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An efficient plasmid-driven system for the generation of influenza virus-like particles for vaccine
International Congress Series 1219 (2001) 1015 – 1017
An efficient plasmid-driven system for the generation of inf luenza virus-like particles for vaccine Tokiko Watanabea,b, Shinji Watanabea, Gabriele Neumanna, Hiroshi Kidab, Yoshihiro Kawaokaa,c,* a
Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, 2015 Linden Drive West, Madison, WI 53706, USA b Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan c Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
Abstract We established an efficient plasmid-driven system for the generation of infectious influenza VLPs containing a virus-like RNA segment entirely from cDNAs. To generate influenza VLPs, we used the RNA polymerase I system for the intracellular synthesis of influenza virus RNAs. Human embryonic kidney cells (293T) were transfected with plasmids encoding the influenza A virus structural proteins and with a plasmid encoding an influenza virus-like viral RNA (vRNA), which contained an antisense copy of the cDNA for green fluorescence protein (GFP) flanked by an RNA polymerase I promoter and terminator (pPolI-GFP). Influenza virus-like particles containing GFP vRNA that were infectious and expressed GFP in infected cells were generated. We also generated VLPs lacking the NS gene by eliminating the plasmid for the NS RNA segment from the set of plasmids required for infectious influenza virus production. Because of its efficiency, this system would be useful in studies of influenza virus replication and particle formation as well as for production of vaccines. D 2001 Elsevier Science B.V. All rights reserved. Keywords: Reverse genetics; NS; Vaccine vector
*
Corresponding author. Tel.: +1-608-265-4925; fax: +1-608-265-5622. E-mail address: [email protected] (Y. Kawaoka).
0531-5131/01/$ – see front matter D 2001 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 1 ) 0 0 3 8 4 - 3
1016
T. Watanabe et al. / International Congress Series 1219 (2001) 1015–1017
1. Introduction Influenza A viruses possess a genome of eight single-stranded negative-sense viral RNAs (vRNAs) that encode a total of 10 proteins. Mena et al. [1] established a vaccinia virus-based system for generation of influenza virus-like particles (VLPs). In this system, an influenza virus-like vRNA carrying a reporter gene is transcribed in vitro and transfected into eukaryotic cells. All 10 influenza virus proteins are expressed from plasmids under the control of a T7 RNA polymerase promoter. When the transfected cells are infected with recombinant vaccinia virus that expresses T7 RNA polymerase, they produce influenza VLPs containing the vRNA of an artificial reporter gene [1]. However, vaccinia virus expresses more than 80 proteins, any of which could affect the influenza virus life cycle. We therefore generated infectious influenza VLPs containing a virus-like RNA segment without using vaccinia virus. In addition, we explored the possibility of using the reverse genetics system we recently established [2,3] for vaccine purposes.
2. Materials and methods To generate influenza VLPs, nine plasmids for nine structural proteins and an RNA polymerase I reporter gene construct (pPolI-GFP) were mixed with transfection reagent (Trans IT LT-1; Panvera, Madison, WI), incubated at room temperature for 15 min, and added to 1106 293T cells. Six hours later, the DNA-transfection reagent mixture was replaced with Opti-MEM (GIBCO/BRL) containing 0.3% BSA and 0.01% FCS. Fortyeight hours later, MDCK cells were infected with the VLPs in the culture supernatant, along with A/WSN/33 virus. At 10-h post-infection, we observed the level of GFP expression. To generate influenza VLPs lacking one of the influenza gene segments, we followed the procedure for the generation of infectious influenza virus as described, but eliminated the plasmid for the NS RNA segment.
3. Results and discussion To generate influenza VLPs, we used the RNA polymerase I system for the intracellular synthesis of influenza virus RNAs [4]. In this system, a cDNA carrying a reporter gene in antisense orientation is flanked by the 50 and 30 noncoding regions of an influenza virus RNA. This cassette is inserted between an RNA polymerase I promoter and terminator. Transfection of such constructs into eukaryotic cells leads to transcription of the reporter gene by cellular RNA polymerase I, thereby generating influenza virus-like RNAs [4]. Upon influenza virus infection, artificial vRNAs are replicated and transcribed by the viral polymerase complex, resulting in the expression of the reporter gene [4]. To generate VLPs, we transfected 293T cells with pPolI-GFP and plasmids for expression of nine influenza viral proteins: PB2, PB1, PA, HA, NP, NA, M1, M2, and NS2. Culture supernatants were harvested 48 h after transfection and mixed with A/WSN/ 33 virus to provide the virus polymerase proteins and NP protein required for replication and transcription of GFP vRNA. The mixture was then inoculated into MDCK cells. At
T. Watanabe et al. / International Congress Series 1219 (2001) 1015–1017
1017
10-h post-infection, we detected GFP-positive MDCK cells, corresponding to 104 particles per ml of supernatant. Thus, plasmid-driven expression of all influenza virus structural proteins resulted in the efficient formation of infectious influenza VLPs containing GFP vRNA that could be delivered into subsequent cells. We next attempted to generate influenza VLPs lacking the NS gene. To this end, we followed the procedure for influenza virus production, but eliminated the plasmid for the NS gene segment. When the supernatant of 293T cells transfected with the plasmids were incubated with MDCK cells, NP was immunologically detected 16-h post-inoculation. The number of NP-positive cells corresponded to 104 infectious VLPs/ml. The plasmid-based system for producing influenza VLPs we established is highly efficient and would be useful in studies of influenza virus replication and particle formation. In addition, one can now generate VLPs that contain vRNAs encoding the proteins required for transcription and replication (i.e., the NP and the polymerase), as well as a vRNA encoding the protein of interest. Although these particles are infectious and some viral proteins are expressed in these infected cells, they cannot produce infectious progeny virus since they do not contain a full complement of viral genes. Therefore, we suggest that these VLPs may be effective vaccines for influenza and useful as virus vectors expressing foreign proteins.
Acknowledgements We thank members of our laboratory for the production of data presented in this manuscript. Support for this work was provided by NIAID Public Health Service research grants and from the Japan Health Science Foundation and the Ministry of Education and Culture of Japan. T.W. is the recipient of a Research Fellowship for Young Scientists from the Japan Society for the Promotion of Science. S.W. is the recipient of the Japan Society for Promotion of Science Postdoctoral Fellowship for Research Abroad.
References [1] I. Mena, A. Vivo, E. Perez, A. Portela, Rescue of a synthetic chloramphenicol acetyltransferase RNA into influenza virus-like particles obtained from recombinant plasmids, J. Virol. 70 (1996) 5016 – 5024. [2] G. Neumann, T. Watanabe, H. Ito, S. Watanabe, H. Goto, P. Gao, H. Hughes, D.R. Perez, R. Donis, E. Hoffmann, G. Hobom, Y. Kawoaka, Generation of influenza A viruses entirely from cloned cDNAs, PNAS 96 (1999) 9345 – 9350. [3] E. Fodor, L. Devenish, O.G. Engelhardt, P. Palese, G.G. Brownlee, A. Garcia-Sastre, Rescue of influenza A virus from recombinant DNA, J. Virol. 73 (1999) 9679 – 9682. [4] G. Neumann, A. Zobel, G. Hobom, RNA polymerase I-mediated expression of influenza viral RNA molecules, Virology 202 (1994) 477 – 479.
An efficient plasmid-driven system for the generation of inf luenza virus-like particles for vaccine Tokiko Watanabea,b, Shinji Watanabea, Gabriele Neumanna, Hiroshi Kidab, Yoshihiro Kawaokaa,c,* a
Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, 2015 Linden Drive West, Madison, WI 53706, USA b Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan c Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
Abstract We established an efficient plasmid-driven system for the generation of infectious influenza VLPs containing a virus-like RNA segment entirely from cDNAs. To generate influenza VLPs, we used the RNA polymerase I system for the intracellular synthesis of influenza virus RNAs. Human embryonic kidney cells (293T) were transfected with plasmids encoding the influenza A virus structural proteins and with a plasmid encoding an influenza virus-like viral RNA (vRNA), which contained an antisense copy of the cDNA for green fluorescence protein (GFP) flanked by an RNA polymerase I promoter and terminator (pPolI-GFP). Influenza virus-like particles containing GFP vRNA that were infectious and expressed GFP in infected cells were generated. We also generated VLPs lacking the NS gene by eliminating the plasmid for the NS RNA segment from the set of plasmids required for infectious influenza virus production. Because of its efficiency, this system would be useful in studies of influenza virus replication and particle formation as well as for production of vaccines. D 2001 Elsevier Science B.V. All rights reserved. Keywords: Reverse genetics; NS; Vaccine vector
*
Corresponding author. Tel.: +1-608-265-4925; fax: +1-608-265-5622. E-mail address: [email protected] (Y. Kawaoka).
0531-5131/01/$ – see front matter D 2001 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 1 ) 0 0 3 8 4 - 3
1016
T. Watanabe et al. / International Congress Series 1219 (2001) 1015–1017
1. Introduction Influenza A viruses possess a genome of eight single-stranded negative-sense viral RNAs (vRNAs) that encode a total of 10 proteins. Mena et al. [1] established a vaccinia virus-based system for generation of influenza virus-like particles (VLPs). In this system, an influenza virus-like vRNA carrying a reporter gene is transcribed in vitro and transfected into eukaryotic cells. All 10 influenza virus proteins are expressed from plasmids under the control of a T7 RNA polymerase promoter. When the transfected cells are infected with recombinant vaccinia virus that expresses T7 RNA polymerase, they produce influenza VLPs containing the vRNA of an artificial reporter gene [1]. However, vaccinia virus expresses more than 80 proteins, any of which could affect the influenza virus life cycle. We therefore generated infectious influenza VLPs containing a virus-like RNA segment without using vaccinia virus. In addition, we explored the possibility of using the reverse genetics system we recently established [2,3] for vaccine purposes.
2. Materials and methods To generate influenza VLPs, nine plasmids for nine structural proteins and an RNA polymerase I reporter gene construct (pPolI-GFP) were mixed with transfection reagent (Trans IT LT-1; Panvera, Madison, WI), incubated at room temperature for 15 min, and added to 1106 293T cells. Six hours later, the DNA-transfection reagent mixture was replaced with Opti-MEM (GIBCO/BRL) containing 0.3% BSA and 0.01% FCS. Fortyeight hours later, MDCK cells were infected with the VLPs in the culture supernatant, along with A/WSN/33 virus. At 10-h post-infection, we observed the level of GFP expression. To generate influenza VLPs lacking one of the influenza gene segments, we followed the procedure for the generation of infectious influenza virus as described, but eliminated the plasmid for the NS RNA segment.
3. Results and discussion To generate influenza VLPs, we used the RNA polymerase I system for the intracellular synthesis of influenza virus RNAs [4]. In this system, a cDNA carrying a reporter gene in antisense orientation is flanked by the 50 and 30 noncoding regions of an influenza virus RNA. This cassette is inserted between an RNA polymerase I promoter and terminator. Transfection of such constructs into eukaryotic cells leads to transcription of the reporter gene by cellular RNA polymerase I, thereby generating influenza virus-like RNAs [4]. Upon influenza virus infection, artificial vRNAs are replicated and transcribed by the viral polymerase complex, resulting in the expression of the reporter gene [4]. To generate VLPs, we transfected 293T cells with pPolI-GFP and plasmids for expression of nine influenza viral proteins: PB2, PB1, PA, HA, NP, NA, M1, M2, and NS2. Culture supernatants were harvested 48 h after transfection and mixed with A/WSN/ 33 virus to provide the virus polymerase proteins and NP protein required for replication and transcription of GFP vRNA. The mixture was then inoculated into MDCK cells. At
T. Watanabe et al. / International Congress Series 1219 (2001) 1015–1017
1017
10-h post-infection, we detected GFP-positive MDCK cells, corresponding to 104 particles per ml of supernatant. Thus, plasmid-driven expression of all influenza virus structural proteins resulted in the efficient formation of infectious influenza VLPs containing GFP vRNA that could be delivered into subsequent cells. We next attempted to generate influenza VLPs lacking the NS gene. To this end, we followed the procedure for influenza virus production, but eliminated the plasmid for the NS gene segment. When the supernatant of 293T cells transfected with the plasmids were incubated with MDCK cells, NP was immunologically detected 16-h post-inoculation. The number of NP-positive cells corresponded to 104 infectious VLPs/ml. The plasmid-based system for producing influenza VLPs we established is highly efficient and would be useful in studies of influenza virus replication and particle formation. In addition, one can now generate VLPs that contain vRNAs encoding the proteins required for transcription and replication (i.e., the NP and the polymerase), as well as a vRNA encoding the protein of interest. Although these particles are infectious and some viral proteins are expressed in these infected cells, they cannot produce infectious progeny virus since they do not contain a full complement of viral genes. Therefore, we suggest that these VLPs may be effective vaccines for influenza and useful as virus vectors expressing foreign proteins.
Acknowledgements We thank members of our laboratory for the production of data presented in this manuscript. Support for this work was provided by NIAID Public Health Service research grants and from the Japan Health Science Foundation and the Ministry of Education and Culture of Japan. T.W. is the recipient of a Research Fellowship for Young Scientists from the Japan Society for the Promotion of Science. S.W. is the recipient of the Japan Society for Promotion of Science Postdoctoral Fellowship for Research Abroad.
References [1] I. Mena, A. Vivo, E. Perez, A. Portela, Rescue of a synthetic chloramphenicol acetyltransferase RNA into influenza virus-like particles obtained from recombinant plasmids, J. Virol. 70 (1996) 5016 – 5024. [2] G. Neumann, T. Watanabe, H. Ito, S. Watanabe, H. Goto, P. Gao, H. Hughes, D.R. Perez, R. Donis, E. Hoffmann, G. Hobom, Y. Kawoaka, Generation of influenza A viruses entirely from cloned cDNAs, PNAS 96 (1999) 9345 – 9350. [3] E. Fodor, L. Devenish, O.G. Engelhardt, P. Palese, G.G. Brownlee, A. Garcia-Sastre, Rescue of influenza A virus from recombinant DNA, J. Virol. 73 (1999) 9679 – 9682. [4] G. Neumann, A. Zobel, G. Hobom, RNA polymerase I-mediated expression of influenza viral RNA molecules, Virology 202 (1994) 477 – 479.