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Journal of General Virology (2006), 87, 21–27 Short Communication DOI 10.1099/vir.0.81479-0 Modified vaccinia virus Ankara multiplies in rat IEC-6 cells and limited production of mature virions occurs in other mammalian cell lines Malachy Ifeanyi Okeke,1 Øivind Nilssen2 and Terje Traavik1,3 1 Department of Microbiology and Virology, Faculty of Medicine, University of Tromsø, N-9037 Tromsø, Norway Correspondence Terje Traavik 2 [email protected] 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 Received 31 August 2005 Accepted 28 September 2005 Recombinant viruses based on modified vaccinia virus Ankara (MVA) are vaccine candidates against infectious diseases and cancers. Presently, multiplication of MVA has been demonstrated in chicken embryo fibroblast and baby hamster kidney (BHK-21) cells only. The multiplication and morphogenesis of a recombinant (MVA-HANP) and non-recombinant MVA strain in BHK-21 and 12 other mammalian cell lines have now been compared. Rat IEC-6 cells were fully permissive to MVA infection. The virus yield in IEC-6 cells was similar to that obtained in BHK-21 cells at low as well as high multiplicities of infection. Vero cells were semi-permissive to MVA infection. Mature virions were produced in supposedly non-permissive cell lines. The multiplication and morphogenesis of non-recombinant MVA and MVA-HANP were similar. These results are relevant to the production and biosafety of MVA-vectored vaccines. Modified vaccinia virus Ankara (MVA) is a highly attenuated vaccinia virus (VACV) strain derived from VACV Ankara. MVA was developed by more than 570 serial passages in chicken embryo fibroblast (CEF) cultures during which it incurred multiple DNA deletions (Meyer et al., 1991). 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 Supplementary figures are available in JGV Online. 0008-1479 G 2006 SGM 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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 19 Jun 2017 03:09:22 Printed in Great Britain 21 M. I. Okeke, Ø. Nilssen and T. Traavik 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. Antiinfluenza 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). To determine whether other mammalian cell lines, besides BHK-21, were permissive to MVA infection, different cell lines were infected with MVA-HANP and MVAnr at an m.o.i. of 0?05 IU per cell. After adsorption for 1 h, cell monolayers were washed twice with PBS and incubated for 72 h post-infection (p.i.). Multiplication (fold increase in virus titre) was determined by dividing virus yield at 72 h p.i. by virus titre after adsorption. Permissiveness and cytopathic effect (CPE) were defined according to Caroll & Moss (1997). 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 BSC-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 MVAHANP, 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 hostrange 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). To evaluate further the multiplication of MVA in IEC-6 and BHK-21 cells, the kinetics of virus multiplication were Table 1. Screening of mammalian cell lines for productive infection of MVA-HANP and MVA Virus multiplication (fold increase in virus titre) was determined by dividing virus yield at 72 h by virus titre after adsorption. The values are the mean of two independent experiments titrated in duplicate. Letters in parentheses refer to permissiveness (P), semi-permissiveness (SP) and non-permissiveness (NP). Cell line IEC-6 BHK-21 Caco-2 H411E FHs74int Hutu-80 Vero RK-13 CHO-K1 A549 PK15 NMULI 293 22 ATCC code CRL-1592 CCL-10 HTB-37 CRL-1548 CCL-241 HTB-40 CCL-81 CCL-37 CCL-61 CCL-185 CCL-33 CRL-1638 CRL-1573 Species Rat Hamster, syrian Human Rat Human Human African green monkey Rabbit Hamster, chinese Human Pig Mouse Human CPE Multiplication MVA-HANP MVA ++ +++ 2 + 2 2 + + 2 2 + 2 2 +++ ++++ 2 + 2 2 ++ + 2 + + ++ 2 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 19 Jun 2017 03:09:22 MVA-HANP 370 477 <1?0 <1?0 <1?0 <1?0 2?0 <1?0 <1?0 <1?0 <1?0 <1?0 <1?0 (P) (P) (NP) (NP) (NP) (NP) (SP) (NP) (NP) (NP) (NP) (NP) (NP) MVA 2218 6068 <1?0 <1?0 <1?0 <1?0 10?0 <1?0 <1?0 1?8 <1?0 6?0 <1?0 (P) (P) (NP) (NP) (NP) (NP) (SP) (NP) (NP) (SP) (NP) (SP) (NP) Journal of General Virology 87 Productive MVA infections 24 h p.i. 48 h p.i. 72 h p.i. (a) (b) 8 (d) 8 7 7 6 6 _ Virus titre (log10 IU ml 1) _ Virus titre (log10 IU ml 1) (c) 5 4 3 2 MVA-HANP/cell virus 1 MVA-HANP/medium virus 0 MVA/cell virus 0 12 24 36 48 60 4 MVA-HANP/cell virus 3 MVA-HANP-medium virus 2 MVA/cell virus MVA/medium virus 1 0 MVA/medium virus _1 5 _1 72 84 0 12 24 (f) 8 7 7 48 60 72 84 _ _ Virus titre (log10 IU ml 1) (e) 8 Virus titre (log10 IU ml 1) 36 Time (h) Time (h) 6 5 MVA-HANP/cell virus 4 MVA-HANP/medium virus MVA/cell virus 3 6 5 MVA-HANP/cell virus 4 MVA-HANP/medium virus MVA/cell virus 3 MVA/medium virus MVA/medium virus 2 2 0 10 20 30 40 0 10 Time (h) 20 30 40 Time (h) Fig. 1. Cell-to-cell spread and multiplication of recombinant and non-recombinant MVA in mammalian cells. Cell spread of MVA-HANP (a) and MVA (b) in IEC-6 cells. The panels show representative fields at approximately6200 magnification. Arrowheads point to foci containing many stained cells while arrows point to singly stained cells. Low multiplicity infection (0?05 IU per cell) of IEC-6 (c) and BHK-21 (d) cells with MVA-HANP and MVA. Multiplication of MVA-HANP and MVA in IEC-6 (e) and BHK-21 (f) cells at a high m.o.i. (5 IU per cell). Values are the mean of two independent experiments titrated in duplicate. http://vir.sgmjournals.org Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 19 Jun 2017 03:09:22 23 M. I. Okeke, Ø. Nilssen and T. Traavik studied at low as well as at high m.o.i. values. Cell monolayers in 25 cm2 tissue culture flasks were infected with MVA-HANP and MVAnr at an m.o.i. of 0?05 IU per cell (low) and 5 IU per cell (high), respectively. After adsorption for 1 h at 37 uC, infected cells were washed twice with PBS and incubated in appropriate medium supplemented with 2?5 % fetal bovine serum at 37 uC in a 5 % CO2 atmosphere. At multiple time points post-infection, cells and medium (culture supernatant) were harvested. Intracellular virions were released by three cycles of freeze–thawing and brief sonication. Virus titre in the cell lysate and culture medium was determined by back titration onto BHK-21 cell monolayers. Infected cell-foci were visualized by immunostaining after 24 h. The kinetics of MVA-HANP multiplication at low and high m.o.i. in IEC-6 and BHK-21 cells were similar (Fig. 1c–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 MVAHANP 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 nonpermissive 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 uC, the cells were washed three times with PBS and incubated with fresh medium at 37 uC 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 24 (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 BHK21 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 MVAHANP. 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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 19 Jun 2017 03:09:22 Journal of General Virology 87 Productive MVA infections Table 2. Quantification of viral forms produced in various mammalian cell lines infected for 24 h with MVA-HANP Different viral forms were counted in 50 section profiles of cells that were clearly infected. Absolute and relative amounts of various viral forms were calculated. The values in parentheses refer to the relative amount of viral forms as a percentage. Relative amount of each viral form made at 24 h p.i. was calculated by dividing the number of each viral form in 50 cell sections by the total of all the viral forms counted. C, Crescent; IV1, incomplete immature virus; IV2, complete immature virus; IV3, complete immature virus with DNA spot; DP, dense particle (this includes forms that are naked or surrounded by single or double membranes); IMV, intracellular mature virus; IEV, intracellular enveloped virus; and CEV, cell-associated enveloped virus. Cell line IEC6 BHK-21 Caco-2 H411E FHs74int Hutu-80 Vero RK-13 CHO-K1 A549 PK15 NMULI 293 C 108 64 33 178 104 0 244 12 0 165 98 124 85 (7?4) (4?2) (5?9) (16?7) (15?8) (0?0) (23?1) (19?1) (0?0) (14?2) (12?7) (9?0) (11?3) IV1 126 87 118 256 143 0 255 24 0 265 118 212 101 IV2 (8?6) (5?7) (20?9) (24?0) (21?7) (0?0) (24?2) (38?1) (0?0) (23?0) (15?3) (15?3) (13?5) 465 377 278 538 398 0 530 22 0 481 518 692 371 (31?8) (24?8) (49?3) (50?4) (60?4) (0?0) (50?3) (34?9) (0?0) (41?7) (67?2) (50?0) (49?5) IV3 73 121 10 24 0 0 7 5 0 17 3 71 4 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). MVA is considered a safe vaccine vector because mature virions are supposedly not produced in cells defined as semi- or non-permissive. However, the results of our EM analysis indicate that mature virions were produced in semiand non-permissive cell lines infected with MVA-HANP (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 MVAHANP 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 http://vir.sgmjournals.org DP (5?0) (7?9) (1?8) (2?2) (0?0) (0?0) (0?7) (7?9) (0?0) (1?5) (0?4) (5?1) (0?5) 101 70 118 41 12 0 18 0 0 173 34 161 189 (6?9) (4?6) (20?9) (3?8) (1?8) (0?0) (1?7) (0?0) (0?0) (15?0) (4?4) (11?6) (25?3) IMV 245 565 4 31 2 0 0 0 0 29 0 118 0 (16?8) (37?2) (0?7) (2?9) (0?3) (0?0) (0?0) (0?0) (0?0) (2?5) (0?0) (8?5) (0?0) IEV 100 194 3 0 0 0 0 0 0 13 0 6 0 (6?9) (12?8) (0?5) (0?0) (0?0) (0?0) (0?0) (0?0) (0?0) (1?1) (0?0) (0?4) (0?0) CEV 243 42 0 0 0 0 0 0 0 11 0 1 0 (16?6) (2?8) (0?0) (0?0) (0?0) (0?0) (0?0) (0?0) (0?0) (1?0) (0?0) (0?1) (0?0) 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 nonpermissive. Further research is required to unravel the molecular and cellular basis for these observations. Acknowledgements This work was supported by the Norwegian Research Council (project no. 148535/V10) and the University of Tromsø, Norway. The authors acknowledge with thanks the technical assistance of Randi Olsen, Electron Microscopic Unit, University of Tromsø, Norway. References Ali, A. N., Turner, P. C., Brooks, M. A. & Moyer, R. W. (1994). 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