1 GSF Institute of Molecular Virology and Institute of Virology, Technical University of Munich, Trogerstrasse 4b, 81675 München, Germany
2 Department of Virology, Paul-Ehrlich-Institute, Paul-Ehrlich-Strasse 51-59, 63225 Langen, Germany
Correspondence
Gerd Sutter
sutge{at}pei.de
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ABSTRACT |
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INTRODUCTION |
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MVA was obtained by long-term serial passage in chicken embryo fibroblast (CEF) tissue cultures, which resulted in much loss of genomic information, including many genes regulating virushost interactions (Meyer et al., 1991; Antoine et al., 1998
). The MVA homologues of genes encoding recognized poxvirus immune-evasion molecules (reviewed by Moss & Shisler, 2001
; Alcami, 2003
) including the viral IFN type I and type II receptors, the interleukin 1
(IL1
) converting enzyme inhibitor SPI-2, the vaccinia complement-binding protein, the vaccinia semaphorin, the 35 kDa chemokine-binding protein and the tumour necrosis factor (TNF)-
receptor are deleted or fragmented. Interestingly, some viral genes with immunomodulatory function are maintained in the MVA genome and their possible relevance for the use of MVA-based vaccines remains to be determined. One such example is the coding sequence for the viral IL1
receptor (IL1
R), which is highly conserved in the MVA genome (Antoine et al., 1998
), and expression of IL1
R in MVA-infected cells has been described (Blanchard et al., 1998
). IL1 is a cytokine that plays an important role in the regulation of inflammatory processes and the host innate immune response against infectious agents (reviewed by Sims, 2002
). In contrast to its cellular counterpart, soluble viral IL1
R has a specific affinity only for IL1
(Alcami & Smith, 1992
), the major endogenous pyrogen (Alcami & Smith, 1996
). During vaccinia virus infection of mice, IL1
R was shown to prevent fever by interaction with IL1
. Furthermore, deletion of the IL1
R gene in vaccinia virus accelerated the appearance of symptoms of illness and mortality in intranasally infected mice, suggesting that the blockade of IL1
by vaccinia virus can diminish the systemic acute-phase response to infection and modulate the severity of the disease (Alcami & Smith, 1996
). In contrast, virulence was reduced following intracranial infection of mice with IL1
R-deficient vaccinia virus, possibly reflecting a beneficial role of IL1
on brain-tissue infection (Spriggs et al., 1992
). Therefore, the viral IL1
R gene represents an interesting target for molecular engineering in the search for optimized vaccinia viruses as candidate second-generation vaccines. Here, we evaluated the effects of deletion of the IL1
R gene from the MVA genome. The construction of an MVA IL1
R deletion mutant allowed us to analyse the significance of IL1
R synthesis following in vitro and in vivo infection with MVA. Our data suggested that inactivation of the IL1
R gene may be beneficial for the development of future MVA vaccines.
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METHODS |
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Plasmids.
The transfer plasmid pK1L-184R carries two DNA fragments that represent flanking sequences of MVA ORF 184R (nt 162021163001, GenBank accession no. U94848) that have been inserted into multiple cloning sites 1 and 2 of plasmid p
K1L (Staib et al., 2000
). One fragment, designated flank 184R-I, consists of a 486 bp MVA DNA sequence starting in the 5'-intergenic region of ORF 184R and ending at the start codon for translation of ORF 184R; the other fragment, flank 184R-II, is a 544 bp PCR fragment of MVA DNA extending from the codon for 184R translation termination into the 3'-intergenic region of the 184R gene.
Genetic modification of MVA.
Mutant MVAs were obtained following the transient K1L-based host-range selection protocol as described previously (Staib et al., 2000; Staib & Sutter, 2003
). Briefly, for the generation of deletion mutant viruses, monolayers of 1x106 confluent CEFs were infected with MVA at an m.o.i. of 0·01 infectious units (IU) per cell. At 90 min post-infection (p.i.), cells were transfected with 1·5 µg plasmid p
K1L-184R DNA using FuGENE (Roche) as recommended by the manufacturer. At 48 h p.i., transfected cells were harvested and plated on RK-13 cell monolayers for growth selection. Mutant viruses were isolated by plaque cloning on RK-13 cells and then passaged on CEF cells to remove the selectable marker gene K1L. For control purposes, we generated revertant virus MVA-IL1
Rev using a standard methodology (Staib & Sutter, 2003
), reinserting a DNA fragment comprising the complete IL1
R gene sequence under the transcriptional control of its authentic promoter at the site of deletion VI (depicted in Fig. 1
) into the genome of MVA-
IL1
R.
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Alternatively, total DNA isolated from virus-infected cells was digested with EcoRI, separated by gel electrophoresis in 0·8 % agarose, transferred to a Hybond-N membrane (Amersham) and hybridized to a DNA probe consisting of a PCR fragment from flank 184R-I sequences labelled with [-32P]dCTP. Prehybridization and hybridization were performed according to Sambrook et al. (1989)
. Blots were exposed to a Kodak BioMax film.
Radioimmunoprecipitation of virus-infected cell lysates.
CEF cells grown in six-well tissue culture plates were infected with MVA at an m.o.i. of 20 IU per cell. At 6 h p.i., the virus inoculum was replaced with methionine-free MEM containing 5 % FBS and 50 µCi (1·85 MBq) [35S]methionine ml1 and incubated for a further 2 h at 37 °C. Cells were lysed in RIPA buffer containing 0·15 M NaCl, 0·01 M Tris/HCl pH 7·4, 1 % NP-40, 0·1 % SDS, 1 % sodium deoxycholate, 1 mM PMSF and incubated for 16 h at 4 °C with 50 % protein ASepharose coupled to rabbit polyclonal anti-B15R antibody (Alcami & Smith, 1992). Immune complexes were washed in RIPA buffer, dissolved in Laemmli buffer and proteins were separated by 10 % SDS-PAGE.
Analysis of virus growth.
To determine low- or high-multiplicity growth profiles, confluent CEF monolayers (grown on six-well plates) were infected with 0·05 or 10 IU MVA or mutant MVA per cell, respectively. After virus adsorption for 60 min at 37 °C, the inoculum was removed. Cells were washed twice with RPMI 1640 and incubated with fresh RPMI 1640 containing 10 % FBS at 37 °C and 5 % CO2. At multiple time points p.i., infected cells were harvested and virus was released by freezethaw and brief sonication. Virus titres were determined following standard procedures, as described previously (Hornemann et al., 2003).
Cellular vaccinia virus responses.
For ex vivo monitoring of peptide-specific acute- and memory-phase CD8+ T-cell responses, splenocytes from vaccinia virus-immunized HHD mice were prepared and incubated for 5 h with HLA-A*0201-binding peptide (VP35#1; Drexler et al., 2003) at 106 M. After 2 h, brefeldin A was added at a final concentration of 1 µg ml1 (GolgiPlug; BD Biosciences Pharmingen). Splenocytes were live/dead stained with PBS containing 1 % BSA and 1 µg ethidium monoazide bromide (EMA; Molecular Probes) ml1 and blocked for non-specific Fc
III and -II receptor-mediated binding with 5 µg purified anti-CD16/CD32 (Fc Block; BD Biosciences Pharmingen) ml1 for 20 min at 4 °C. Cell-surface staining was performed with phycoerythrin (PE)-conjugated anti-CD8 (clone 53-6.7) and allophycocyanin (APC)-conjugated anti-CD62L (clone Mel-14) for 30 min at 4 °C. After permeabilization of cells (Cytofix/Cytoperm kit; BD Biosciences Pharmingen), intracellular cytokine staining was performed for 30 min at 4 °C using FITC-conjugated anti-IFN-
(clone XMG1.2) or FITC-conjugated anti-TNF-
(clone MP6-XT22) or the respective FITC-labelled IgG1 isotype control (clone R3-34) (all from BD Biosciences Pharmingen). Splenocytes were analysed by four-colour flow cytometry (FACSCalibur) using CellQest software (both from Becton Dickinson). For detection of ex vivo vaccinia virus-specific total CD8+ T-cell responses, a fraction of the splenocytes, isolated from HHD or C57BL/6 mice 6 months after vaccination, was infected for 7 h with MVA prior to the addition of brefeldin A. After a further incubation for 16 h, cells were stained and analysed as described above. Statistical analysis (f- or t-test) was performed using GraphPad Prism 4 software. Vaccination experiments were performed at least three times.
Animal models.
Female 68-week-old transgenic HHD+/+ 2m/ Db/ mice (HHD) (Pascolo et al., 1997
) or female 68-week-old BALB/c or C57BL/6 mice were used for vaccination experiments. HHD mice were inoculated with 0·25 ml virus vaccine by the intraperitoneal route and monitored for HLA-A*0201-restricted T-cell responses at days 10 and 180 post-immunization. For protection assays, animals were vaccinated once with 0·030·25 ml virus vaccine given by the intranasal or intraperitoneal route. At 36 months post-immunization, animals were anaesthetized, infected intranasally with vaccinia virus Western Reserve (107 p.f.u. diluted in 30 µl PBS) and monitored for at least a further 3 weeks for morbidity and mortality, with daily measurement of individual body weights and scoring of signs of illness as described previously (Alcami & Smith, 1996
). Animals suffering from severe systemic infection and having lost >30 % body weight were sacrificed. The mean change in body weight was calculated as the percentage of the mean weight for each group on the day of challenge. Body temperature was determined with an Electronic Laboratory Animal Monitoring System (BioMedic Data Systems) using subcutaneously implanted microchip battery-free transponders and a DAS-5004 Pocket Scanner for data collection. Mean changes in body temperature were calculated by subtracting the pre-challenge (days 3 to 0) baseline temperature of each group from each subsequent time point. Experiments in animal models were performed at least twice.
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RESULTS |
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Molecular characterization and unimpaired in vitro replication of mutant virus MVA-IL1
R
After isolation of the MVA deletion mutants, we first wished to confirm the correct removal of IL1R coding sequences at the genetic level. We analysed viral DNA extracted from CEFs infected with wild-type, mutant or IL1
-revertant MVA (MVA-IL1
-Rev) by PCR using oligonucleotide primers specific for MVA genomic sequences adjacent to the IL1
R gene locus or specific for the site of deletion VI within the MVA genome, the insertion site of the reintroduced IL1
R gene in MVA-IL1
-Rev (Fig. 2
a). The IL1
R gene-specific PCR specifically amplified a 2·1 kb DNA fragment from wild-type templates, whereas the use of DNA from MVA-
IL1
R and revertant MVA-infected cells generated 1·1 kb PCR products corresponding to the expected reduction in size after deletion of ORF 184R. PCR analysis specific for MVA sequences at the site of deletion VI revealed the expected 306 bp DNA fragments for MVA-
IL1
R and MVA DNA, whereas the use of genomic DNA from revertant virus resulted in amplification of a 1·6 kb DNA fragment, suggesting the correct insertion of the complete IL1
R gene expression cassette. Furthermore, we digested viral DNAs with restriction endonuclease EcoRI and revealed DNA fragments containing the IL1
R gene locus by Southern blot analysis. Confirming the PCR data, we detected a 6500 bp EcoRI fragment in the genomic DNA of deletion mutant MVA-
IL1
R compared with a 7800 bp MVA DNA fragment (Fig. 2b
), corresponding to the expected loss of 1·3 kb and again verifying correct deletion of the targeted ORF 184R sequences.
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We also wanted to assess the replicative capacity of mutant MVA-IL1
R in comparison with wild-type MVA. After infection of CEFs, we found comparable amounts of new viral progeny being formed with almost identical kinetics during one-step (Fig. 2d
) and multiple-step (Fig. 2e
) virus growth. These data clearly suggested that inactivation of MVA ORF 184R did not affect the in vitro multiplication of the virus.
Avirulence of MVA-IL1
R following high-dose respiratory infection of mice
An important question was whether the inability to produce the viral IL1R protein would influence the outcome of MVA infection in vivo. Previous work in mice with vaccinia virus Western Reserve deletion mutants revealed either enhancement of respiratory disease after intranasal infection (Alcami & Smith, 1992
) or reduced virulence after intracranial infection (Spriggs et al., 1992
). The more severe respiratory infection appeared to be linked to induction of fever response and the functional activity of the viral IL1
R neutralizing IL1
as the major endogenous pyrogen (Alcami & Smith, 1996
). Therefore, we tested mutant virus MVA-
IL1
R following intranasal infection of mice. Severity of disease in this mouse model is well reflected by changes in body weight and the appearance of characteristic signs of illness (Williamson et al., 1990
; Alcami & Smith, 1996
; Drexler et al., 2003
; Reading et al., 2003
; Reading & Smith, 2003b
). Additionally, we wished to monitor changes in body temperature because of the possible onset of febrile reactions. We transplanted BALB/c mice with subcutaneous microchip transponders to allow computable readings, and 1 week later infected the animals with 108 IU MVA or MVA-
IL1
R, or with 5x105 p.f.u replication-competent vaccinia virus CVA or 3x104 p.f.u Western Reserve as a control. Animals were monitored daily over a period of 3 weeks (Fig. 3
). Infection of mice with MVA or mutant MVA-
IL1
R did not result in any obvious disease. In contrast, infection with the replication-competent viruses CVA and WR caused a drastic loss of body weight (Fig. 3a
) and severe signs of illness, also reflected by a reduced body temperature (Fig. 3b
). In MVA-infected animals, body temperature remained stable over the observation period. Taken together, these data suggested preservation of the attenuated phenotype of MVA after deletion of the IL1
R gene from its genome.
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To investigate whether inactivation of viral IL1R influenced the formation of memory T-cell responses, we vaccinated groups of HHD mice once with 108 IU MVA-
IL1
R, MVA or MVA-IL1
-Rev and monitored the presence of virus-specific T cells for more than 6 months after this primary immunization. We detected clearly higher levels of approximately 1·7 % VP35#1-reactive and IFN-
-releasing splenic CD8+ memory T cells in MVA-
IL1
R-immunized animals compared with vaccination with non-recombinant MVA or MVA-IL1
-Rev, which resulted in approximately 0·4 and 0·5 % epitope-specific IFN-
-secreting CD8+ T cells, respectively (Fig. 5
a). This difference in favour of MVA-
IL1
R vaccination was statistically significant (P<0·005) and, interestingly, splenocytes from vaccinees of this group also contained significantly higher amounts of total vaccinia-specific CD8+ memory T cells (P<0·05; Fig. 5a
).
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The HHD mouse model allows convenient analysis of epitope-specific HLA-A*0201-restricted CD8+ T-cell responses; however, possibly because of their knockout phenotype for mouse MHC class I, these mice develop unusually low numbers of total CD8+ T cells (Pascolo et al., 1997). As this phenotype might influence the analysis of total vaccinia-specific T-cell responses, we also assessed the number of total CD8+ memory T cells induced by MVA-
IL1
R or MVA after vaccination of normal C57BL/6 mice (Fig. 6
a). Again, in comparison with conventional MVA vaccination, we found significantly (P=0·003) higher numbers of vaccinia-specific CD8+ T cells in animals immunized with MVA-
IL1
R. These data strongly suggested an improved capacity of MVA-
IL1
R to elicit or maintain vaccinia virus-specific CD8+ T-cell memory.
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DISCUSSION |
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Despite using molecular engineering techniques allowing precise mutagenesis (Staib et al., 2000, 2003
), the resulting phenotypes of mutant viruses are unpredictable, and inactivation of the MVA IL1
R gene may serve as further example. Our finding that inactivation of the 184R ORF had no impact on the in vitro replicative capacity of the MVA mutant virus was not surprising, since growth deficiencies have not been reported with corresponding mutants derived from vaccinia virus Western Reserve (Alcami & Smith, 1992
). Yet the capacity for high-level amplification is of the utmost importance for a virus to be used in vaccine production. Additionally, it should be noted that, with another MVA mutant defective in expression of the viral IFN response gene E3L, we recently found a very unexpected host range phenotype in CEFs, the preferred cell type for MVA vaccine production (Hornemann et al., 2003
).
In further experiments, we characterized the in vivo properties of MVA-IL1
R. In vivo, the removal of an immunomodulator such as the viral IL1
R could have different consequences. On the one hand, it was possible that an unhampered IL1
activity elicited by infection with MVA-
IL1
R would have triggered strong inflammation reactions and febrile responses that resulted in adverse effects of vaccination. On the other hand, we speculated that IL1
action could be locally restricted and might influence the potency of MVA immunization in an adjuvant-like manner. Following intranasal infection of BALB/c mice with MVA-
IL1
R, we did not detect signs of respiratory illness, despite using high-dose inoculations and the fact that this mouse model system appears to be particularly suitable to assess potential pathogenic consequences of inflammatory responses to viral infection (Alcami & Smith, 1996
; Reading et al., 2003
; Reading & Smith, 2003a
). These findings confirmed that the avirulent phenotype of MVA had been conserved, even after the removal of IL1
R as a vaccinia viral factor having the potential to attenuate the pathogenicity of infection. Maintenance of attenuation is of utmost importance, especially with regard to MVA vaccine development, and may be a consequence of the particular MVA genotype, with other vaccinia virus regulatory or immunomodulatory genes being fragmented or deleted (Antoine et al., 1998
). Alternatively, it could be that the disease enhancement observed with vaccinia virus IL1
R deletion mutants requires active in vivo replication of the virus after intranasal infection. As there is good recent evidence confirming that MVA is unable to replicate in vivo in mice (Ramirez et al., 2000
) or macaques (Stittelaar et al., 2001
), our data might suggest that transient one-step infection with MVA-
IL1
R is simply not sufficient to result in IL1
activities inducing adverse systemic fever or inflammation reactions.
In first-vaccination experiments, we found a slight benefit of MVA-IL1
R immunization when monitoring for acute vaccinia virus epitope-specific T-cell responses. The H3L gene product-derived epitope VP35#1 is the target of an immunodominant HLA-A*0201-restricted T-cell specificity and can be used to examine the induction of virus-specific CD8+ T cells in an epitope-specific manner (Di Nicola et al., 2003
; Drexler et al., 2003
). In contrast, bulk analysis of total vaccinia virus-specific responses can provide a representative picture based on a multitude of different T-cell specificities, but might not allow us to assess subtle changes in the activation of single T-cell populations. Suspecting a possible effect on T-cell activation, we opted also to assess VP35#1-specific T-cell memory responses, which we had previously found detectable for more than 6 months after vaccination of HLA-A*0201-transgenic mice (Drexler et al., 2003
). Indeed, we observed a significant enhancement of VP35#1-specific T-cell responses after vaccination with MVA-
IL1
R (P<0·005). We noticed for the first time a significantly increased total CD8+ T-cell response in transgenic HHD mice (P<0·05) and, more importantly, we further corroborated this result following vaccination of non-transgenic mice (P=0·003) harbouring a bona fide pool of CD8+ T cells and allowing for higher variety of MHC class I-presented peptide epitopes in comparison with HHD mice. These additional findings appeared to confirm the long-term beneficial effect of MVA-
IL1
R immunization. In addition, we found higher protective capacities against lethal respiratory challenge with vaccinia virus Western Reserve in both transgenic HHD and normal mice.
How can the enhanced vaccine efficacy in the context of IL1 function be explained? Interestingly, results from two recent studies investigating Leishmania major infection of susceptible and resistant mice suggested that the ability of dendritic cells (DCs) to secrete IL1
or IL1
is specifically associated with the induction of protective Th1 immunity (Filippi et al., 2003
; Von Stebut et al., 2003
). In addition, there is recent evidence for IL1
being an essential mediator of Fas-ligation-induced maturation of murine DCs (Guo et al., 2003
). The same work demonstrated that maturation of murine DCs could be completely abrogated by the use of IL1
-neutralizing antibodies, which may function in a similar manner as would be expected with the soluble vaccinia virus IL1
R molecule. Therefore, the lack of IL1
neutralization following vaccination with MVA-
IL1
R may lead to improved functionality of DCs to serve as antigen-presenting cells, which might result in better T-cell memory responses.
Similarly, it has been shown that stimulation of endothelial cells with IL1 resulted in human inducible co-stimulator-ligand-mediated activation of memory T cells (Khayyamian et al., 2002
). Such activity of IL1
might be the functional basis for our finding that vaccination with MVA-
IL1
R appeared predominantly to improve memory T-cell responses, suggesting that the viral IL1
R could have a specific role in abrogating anti-viral memory T-cell responses. This appears alluring in view of recent data either demonstrating an increased persistence of CD4+ compared with CD8+ T cells upon analysis of vaccinia virus-specific memory T cells in humans (Amara et al., 2004
) or suggesting that recombinant poxvirus vaccines may potently amplify peak but not necessarily memory T-cell responses following vaccination experiments in the human simian immunodeficiency virus/rhesus macaque model (Santra et al., 2004
).
In summary, our analysis recommends deletion of the viral IL1R gene as a first step towards the development of a new generation of MVA-based vaccines. High virus titres were obtained following in vitro propagation of the deletion mutant MVA-
IL1
R, and the avirulent phenotype of MVA was well conserved in MVA-
IL1
R, even after high-dose in vivo infection. Our finding of improved vaccine properties of MVA-
IL1
R is particularly promising, since it demonstrates for the first time the possibility of obtaining more efficacious MVA vaccines through rational genetic engineering.
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ACKNOWLEDGEMENTS |
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Received 28 September 2004;
accepted 12 April 2005.