CSIRO Australian Animal Health Laboratory, PO Bag 24, Geelong, Victoria 3220, Australia1
Author for correspondence: Barbara Coupar. Fax +61 3 5227 5555. e-mail barbara.coupar{at}dah.csiro.au
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Abstract |
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Introduction |
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In experiments reported previously (Andrew & Coupar, 1992 ) we have expressed murine interleukin 2 (IL-2) and IL-4 from either the tk insertion site or the HindIII F insertion site. These two cytokines had quite different effects in mice. IL-2 had an attenuating effect in that it allowed immunocompromised (nude or sublethally irradiated) mice to survive an otherwise lethal dose of vaccinia virus (Ramshaw et al., 1987
; Flexner et al., 1987
). In contrast, IL-4 expressed from a recombinant vaccinia virus resulted in shorter mean times to death in immunocompromised mice and, at high enough doses, was able to kill normal female mice (Andrew & Coupar, 1992
). Coexpression of IL-2 with IL-4 improved the survival rates of the mice. However, IL-2 expressed from the HindIII F insertion site was more effective in overcoming the effects of IL-4 than when the same gene, regulated by the same promoter, was expressed from the tk insertion site with IL-4 in the HindIII F region (Andrew & Coupar, 1992
; unpublished observations). These effects were observed in vivo despite no detectable difference in the level of either cytokine expressed from each of the constructs in infected cell cultures.
In order to investigate potential insertion site effects more thoroughly and with a more readily quantifiable product, we have constructed a number of vaccinia virus recombinants where the firefly luciferase gene has been inserted into the tk gene or into the HindIII F region of the viral genome. In each case regulation of luciferase expression was under the control of the vaccinia 7·5 kDa promoter. Here we report the comparative levels of luciferase activity from different recombinant virus infections in cell culture and in a range of mouse tissues after intravenous inoculation.
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Methods |
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Plasmids and construction of recombinant vaccinia viruses.
Firefly luciferase coding sequence was subcloned from pFC05 (Cameron & Jennings, 1989 ) into pBCB06 (Boyle et al., 1985
) and pBCB07 (Andrew et al., 1987
) for insertion into the vaccinia virus tk, and into pTK7.5A and pTK7.5B (Coupar et al., 1988
) for HindIII F insertions. In each case the luciferase sequence was 3' to the vaccinia virus 7·5 kDa promoter. Thymidine kinase negative vaccinia virus recombinants (VV-104 and VV-105) were constructed using VV-WR-L929 as described previously (Boyle et al., 1985
) with insertion of the promoter-gene cassette between residues 92 and 93 of the 177 amino acid tk sequence and in both orientations relative to the tk sequence. Recombinants with the luciferase gene inserted into the HindIII F region (VV-128 to VV-131) were constructed using VV-PR8-HA6 (VV-128 and VV-129) or VV-PR8-NP6 (VV-130 and VV-131) (Andrew et al., 1986
) as parental virus. Control recombinants (VV-163 and VV-164) resulted from the insertion of the HSV tk sequence alone into the HindIII F region of VV-104 or VV-105 using pFB-TK (Coupar et al., 1988
). The F7L ORF was disrupted by insertion of a 1·8 kb fragment containing the HSV tk coding sequence, followed by the promoter-gene cassette in either orientation relative to F7L and HSV tk, after the 6th residue of the F7L putative product.
Recombinant viruses expressing murine cytokines IL-2 and IL-4 and a control virus VV-HA-TK have been described previously (Ramshaw et al., 1987 ; Andrew & Coupar, 1992
).
Mice.
CBA/H mice were inoculated intravenously (i.v.) with 107 p.f.u. of virus and after 3 days organs were removed for luciferase assay and for vaccinia virus titrations.
Luciferase assays.
Infection of 143B cells with vaccinia virus recombinants was carried out in 96-well plates at a range of m.o.i.s and for various times up to 9 h. Samples from quadruplicate wells were assayed for luciferase activity using a Promega luciferase assay kit. Assays were carried out using the luciferase assay reagent at a dilution of 1:20 and 1 min reaction time in a Berthold luminometer. A standard curve was prepared using dilutions of luciferase (Sigma) before each batch of samples was tested. Luciferase assays on mouse tissue samples were carried out using a 1:10 dilution of assay reagent for ovary samples and undiluted reagent for all other tissues. Duplicate assays were performed on each tissue sample. Comparisons of levels of luciferase activity were confined to data generated on the same day with the same batch of reagents and conditions. Luciferase activity is expressed in relative light units (RLU).
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Results |
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Attempts to quantify the levels of IL-2 and IL-4 expressed in infected cell culture supernatants from these recombinants showed no significant differences between any of the constructs for the expression of either cytokine (data not shown). Levels of virus replication as measured by one-step growth curves were not significantly different (data not shown).
Construction and characterization of vaccinia virus recombinants expressing luciferase
Vaccinia virus recombinants with the coding sequence for firefly luciferase inserted downstream from the vaccinia virus 7·5 kDa promoter, in either of two sites in the viral genome, are shown schematically in Fig. 1. Insertion into the tk gene provided recombinants with the luciferase coding sequence in either orientation relative to the tk gene (VV-104 and VV-105). In each case, four separate recombination reactions were carried out and recombinant viruses were plaque purified from each (AD in each case). Recombinant viruses expressing luciferase from insertions in the HindIII F fragment were constructed using two tk- viruses expressing influenza virus HA (VV-128 and VV-129) or NP (VV-130 and VV-131) antigens. Once again both orientations of the insertion were used and recombinants were constructed in duplicate (A and B). Control viruses (VV-163AD and VV-164AD) with the HSV tk sequence inserted into the HindIII F region of VV-104AD and VV-105AD were constructed to provide the same tk phenotype as VV-128 to VV-131. The genome arrangement and homogeneity of each of the recombinants were examined by Southern blotting and probing with a number of radioactively labelled fragments of DNA, including the luciferase coding region and the wild-type vaccinia virus fragments covering each of the two insertion sites. All constructs were shown to have the expected patterns of hybridization and wild-type or parental vaccinia virus was not detected (data not shown).
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Expression of luciferase activity in recombinant vaccinia virus-infected cells
In an initial series of experiments the levels of luciferase activity in lysates of 143B cells infected with vaccinia virus recombinants were measured at a range of m.o.i. values (0·510 p.f.u. per cell) and times post-infection (p.i.) up to 9 h. Further experiments were carried out using quadruplicate wells infected at 5 and 1 p.f.u. per cell for 78 h. Fig. 2(a) shows no significant difference in the level of activity produced by the four recombinants VV-104AD, where the luciferase coding sequence is inserted into the tk gene of vaccinia virus. However, VV-105B consistently showed very low levels of luciferase activity when compared with the other three recombinants constructed in parallel (VV-105A, C and D). Southern blotting and probing of the DNA from VV-105B did not reveal any obvious reason for this low level of luciferase expression and further investigation was not carried out. Data derived using VV-105B were excluded from the calculation of means for any particular group of recombinants. Orientation of the inserted DNA relative to tk did not appear to affect the level of expression of luciferase (VV-104 versus VV-105).
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In order to provide recombinants with the same tk phenotype and equivalent manipulations of the viral genome, the eight recombinants with luciferase insertions in the tk locus (VV-104AD and VV-105AD) were used for insertion of the selectable marker HSV tk into the HindIII F region. When the resultant recombinants, VV-163AD and VV-164AD, were compared with their parental viruses, surprisingly, the results showed a significant drop in the level of luciferase expressed in the double recombinants. Results from a representative experiment are shown in Fig. 3. Apart from VV-164B, which was constructed using VV-105B (already shown to express abnormally low levels of luciferase), each recombinant with an insertion into and interruption of F7L and expressing HSV tk expressed significantly lower levels of luciferase than its tk negative parent. The reduction in activity ranged from 43% for VV-104D/VV-163D to 76% for VV-105D/VV-164D.
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Discussion |
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Using luciferase as a quantifiable reporter gene we have demonstrated that in infected cells the level of expression from the HindIII F insertion site is higher than from the tk insertion site, provided that the tk phenotype is the same (Figs 4 and 5
). In tk insertion site constructs the orientation of the inserted sequence relative to the tk gene and its promoter does not appear to have a significant influence on the level of luciferase expressed. Although in VV-105, where the possibility of readthrough transcription from the tk promoter exists, the levels are marginally higher (Fig. 2a
) the increased levels of expression observed by Davison & Moss (1989)
when the promoter-gene insert was in the same, rather than the opposite, orientation compared with tk were not observed. Recombinant viruses with luciferase expression from the HindIII F insertion site did show higher levels of expression when the promoter-luciferase gene cassette was in the same orientation as F7L (Fig. 2b
); however, the F7L promoter has been shown to be relatively weak (Coupar et al., 1987
) and is located more than 2 kb upstream of the luciferase coding sequence.
When vaccinia virus recombinants which differed only in the insertion site for the luciferase coding sequence were compared in mice, once again there was a significantly higher level of expression from the HindIII F site in both ovaries and lungs (Fig. 6). In other organs tested the levels of luciferase detected were relatively low and no significant differences were seen between the constructs.
An unexpected result was the decreased level of luciferase expressed from the tk insertion site when a second insertion was made into the HindIII F site (Fig. 3). In eight pairs of recombinants where the only modification was the insertion of HSV tk into the HindIII F site, with consequent interruption of the F7L ORF, the level of luciferase from a coding sequence inserted into the vaccinia virus tk gene was reduced, despite the two sites being more than 45 kb apart (Fig. 3
). This suggests an effect on expression from the tk locus either due to the expression of HSV tk or to the lack of expression of the F7L gene product. At present we have no data to support or exclude either hypothesis, nor has a function for the F7L product been demonstrated. No significant differences in the levels of virus replication in cell culture for any of the recombinants or wild-type virus were detected in one-step growth curves. In vivo levels of virus replication vary with tk phenotype, with the HSV tk+ constructs having a level of virulence intermediate between the wild-type vaccinia tk and the attenuated tk- viruses (Andrew et al., 1989
), thus emphasizing the importance of comparisons between recombinants with the same tk phenotype.
A number of studies have included data for expression of multiple products from a single recombinant vaccinia virus (Perkus et al., 1985 ; Coupar et al., 1988
; Morrison et al., 1990
; Wild et al., 1992
) but with no direct comparison of effects of insertion site on the expression of a particular product. However, in cases where the same gene has been expressed from different loci within the vaccinia virus genome (e.g. Kunke et al., 1993
; Flexner et al., 1987
) no significant effect of insertion site was demonstrated. Flexner et al. (1987)
inserted the coding sequence for human IL-2 at three separate sites (HindIII C, tk and vaccinia virus HA) within the viral genome and found that the site of insertion was not critical to the attenuating effects observed in mice. Levels of expression of IL-2 produced by these recombinants were not compared. Kunke et al. (1993)
constructed vaccinia virus recombinants coexpressing hepatitis B virus surface (HBsAg) and core (HBcAg) antigens in two sites, interrupting the tk gene and the K1L host-range gene. Reversal of the locations of HBsAg and HBcAg in a second construct did not significantly affect the level of expression of either antigen as determined by ELISA or immunoblot analysis (Kunke et al., 1993
). On the other hand, Bembridge et al. (1998)
did find differences in the levels of expression of respiratory syncytial virus (RSV) F protein and murine cytokines IL-2, IL-4 or IFN-
which were coexpressed from the tk locus or the VP37 locus. Levels of expression assessed by ELISA were lower from the tk insertion site than from the VP37 site in each case (Bembridge et al., 1998
). However, as different promoters were associated with each locus (expression from the tk site being regulated by the 7·5 kDa promoter and that from the VP37 by a synthetic early/late promoter), direct comparison of the insertion sites is difficult. More recently, Bennett et al. (1999)
have shown differences in the levels of expression of the Clostridium perfringens
-toxin expressed from the tk or the B13R (serpin-2 gene) loci. In each case expression was regulated by the 7·5 kDa promoter and in the tk locus, where higher levels of expression were detected, the insert was in the same orientation as the tk gene, with the potential for enhanced transcription.
The data presented here suggest than in the construction of vaccinia virus recombinants, particularly for the coexpression of antigens or immunomodulators as vaccines, the selection of sites for insertion of the foreign genes may be critical to the outcome in vivo.
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Acknowledgments |
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References |
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Received 27 July 1999;
accepted 3 November 1999.