pathogenesis of both strains was similar. We concluded that incorporating E1 of HCV into the gX locus of PRV did not change cell or host tropism, nor did it change the virulence of either non-virulent or virulent PRV.