Non-replicating recombinant vaccinia virus encoding ... - CiteSeerX

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Non-replicating recombinant vaccinia virus encoding murine B-7 molecules elicits effective costimulation of naive CD4 ÷ splenocytes in vitro Daniel Oertli, 2 Walter R. Marti, 2 Jeffrey A. N o r t o n 1 a n d K a n g l a T s u n g 1 1Laboratory of Biological Therapy, Department of Surgery, Washington University School of Medicine, 660 S. Euclid Ave, CSRB 3316, St Louis, MO 63110, USA 2Research Unit, Department of Surgery, University Hospital of Basel, Laboratory 404, Hebelstrasse 20, CH-4031 Basel, Switzerland

Using a series of new insertion/expression vectors, we constructed a set of recombinant vaccinia viruses (recVV) encoding the murine T cell costimulatory molecules roB7-1 or mB7-2, or both together in the same construct. On infection with replication incompetent and non-cytopathic recW, several tumour cell lines expressed the respective molecules and bound to CTLA-4. The highest binding capacity was found when both roB7 molecules were co-expressed. Mouse B16.F10 melanoma cells expressing mBT-1 or mB7-2 provided effective costimulation for proliferation of resting CD4 ÷ T cells in the presence of concanavalin A and plate-bound anti-T cell receptor antibodies, respectively. If mB71 and roB7-2 were delivered together on the same cell, the proliferative response of CD4 ÷ T cells increased further. The costimulatory effect could be blocked with CTLA-4, the soluble ligand for B7 molecules. The possibility of engineering tumour cells using recW holds implications for the future design of vaccination strategies.

At least two signals must be provided by an antigen presenting cell (APC) in order to induce activation and clonal expansion of CD4 + cells (Gimmi et al., 1991). The first signal consists of the engagement of the T cell receptor (TCR) by the major histocompatibility class II antigen-peptide complex. The second signal is usually provided by the members of the IgG superfamily B7-1 (CDSO) and/or B7-2 (CD86) on the surface of the APC. They bind to both the CD28 and the CTLA-4 receptors on the responding CD4 + T cell (Freeman et al., 1989, 1993; Linsley eta]., 1994). Optimal stimulation of CD4 + cells

Authorfor correspondence:Daniel Oertli. Fax +41 61 265 3990.

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is produced when the specific ligand and costimulatory activity are provided by the same cell (Liu & Janeway, 1992) and CD4 + cell anergy can be induced if the B7 costimulatory pathway is missing or has been blocked (Harding et al., 1992; Chen & Nabavi, 1994). We investigated recombinant vaccinia virus (recVV) as a gene expression vector system for delivery of the costimulation function in vitro. The choice of this vector system is based on the efficient infection of mouse and human cells by VV and the capacity of the viral vector to carry multiple genes for simultaneous expression in infected cells. Furthermore, VV has been used as a vaccine vector in many experimental systems and studies. Treatment of VV with Psoralen and irradiation with long-wave UV light renders the virus nonreplicating and abrogates its cytopathic effect. Such a treatment protocol, established in our laboratory (Tsung et al., 1996), shows that despite complete inactivation of virus replication and absence of cytopathic effect after Psoralen/UV treatment of recVV, sufficient gene expression can be achieved if the inserted gene is driven by early promoters. In a first set of experiments, we constructed a series of plasmid vectors (pKT1630) for insertion of genes into the 3flhydroxy-5-ene steroid dehydrogenase locus (Goebel etal., 1990) of the VV genome. The plasmids used in these experiments contain multiple expression/insertion cassettes containing early promoters (Davison & Moss, 1989) and a multiple cloning site with the downstream located VV early transcriptional termination sequence (TTTTTNT). The expression/insertion cassettes are flanked by sequences that are identical to different viral loci allowing homologous recombination and production of recVV. The gene for E. coli guanine phosphoribosyltransferase (gpt) is coexpressed outside and downstream of the homologous sequences flanking the expression/insertion cassette and is used as a transient marker for selection of r e c W after homologous recombination (Falkner & Moss, 1990). The genes encoding costimulatory molecules mBT-1 (GenBank accession no. X60958; generous gift from G. Freeman, Boston, Mass., USA) and mBT-2 (GenBank accession no. L25606; generous gift from J. Schlom,

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log f l uorescence ,,.."~ Fig. 1. Flow cytometric analysis of cell surface expression of mB7-1 and mB7-2 and binding capacity to CTLA-4 fusion protein. B16.F10 melanoma cells (I-A b) were infected with the respective replicating recW or with replication-incompetent recW (Psoralen/Iong-wave UV treated; PLWUV) at an m.o.i, of 10 for 4 0 - 6 0 min at 37 °C on a constant rocking device and cultured overnight. As a negative control, cells were infected with recW lacking the A44L locus. Upper two rows: cells were stained with FITC labelled anti-mB7-1 MAb or with FITC labelled anti-mB7-2 NIAb, washed, fixed with paraformaldehyde, and analysed. Lower two rows: cells were incubated with soluble CTLA-4-1gG fusion protein, washed twice, incubated with FITC conjugated goat anti-human (Fc specific) IgG, fixed with paraformaldehyde, and subsequently analysed.

NIH, Bethesda, Md., USA) were cloned into the insertion cassette of pKT1630 resulting in pKT1630-B7-1, pKT1630B7-2 and pKT1630-BT-1,B7-2. As negative controls, we constructed a plasmid, pKT1601, which contains the flanking sequences of the A44L locus only, resulting in an A44L-deleted recVV. For homologous recombination, we used derivatives of the Western Reserve (ATCC VR 119) laboratory VV strain. Briefly, CV-1 African Green monkey kidney cells were infected with wild-type VV and the expression plasmids in the presence of lipofectin and Opti-MEM I. Recombinant virus clones were selected according to their transient expression of the E. coli gpt

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marker under the selection pressure of mycophenolic acid, xanthine and hypoxanthine, as described (Earl & Moss, 1995). Candidate clones were amplified and checked for the presence of the desired inserts in the different loci of the VV genome by PCR. After large scale amplification on BSC-40 African Green monkey kidney cells, the recVV were purified using ultracentrifugation over 36% sucrose. The virus titre was determined using the plaque-forming assay (Earl & Moss, 1995). For inactivation of replication, the viruses were aliquoted at a dose of 5 x 108 p.f.u, in I ml Hanks' balanced salt solution supplemented with 0"1% (v/v) BSA fraction V in a 35 mm

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Fig. 2. Proliferation of isolated naive CD4 + T ceils in the presence of costimulatory molecules• Erythrocyte-depleted murine splenocytes from C57BL/6 mice were passed over nylon wool columns. CD4 + T cells were isolated after the non-adherent splenocytes were depleted of CD8 + and l-Ab+ cells using MAbs and magnetic cell sorting (MACS). Microcultures were set up in triplicate from the CD4 + fraction in 9G-well plates. Briefly, 2 x 105 effector T cells were cultured with stimulator cells in RPMI 1 640 medium supplemented with 10% (v/v) fetal calf serum, 2 mM-L-glutamine, 50 mM-2-mercaptoethanol, I mM-sodium pyruvate, non-essential amino acids (0-I mM each), streptomycin (I O0 pglml), gentamicin (500 l~glml) and penicillin (I O0 U/ml) at 37 °C with 5% CO2. As stimulator cells, BI 6.FI 0 melanoma cells were infected with non-replicating recVV encoding roB7-1 ( 0 ) , mB7-2 (I-]), or both together in the same construct (II), or with non-replicating control virus r W A44L- (O) and cultured overnight. The stimulator cells were then irradiated (5000 rad) and plated together with 2 x I 0 s CD4 ÷ effector cells per well either in the presence of concanavalin A (a) or plate-bound anti-TCR Abs (b) for 48 h ; I pCi (37 kBq) [methyl-3H]thymidineper well was then added for the subsequent 16 h of the incubation period. The cultures were harvested onto filter paper and the incorporated radioactivity was determined with a liquid scintillation counter. Background radioactivity of the infected turnout cells was always negligible (less than 4000 c.p.m.) indicating that the tumour cells were not growing after irradiation and there was no intracellular replicating VV. Error bars represent the standard deviation of the mean c.p.m. We found statistically significant differences between the four groups at a concanavalin A dose of 1.5 l~g/ml (P < 0-008, ANOVA test), and at concanavalin A doses of I "0 pglml and 2.0 laglml (P < 0.034, ANOVA test). We found statistical differences between the single B7 constructs and the B7-I, B7-2 double construct at anti-TCR Abs doses of 316 ng/ml (P < 0.01 5, ANOVA test). At a dose of 1000 ng/ml, statistically significant differences were found between all B7 constructs compared with control virus (P < 0.0075, ANOVA test), and between the single B7-I construct and the B7-I, B7-2 double construct (P < 0.01 5, ANOVA test). These experiments were repeated with similar results.

Petri dish. Subsequently, 1"0 rtg Psoralen (4'-aminomethylTrioxalen) was added and the solution was incubated at room temperature for 1 0 - I 5 min and irradiated in a 365 nm longwave UV cross-linking unit. Infection of various murine tumour cell lines with replication-incompetent recVV revealed them as non-cytopathic and no plaques could be detected by titrating the recVV on a BSC-40 monolayer. Secondly, we investigated the surface expression of the B7 molecules and their ability to bind to their natural ligand CTLA-4. As shown by FACS analysis, mB7-I and roB7-2 were constitutively expressed on the surface of BI6.F10 melanoma cells (Fig. i). The respective expression profiles were similar; this was also found to be the case in all other routine cell lines investigated - K1736 melanoma, Pan02 pancreatic cancer and 4T1 breast carcinoma - and in BSC-40 African Green monkey kidney cells (data not shown). However, expression after infection with replicating recVV was higher than that observed in cells with replication-incompetent recVV. All cells infected with replicating r e c W showed uniform binding to MAbs and

high mean fluorescence intensities in the FACS analysis• In contrast, cells infected with replication incompetent recVV stained less abundantly with the MAbs. This is most likely due to the partial inhibition of transcription of large genes by genome cross-linking with Psoralen (Tsung et at., I996). Strong binding of mB7-1 and roB7-2 molecules to soluble CTLA-4 IgG fusion protein (a generous gift from P. Linsley; Linsley et aL, 199I) was also demonstrated (Fig. I). The molecules roB71 and roB7-2 bound similarly to CTLA-4 with the mean relative fluorescence intensities for roB7-1 and mB7-2 being 1175"5 and 972"& respectively, for replicating recVV. In the case of replication-incompetent recVV, the relative intensities for roB7-1 and mB7-2 were 189"9 and 371"5, respectively• However, if the cells were infected with r e c W encoding both genes mB7-1 and roB7-2 together, the mean relative fluorescence nearly doubled (1765"5 for replicating rVV and 511"2 for replication-incompetent recVV) suggesting the expression of a double amount of CTLA-4 IgG binding surface molecules. In a third set of experiments, the bioactivitity of the

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Fig, 3. Blocking of the costimulatory function by preincubating engineered BI 6.FI O cells with CTLA-4-1gG fusion protein. CD4 + cells were isolated and the proliferation assay was performed under the same conditions as described in the legend to Fig. 2. As stimulator cells, BI 6.FI 0 melanoma cells were infected with non-replicating recW encoding mB7-1 ( l l ) , roB7-2 (I-I), or both together in the same construct (II). After overnight culture, the cells were incubated without or with various doses of CTLA4-1gG fusion protein for 60 min at 37 °C, washed, irradiated (5000 rad), and eventually co-cultured with the CD4 ÷ effector cells for the proliferation assay. Error bars represent the standard deviation of the mean c.p.m. This experiment was repeated with similar results.

References Chen, C. & Nabavi, N. (1994). In vivo induction of T cell anergy by

blocking B7 and early T cell costimulatory molecule ETC-I/B7-2. Immunity 1, 147-154. Davison, A.J. & Moss, B. (1989). Structure of vaccinia virus early promoters. Journal of Molecular Biology 210, 749-769. Earl, P. L. & Moss, B. (1995). Expression of proteins in mammalian cells using vaccinia. In Current Protocols in Molecular Biology, ist edn, pp. 16.15.I-16.18.10. Edited by F. M. Ausubel, R. Brent & R. E. Kingston. New York: Greene Publishing Association. Falkner, F.G. & Moss, B. (1990). Transient dominant selection of recombinant vaccinia virus. Journal of Virology 64, 3108-31I I. Fearon, E. R., Pardoll, D. M., Itaya, T., Golumbek, P. T., Levitsky, H. I., Simons, J.W., Karasuyana, H., Vogelstein, B. & Frost, P. (1990).

expressed costimulatory molecules was evaluated in vitro. As shown in representative experiments (Fig. 2), strong proliferative responses of isolated resting CD4 + splenocytes from naive mice were observed when syngeneic B16.F10 cells modified by non-replicating recVV encoding either mB7-1 or mB7-2 were used to provide costimulation in both cases of primary TCR stimulation by concanavalin A (Fig. 2 a) or by anti-TCR Ab (Fig. 2 b). By coexpression of mB7-1 and roB7-2 molecules, the proliferation was further enhanced. We then showed that CD4 + cell proliferation could be blocked by preincubating the recVV infected B16.F10 cells with CTLA-4 before they were cocultured with CD4 + in the presence of plate bound anti-TCR Abs (Fig. 3). In the present studies, we used non-replicating recVV as expression vectors for costimulatory molecules. The inactivation of virus replication and cytopathic effect might have great advantages over replicating recVV, mainly because the non-replicating recVV used in our study allowed for continued host gene expression. This may be especially critical for introducing costimulatory functions, for example into tumour cells, with Vv-based vectors. A defective immune response has often been attributed to a lack of helper cell triggering, which would prevent the expansion of class I restricted CD8 + CTL (Fearon et al., 1990; Golumbek et al., 1991; Topalian et al., 1994). Therefore, augmentation of T cell costimulation by transfer of the B7 genes may be therapeutically beneficial. Our

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Interleukin-2 production by tumor cells bypasses T helper function in the generation of an antitumor response. Cell 60, 397-403. Freeman, G. J., Freedman, A. S., Segil, J. M., Lee, G., Whitman, J. F. & Nadler, L. M. (1989). B7, a new member of the Ig superfamily with

unique expression on activated and neoplastic B cells. Journal of Immunology 143, 2714-2722. Freeman, G. l., Grlbben t I. G., Boussiotis, V. A., Ng, J. W., Restivo, V. A., Lombard, L A., Gray, G. S. & Nadler, L. M. (1993). Cloning of B7-

2: a CTLA-4 counter-receptor that costimulates human T cell proliferation. Science 262, 909-911. Gimmi, C. D., Greeman, G.J., Gribben, J. G., Sugita, K., Freedman, A. S., Morimoto, C. & Nadler, L. M. (1981). B-cell surface antigen B7

provides a costimulatory signal that induces T cells to proliferate and secrete interleukin 2. Proceedings of the National Academy of Sciences, USA 88, 6575-6579. Goebel, S. J., Johnson, G. P., Perkus, H. E., Davis, S. W., Winslow, J. P. & Paoletti, E. (1990). The complete DNA sequence of vaccinia virus.

Virology 179, 247-266. Golumbek, P.T., Lazenby, A.J., Levitsky, H. I., Jaffee, L. M., Karasuyama, H., Baker, M. & Pardoll, D. M. (1991 ). Treatment of established

renal cancer by tumor cells engineered to secrete interleukin-4. Science 254, 713-716. Harding, F. A., McArthur, J. G., Gross, J. A., Raulet, D. H. & Allison, J. P. (1992). CD28 mediated signalling co-stimulates murine T cells and

prevents induction of allergy in T cell clones. Nature 356, 607-609. Linsley, P. S., Brady, W., Grosmaire, L., Aruffo, A., Damle, N. K. & Ledbetter, J. A. (1991). Binding of the B cell activation antigen B7 to

CD28 costimulates T cell proliferation and interleukin 2 mRNA accumulation. Journal of Experimental Medicine 173, 721-730.

ijii iiiiiii!iiiiiiiiiii ii; i i ; i ili i i ili i iiiiiiiiiiiiiiiiiiiiiiiii!iiiiiiiiiiiiiiiii iiii Linsley, P. S., Greene, J. L., Brady, W., Bajorath, J., Ledbetter, J. A. & Peach, R. (1994). Human B7-1 (CDS0) and B?-2 (CD~6) bind with similar avidities but distinct kinetics to CD28 and CTLA-4 receptors. Immunity 1, 793-801.

Liu, Y. & Janeway, C. A. (1992). Cells that present both specific ligand and costimulatory activity are most efficient inducers of clonal expansion of normal CD4 T cells. Proceedingsof the NationalAcademy of Sciences, USA 89, 3845-3849. Topa|ian, S. L., Rivoltini, L., Hancini, H., Harkus, N. R., Robbins, P. F.,

Kawakami, Y. & Rosenberg, S.A. (1994). Human CD4 + T ceils specifically recognize a shared melanoma-associated antigen encoded by the tyrosinase gene. Proceedingsof the National Academy of Sciences, USA 91, 9461-9465. Tsung, K., Yim, I. H., Marti, W. R., Bullet, M. L. & Norton, J. A. (1996). Gene expression and cytopathic effect of chemically inactivated vaccinia virus. Journal of Virology 70, I65-171.

Received 4 July 1996; Accepted 12 August 1996