| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Short Communication |
1 Department of Microbiology and Virology, Faculty of Medicine, University of Tromsø, N-9037 Tromsø, Norway
2 Department of Medical Genetics, University Hospital of North Norway, N-9038 Tromsø, Norway
3 GENOK-Norwegian Institute of Gene Ecology, Tromsø Science Park, N-9294 Tromsø, Norway
Correspondence
Terje Traavik
terjet{at}genok.org
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
Supplementary figures are available in JGV Online.
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
). Genes that encode host-range and immune-evasion factors are either deleted or fragmented in MVA (Antoine et al., 1998; Blanchard et al., 1998). MVA multiplies efficiently in CEF and baby hamster kidney (BHK-21) cells. In other mammalian cell lines incomplete morphogenesis occurs (Caroll & Moss, 1997; Drexler et al., 1998). Recombinant and non-recombinant MVA have been reported apathogenic in several in vivo models even when administered in high doses to immune deficient animals (Stittelaar et al., 2001; Ramirez et al., 2003; Hanke et al., 2005). A recent report indicated, however, that immunization of ferrets with MVA-vectored vaccine against severe acute respiratory syndrome (SARS) is associated with enhanced hepatitis (Weingart et al., 2004). Overall, the extreme attenuation and un-impaired gene expression in non-permissive cells recommend MVA as a promising vaccine vector. Presently, several recombinant MVA constructs are being evaluated for use as vaccine candidates against infectious diseases and cancers (Sutter et al., 1994; Corona Gutierrez et al., 2002; Drexler et al., 2004; Wyatt et al., 2004; Smith et al., 2005). In the future, it is likely that MVA-vectored vaccines will be licensed for treatment of human and animal diseases. Thus, continued evaluation of MVA morphogenesis and multiplication in mammalian cells is essential to the production of safe MVA-vectored vaccines. Assembly of MVA in the only known permissive cell lines (CEF and BHK-21) is similar to the assembly of other VACV strains (Sancho et al., 2002; Gallego-Gomez et al., 2003). VACV assembly results in the formation of four forms (Sodeik & Krinjse-Locker, 2002; Smith & Law, 2004). Details of the processes leading to these morphological forms have been reported in a number of articles (e.g. Tooze et al., 1993; Schmelz et al., 1994; Hollinshead et al., 2001; Risco et al., 2002; Meiser et al., 2003b; Carter et al., 2005).
MVA assembly in mammalian cells, except BHK-21, seems to be blocked at the immature virus stages (Caroll & Moss, 1997). However, this fact is based on the limited number of mammalian cell lines studied so far. MVA-based vectors and vaccines are currently being produced in CEF and BHK-21 cell lines. The establishment and maintenance of CEF cultures require experience in preparing primary tissue cultures. CEF cultures survive few passages and weekly de novo preparations are required (Drexler et al., 1998). During serial passages, BHK-21 cultures rapidly deteriorate on reaching confluence, and will hence be inadequate for large-scale production purposes, especially in batch systems that require high-density viable cells. Our aim was to identify alternative cell lines that support efficient MVA multiplication. Thus, we investigated the multiplication and morphogenesis of recombinant and non-recombinant MVA in 13 mammalian cell lines.
The cell lines (Table 1) used in this study were purchased from, and grown under conditions suggested by, ATCC. The recombinant MVA (MVA-HANP) was kindly provided by Dr Bernard Moss, National Institute of Health, USA. The MVA-HANP genome contains the influenza virus (A/PR/8/34) haemagglutinin (HA) and nucleoprotein (NP) cDNA inserts (Sutter et al., 1994). MVAnr (non-recombinant MVA; ATCC VR-1508) was purchased from ATCC. Anti-influenza virus HA mouse monoclonal antibody, H28E23, was also a gift from Dr Bernard Moss. Both MVA-HANP and MVAnr-infected foci were visualized by immunostaining as described previously (Hansen et al., 2004; Hornemann et al., 2003, respectively).
|
. Rat IEC-6 cells supported efficient multiplication of MVA-HANP as well as MVAnr. IEC-6 and BHK-21 cells were permissive to MVA-HANP infection with a fold increase in virus titre of 370 (2·56 log) and 477 (2·67 log), respectively (Table 1
). MVAnr multiplied more efficiently than MVA-HANP in both cell lines. The multiplication of MVAnr in IEC-6 and BHK-21 cells was 2218 (3·34 log) and 6068 (3·78 log), respectively (Table 1). Vero, NMULI and A549 cells were semi-permissive to MVAnr, whereas only Vero cells was semi-permissive to MVA-HANP (Table 1). Other African green monkey kidney cell lines (CV-1 and BS-C-1) have been reported previously to be semi-permissive to MVA (Caroll & Moss, 1997). Host-range restriction could result from inhibition of infectious virus formation or spread (Caroll & Moss, 1997). To detect cell spread, cells were infected with MVA-HANP and MVAnr at an m.o.i. of 0·01. At 24, 48 and 72 h p.i., the cells were immunostained. The number of cells per infected focus at different time points post-infection was used to quantify cell spread. IEC-6 cells supported efficient cell-to-cell spread of MVA-HANP (Fig. 1a) and MVAnr (Fig. 1b). Similar results were obtained with infected BHK-21 cells (data not shown). MVAnr had enhanced cell spread and CPE compared with recombinant MVA in IEC-6 cells (Fig. 1a and b) and BHK-21 cells (data not shown). Limited cell spread was present in NMULI and 293 cells infected with MVA-HANP. Other non-permissive cell lines formed exclusively single-cell immunostained foci; an observation indicating lack of cell spread (data not shown). Vero cells infected with MVAnr, but not MVA-HANP, showed significant cell spread although not as pronounced as in IEC-6 or BHK-21 cells (data not shown). The deletion or disruption of host-range genes may explain the inability of MVAnr and MVA-HANP to multiply and spread in most non-permissive cell lines. Except for MVA O18L (an orthologue of VACV-Copenhagen), orthopoxvirus host-range genes (Gillard et al., 1985; Perkus et al., 1990; Ali et al., 1994; Beattie et al., 1996) are either deleted or disrupted in MVA strains (Antoine et al., 1998).
|
–f). At both m.o.i. values, there were fast kinetics of MVA-HANP in both IEC-6 and BHK-21 cells at early time points post-infection. The initial faster replication kinetics of MVAnr have also been reported by other investigators (Caroll & Moss, 1997; Meiser et al., 2003a), and this phenomenon may be due to faster entry kinetics (Sancho et al., 2002). At high m.o.i., medium virus titre of MVA-HANP in BHK-21 but not IEC-6 cells was similar to the cell virus titre (Fig. 1e and f). A reasonable explanation is that a high amount of intracellular virus was liberated into the medium following cell lysis, since extensive CPE occurred in BHK-21 cells infected with MVA-HANP at high m.o.i. Consistent with the results in Table 1, MVAnr multiplied to higher levels at low m.o.i. in both IEC-6 and BHK-21 cells rather than MVA-HANP (Fig. 1c and d). The difference was more pronounced in BHK-21 cells (Fig. 1d). At high m.o.i., the kinetics of cell-associated and medium virus of MVAnr are virtually identical to those of MVA-HANP in IEC-6 cells (Fig. 1e). However, in BHK-21 cells, the cell virus titre of MVAnr is approximately 1·0 log higher than that of MVA-HANP across all time points post-infection (Fig. 1f). Taken together these results demonstrate that the multiplication of MVA-HANP and MVAnr in IEC-6 and BHK-21 cells is comparable. Thus, IEC-6 cells can serve as an alternative to BHK-21 and CEF cells for the production of MVA-based vectors and vaccines. IEC-6 cells have features characteristic of the intestinal epithelium in intact mammals (Quaroni et al., 1979; Wood et al., 2003; Wang et al., 2003) and may serve as a more authentic in vitro model for studying host factors that modulate MVA host-range.
Our cell spread experiment suggested that mature viruses might be present in some of the supposedly semi- or non-permissive cell lines. We performed quantitative electron microscopy (EM) with the aim of defining all viral forms produced in the course of MVA infection. Cell monolayers in six-well plates (Nunc) were infected with MVA-HANP and MVAnr, respectively, at an m.o.i. of 5 IU per cell. After adsorption for 1 h at 4 °C, the cells were washed three times with PBS and incubated with fresh medium at 37 °C in 5 % CO2 atmosphere for 6, 12, 24 and 48 h p.i. At appropriate time points post-infection, infected cells were fixed and processed for EM as described previously (Mckelvey et al., 2002). Morphological forms of MVA were counted in 50 section profiles of cells that were clearly infected. Absolute and relative amounts of each viral form were quantified for each of the 13 mammalian cell lines. Complete morphogenesis of both MVA strains occurred in IEC-6 cells. All four mature virion forms were produced with MVA-HANP (Supplementary Fig. S1 available in JGV Online, Table 2) as well as with MVAnr (Supplementary Fig. S2). Although the morphogenetic structures present in MVA-HANP-infected IEC-6 cells were the same as in BHK-21 cells, there were differences in their abundance. Cell-associated enveloped viruses (CEV) represented 41·3 % (n=243) of mature viruses (IMV, intracellular mature virus; IEV, intracellular enveloped virus; and CEV) produced in IEC-6 as opposed to only 5·2 % (n=42) produced in BHK-21 cells (Table 2). Conversely, in BHK-21 cells, a substantial amount of IMV (70·5 %) and a low amount of CEV (5·2 %) were produced (Table 2). Meiser et al. (2003a) reported that 50 % of mature viruses produced in MVA-infected CEF were CEV. This is similar to what we obtained in IEC-6 cells. Our results hence differ from those of Spehner et al. (2000), which implied that enveloped viruses (IEV and CEV) were the predominant mature viral forms in MVA-infected BHK-21 cells. However, the difference may be a reflection of different methodologies. Quantification by EM in which 50 cell sections were analysed may provide another picture of relative proportion of enveloped particles rather than quantification by CsCl gradient as reported by others. Our EM data also demonstrated the heterogeneity in the block of the MVA-HANP assembly in different cell lines (Table 2). In addition to the normal morphogenetic structures, dense particles (DPs) were produced in non-permissive and to a lesser degree in permissive cell lines (Supplementary Fig. S1, Table 2). There was a large accumulation of DPs in human cell lines (Caco-2, A549 and 293) infected with MVA-HANP. The DPs were slightly smaller in diameter and more electron-dense than typical immature viruses (Supplementary Fig. S1). The DPs were naked or enveloped by single or double membranes (Supplementary Fig. S1). Similar forms of DPs have been observed in HeLa and CEF cells infected with MVA (Meiser et al., 2003b; Caroll & Moss, 1997; Gallego-Gomez et al., 2003). The enwrapment of DPs is similar to the manner in which IMVs are enveloped to form IEV, CEV and extracellular enveloped virus (EEV). This may suggest that DPs encode signals for trans-Golgi network wrapping, intracellular transport and a capacity to egress from the cell. This is in contrast to earlier hypotheses, suggesting that such signals were residing in the IMV only (Krijnse-Locker et al., 2000). Although DPs have been described as the transition between immature virus and IMV (Caroll & Moss, 1997; Gallego-Gomez et al., 2003), it is more likely that they are products of defective virion morphogenesis. This is because DPs produced in 293 cells failed to multiply when inoculated into permissive BHK-21 or IEC-6 cells (data not shown). Our result is consistent with a previous report showing that DPs produced in MVA-infected HeLa cells were non-infectious (Meiser et al., 2003b). The morphogenesis of MVAnr in different mammalian cell lines was similar to MVA-HANP except that higher numbers of morphogenetic structures were present for the former. In addition, mature virions were easily detected in Vero cells infected with MVA, but not MVA-HANP (Supplementary Fig. S3, Table 2).
|
) and MVAnr (Supplementary Fig. S3). Mature virions present in these cell lines were products of virion morphogenesis and not leftover of input virus material. There are at least three reasons for this conclusion. First, mature virions were present within the cell cytoplasm and on the cell surface (Supplementary Fig. S3). Leftover of input virus material will only be on the cell surface. Second, mature virions were not detected in these cell lines at early time points post-infection (6 and 12 h p.i.) (data not shown). Third, mature virions were found in the presence of other products of on-going virion morphogenesis, including viroplasm, immature viruses and DPs (Supplementary Fig. S3). These structures are so fragile that they cannot possibly be found intact in infecting virus material. To test whether the mature virions were infectious, we infected Vero, NMULI, A549, H411E and 293 cells with MVA-HANP and MVAnr, respectively. Cell lysates from these cells were used to infect BHK-21 and IEC-6 cells. In all cases more than a 2 log increase was recorded (data not shown). A549 cells infected with MVAnr, but not MVA-HANP, resulted in more than a 2 log increase in virus titre when passaged in BHK-21 cells. Collectively, these results suggest that mature virions produced in cell lines previously considered to be semi- and non-permissive were infectious. These observations are relevant to the safety of MVA since mature viruses produced in semi- and non-permissive cells can be a source of infection of permissive cells or hosts. Although we have shown that infectivity can be transferred from supposedly semi- or non-permissive cells to permissive cells in vitro, such an outcome may seem far fetched in vivo. This is because MVA infection of some animal species has so far only resulted in abortive infections (Stittelaar et al., 2001; Ramirez et al., 2003; Hanke et al., 2005). In conclusion, we have demonstrated that MVA multiplied efficiently in IEC-6 cells, and that limited production of mature virions occurred in cell lines so far considered to be semi- or non-permissive. Further research is required to unravel the molecular and cellular basis for these observations.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Antoine, G., Scheiflinger, F., Dorner, F. & Falkner, F. G. (1998). The complete genomic sequence of the modified vaccinia Ankara strain: comparison with other orthopoxviruses. Virology 244, 365–396.[CrossRef][Medline]
Beattie, E., Kauffman, E. B., Martinez, H., Perkus, M. E., Jacobs, B. L., Paoletti, E. & Tartaglia, J. (1996). Host-range restriction of vaccinia virus E3L-specific deletion mutants. Virus Genes 12, 89–94.[CrossRef][Medline]
Blanchard, T. J., Alcami, A., Andrea, P. & Smith, G. L. (1998). Modified vaccinia virus Ankara undergoes limited replication in human cells and lacks several immunomodulatory proteins: implications for use as a human vaccine. J Gen Virol 79, 1159–1167.[Abstract]
Caroll, M. W. & Moss, B. (1997). Host range and cytopathogenicity of the highly attenuated MVA strain of vaccinia virus: propagation and generation of recombinant viruses in a nonhuman mammalian cell line. Virology 238, 198–211.[CrossRef][Medline]
Carter, G. C., Law, M., Hollinshead, M. & Smith, G. L. (2005). Entry of the vaccinia virus intracellular mature virion and its interactions with glycosaminoglycans. J Gen Virol 86, 1279–1290.
Corona Gutierrez, C. M., Tinoco, A., Lopez Contreras, M., Navarro, T., Calzado, P., Vargas, L., Reyes, L., Posternak, R. & Rosales, R. (2002). Clinical protocol. A phase II study: efficacy of the gene therapy of the MVA E2 recombinant virus in the treatment of precancerous lesions (NIC I and NIC II) associated with infection of oncogenic human papillomavirus. Hum Gene Ther 13, 1127–1140.[Medline]
Drexler, I., Heller, K., Wahren, B., Erfle, V. & Sutter, G. (1998). Highly attenuated modified vaccinia virus Ankara replicates in baby hamster kidney cells, a potential host for virus propagation, but not in various human transformed and primary cells. J Gen Virol 79, 347–352.[Abstract]
Drexler, I., Staib, C. & Sutter, G. (2004). Modified vaccinia virus Ankara as antigen delivery system: how can we best use its potential? Curr Opin Biotechnol 15, 506–512.[CrossRef][Medline]
Gallego-Gomez, J. C., Risco, C., Rodriguez, D., Cabezas, P., Guerra, S., Carrascosa, J. L. & Esteban, M. (2003). Differences in virus-induced cell morphology and in virus maturation between MVA and other strains (WR, Ankara, and NYCBH) of vaccinia virus in infected human cells. J Virol 77, 10606–10622.
Gillard, S., Spehner, D. & Drillien, R. (1985). Mapping of a vaccinia virus host range sequence by insertion into the viral thymidine kinase gene. J Virol 53, 316–318.
Hanke, T., McMichael, A. J., Dennis, M. J., Sharpe, S. A., Powell, L. A., McLoughlin, L. & Crome, S. J. (2005). Biodistribution and persistence of an MVA-vectored candidate HIV vaccine in SIV-infected rhesus macaques and SCID mice. Vaccine 23, 1507–1514.[CrossRef][Medline]
Hansen, H., Okeke, M. I., Nilssen, Ø. & Traavik, T. (2004). Recombinant viruses obtained from co-infection in vitro with a live vaccinia-vectored influenza vaccine and a naturally occurring cowpox virus display different plaque phenotypes and loss of the transgene. Vaccine 23, 499–506.[Medline]
Hollinshead, M., Rodger, G., van Eijl, H., Law, M., Hollinshead, R., Vaux, D. J. & Smith, G. L. (2001). Vaccinia virus utilizes microtubules for movement to the cell surface. J Cell Biol 154, 389–402.
Hornemann, S., Harlin, O., Staib, C., Kisling, S., Erfle, V., Kaspers, B., Hacker, G. & Sutter, G. (2003). Replication of modified vaccinia virus Ankara in primary chicken embryo fibroblasts requires expression of the interferon resistance gene E3L. J Virol 77, 8394–8407.
Krijnse-Locker, J., Kuehn, A., Schleich, S., Rutter, G., Hohenberg, H., Wepf, R. & Griffiths, G. (2000). Entry of the two infectious forms of vaccinia virus at the plasma membrane is signaling-dependent for the IMV but not the EEV. Mol Biol Cell 11, 2497–2511.
McKelvey, T. A., Andrews, S. C., Miller, S. E., Ray, C. A. & Pickup, D. J. (2002). Identification of the orthopoxvirus p4c gene, which encodes a structural protein that directs intracellular mature virus particles into A-type inclusions. J Virol 76, 11216–11225.
Meiser, A., Boulanger, D., Sutter, G. & Krijnse-Locker, J. (2003a). Comparison of virus production in chicken embryo fibroblasts infected with the WR, IHD-J and MVA strains of vaccinia virus: IHD-J is most efficient in trans-Golgi network wrapping and extracellular enveloped virus release. J Gen Virol 84, 1383–1392.
Meiser, A., Sancho, C. & Krijnse-Locker, J. (2003b). Plasma membrane budding as an alternative release mechanism of the extracellular enveloped form of vaccinia virus from HeLa cells. J Virol 77, 9931–9942.
Meyer, H., Sutter, G. & Mayr, A. (1991). Mapping of deletions in the genome of the highly attenuated vaccinia virus MVA and their influence on virulence. J Gen Virol 72, 1031–1038.
Perkus, M. E., Goebel, S. J., Davis, S. W., Johnson, G. P., Limbach, K., Norton, E. K. & Paoletti, E. (1990). Vaccinia virus host range genes. Virology 179, 276–286.[CrossRef][Medline]
Quaroni, A., Wands, J., Trelstad, R. L. & Isselbacher, K. J. (1979). Epithelioid cell cultures from rat small intestine. Characterization by morphologic and immunologic criteria. J Cell Biol 80, 248–265.
Ramirez, J. C., Finke, D., Esteban, M., Kraehenbuhl, J. P. & Acha-Orbea, H. (2003). Tissue distribution of the Ankara strain of vaccinia virus (MVA) after mucosal or systemic administration. Arch Virol 148, 827–839.[CrossRef][Medline]
Risco, C., Rodriguez, J. R., Lopez-Iglesias, C., Carrascosa, J. L., Esteban, M. & Rodriguez, D. (2002). Endoplasmic reticulum-Golgi intermediate compartment membranes and vimentin filaments participate in vaccinia virus assembly. J Virol 76, 1839–1855.
Sancho, M. C., Schleich, S., Griffiths, G. & Krijnse-Locker, J. (2002). The block in assembly of modified vaccinia virus Ankara in HeLa cells reveals new insights into vaccinia virus morphogenesis. J Virol 76, 8318–8334.
Schmelz, M., Sodeik, B., Ericsson, M., Wolffe, E., Shida, H., Hiller, G. & Griffiths, G. (1994). Assembly of vaccinia virus: the second wrapping cisterna is derived from the trans Golgi network. J Virol 68, 130–147.
Smith, G. L. & Law, M. (2004). The exit of vaccinia virus from infected cells. Virus Res 106, 189–197.[CrossRef][Medline]
Smith, C. L., Dunbar, P. R., Mirza, F. & 9 other authors (2005). Recombinant modified vaccinia Ankara primes functionally activated CTL specific for a melanoma tumor antigen epitope in melanoma patients with a high risk of disease recurrence. Int J Cancer 113, 259–266.[CrossRef][Medline]
Sodeik, B. & Krijnse-Locker, J. (2002). Assembly of vaccinia virus revisited: de novo membrane synthesis or acquisition from the host? Trends Microbiol 10, 15–24.[CrossRef][Medline]
Spehner, D., Drillien, R., Proamer, F., Houssais-Pecheur, C., Zanta, M.-A., Geist, M., Dott, K. & Balloul, J.-M. (2000). Enveloped virus is the major virus form produced during productive infection with modified vaccinia virus Ankara strain. Virology 273, 9–15.[CrossRef][Medline]
Stittelaar, K. J., Kuiken, T., de Swart, R. L. & 8 other authors (2001). Safety of modified vaccinia virus Ankara (MVA) in immune-suppressed macaques. Vaccine 19, 3700–3709.[CrossRef][Medline]
Sutter, G., Wyatt, L. S., Foley, P. L., Bennink, J. R. & Moss, B. (1994). A recombinant vector derived from the host range-restricted and highly attenuated MVA strain of vaccinia virus stimulates protective immunity in mice to influenza virus. Vaccine 12, 1032–1040.[CrossRef][Medline]
Tooze, J., Hollinshead, M., Reis, B., Radsak, K. & Kern, H. (1993). Progeny vaccinia and human cytomegalovirus particles utilize early endosomal cisternae for their envelopes. Eur J Cell Biol 60, 163–178.[Medline]
Wang, Z., Chen, W.-W., Li, R.-R., Wen, B. & Sun, J.-B. (2003). Effect of gastrin on differentiation of rat intestinal epithelial cells in vitro. World J Gastroenterol 9, 1786–1790.[Medline]
Weingart, H., Czub, M., Czub, S. & 18 other authors (2004). Immunization with modified vaccinia virus Ankara-based recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in ferrets. J Virol 78, 12672–12676.
Wood, S. R., Zhao, Q., Smith, L. H. & Daniels, C. K. (2003). Altered morphology in cultured rat intestinal epithelial IEC-6 cells is associated with alkaline phosphatase expression. Tissue Cell 35, 47–58.[Medline]
Wyatt, L. S., Earl, P. L., Eller, L. A. & Moss, B. (2004). Highly attenuated smallpox vaccine protects mice with and without immune deficiencies against pathogenic vaccinia virus challenge. Proc Natl Acad Sci U S A 101, 4590–4595.
Received 31 August 2005;
accepted 28 September 2005.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
