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Development and evaluation of live influenza (LIV) cold-adapted reassortant vaccines in cell culture
International Congress Series 1263 (2004) 551 – 554
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Development and evaluation of live influenza (LIV) cold-adapted reassortant vaccines in cell culture I. Kiseleva a,b, Q. Su a, T. Toner a, C. Szymkowiak a, W.-S. Kwan a, N. Kraiouchkine a, L. Rudenko b, A. Shaw a, R. Youil a,* a
MRL, Merck and Co., Inc., Virus and Cell Biology, 770 Sumneytown Pike, WP44L-206B, West Point, PA 19486, USA b Institute of Experimental Medicine RAMS, St. Petersburg, Russia
Abstract. The Russian influenza A live virus vaccine (LIV) is based on the temperature sensitive (ts) and cold-adapted (ca) A/Leningrad/134/17/57 (H2N2) donor strain (Len/17). Reassortants between current circulating A strains and the ca donor strain are traditionally prepared in eggs. MDCK cells have been used for the commercial manufacture of inactivated whole virus influenza vaccines. There has, as yet, been no transition into cell culture for the development of LIV. In this report we have looked at cell-culture derived reassortants with respect to their ability to maintain their phenotypic and genetic characteristics. D 2004 Elsevier B.V. All rights reserved. Keywords: Influenza; Cold-adapted; Reassortant
1. Introduction Developments made in cell culture will hopefully allow for greater ease in the manufacture of influenza vaccines. Unlike eggs, cell culture is amenable to process modifications allowing for improvements to be made with respect to viral output, as well as purity and clarity in the final vaccine product. The development of reassortants based on the Russian ca influenza donor strain, Len/17, has traditionally taken place in eggs. In this study we have developed type A influenza reassortants between the H2N2 Len/17 and H1N1 A/New Caledonia/20/99 (A/NC). The reassortment was performed entirely in MDCK cells, which were also used to evaluate ts phenotype of isolated reassortants. The Vero cell line was then used to study the growth of two reassortants, which were subsequently genetically characterized.
* Corresponding author. Tel.: +1-215-652-6328; fax: +1-215-993-2096. E-mail address: [email protected] (R. Youil). 0531-5131/ D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2004.02.158
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2. Reassortment in MDCK cells between Len/17 donor strain and A/New Caledonia/ 20/99 wild-type virus Len/17 donor strain and wt A/NC influenza virus were co-infected into MDCK cells at equal ratios (5 log TCID50/ml). Virus stocks were obtained from Dr. A. Klimov (CDC, Atlanta, GA). Five rounds of selective propagation were performed in MDCK cells (passage #55– 75) in the presence of anti-H2 serum (goat) and anti-N2 serum (goat). Anti-serum was derived from A/Singapore/1/57 (H2N2) and obtained from the NIAID, Bethesda, MD. Low temperature propagation (26 jC) was used except for P1 (the initial crossing) and passage 5 (P5) which were carried out at 32 jC. Cloning was performed at P4 and P5. Incubation at each passage ranged between 4 and 7 days. 3. Initial screening of type A reassortants Initial screening for suitable reassortants was performed on MDCK cells [1]. 4. Screening of type A reassortants for their genome composition Clones which displayed the ca/ts phenotype were assayed for either presence of amplicon or by RFLP analysis [2]. Reassortants (6:2) were selected. 5. Subcloning of reassortants Each reassortant was subcloned by plaque assay. Plaques were selected from each clone and propagated in MDCK cells (DMEM media containing 20 Ag/ml trypsin). This passage
Fig. 1. Differential plaque assay of the 38E clone and its subclone 38E/1 following its serial passaging in Vero cells at 32 jC. Plaque assays were performed at 32, 37 and 39 jC. No plaques were observed at 39 jC.
I. Kiseleva et al. / International Congress Series 1263 (2004) 551–554
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Fig. 2. Differential plaque assay of subclone 39E/2 following its serial passaging in Vero cells at 32 jC. Plaque assays were performed at 32, 37 and 39 jC. No plaques were observed at 39 jC.
is referred to as P1. Subclones 38E/1 and 39E/2 were selected for further study. Each subclone was subsequently passaged through several rounds in Vero cells at 32 jC. At each passage plaque assay was performed to measure virus growth at 32, 37 and 39 jC. 6. Passaging of virus in Vero cells FOR each subsequent passage 6 cm dishes containing Vero cells at semi-confluence were prepared (passages #145 –160). Media consisted of DMEM containing 2 mM Table 1 The predicted amino acid changes for Vero-passaged reassortants 38E1 and 39E2 Gene segment
38E/1 (P1 Vero)
38E/1 (P5 Vero)
39E/2 (P1 Vero)
39E/2 (P5 Vero)
PB1 PB2 PA NPa
– – – Gln ! Pro (459) –
– ND ND Gln ! Pro (459) –
M1 M2 NS1 NS2 HA NA
– – – – Asn (203)b –
ND
– – – – – – – – – Glu ! Gly (67) Asp (203)b –
– – – – Asp ! Asn (51) Ser ! Arg (287) His ! Tyr (110) – – Glu ! Gly (67) Asp (203)b –
ND Asn (203)b –
Sequence analysis was performed at passage 1 and passage 5. Numbers in parentheses refer to codon number. a Both 38E/1 and 39E/2 reassortants carried the A/Leningrad/134/47/57 (Leu ! Iso) mutation at codon #341 of the NP gene [2]. b Both residues (Asn and Asp) were identified within the original A/New Caledonia/20/99 viral stock.
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glutamine and 1 Ag/ml trypsin. For propagation, 30 Al of virus supernatant (from P1) was diluted in a total of 1 ml of DMEM and added into each plate. Following a 1 h adsorption at 32 jC, 2 ml of media containing trypsin was added and plates were left to incubate at 32 jC until CPE was reached (48 – 96 h). Trypsin (1 Ag/ml) was added every 24 h. This passage was referred to as P2. A similar procedure was carried out for P3 to P5. MOI for passages P1 –P5 were in the range of 0.1– 1.0. 7. Viral titer determinations Fifty percent egg infectious dose (EID50) and fifty percent tissue culture infectious dose (TCID50) assays were performed as previously described [1]. Plaque assays using MDCK cells were performed as previously described [3]. Plates were incubated at the appropriate temperature with 5% CO2 for 72 h. In this study, 32 jC represents the optimal temperature of viral growth and 37 jC represents the restrictive temperature. 8. Results and discussion Fig. 1 shows results of the differential plaque assay for 38E/1 reassortant following serial passaging at 32 jC in Vero cells. No plaques were observed at 39 jC at any passage. Fig. 2 shows results of the differential plaque assay for subclone 39E/2 following serial passaging in Vero cells at 32 jC. Interestingly, the reduced growth differential observed at passages 4 and 5 could be widened upon re-plaque isolation of the reassortant (data not shown). Table 1 outlines predicted amino acid changes for 38E/1 and 39E/2 at P1 and P5. All mutations described for Len/17 [2] were found to be present. Any changes observed at P1 may be due to the heterogeneous nature of the Len/17 master donor stock [4]. 9. Conclusion MDCK cells provide a good substrate for obtaining reassortments. The Vero cell line is also suitable for their propagation. While the heterogeneous nature of the Len/17 made it difficult to decipher possible cell line-induced changes from variants already existing within the donor stock, genetic characterization confirmed all Len/17 mutations to be present and stable. The ts phenotype was also preserved, as demonstrated by the lack of plaque formation at 39 jC. References [1] I. Kiseleva, et al., Cell based assay for the determination of temperature sensitive and cold adapted phenotype of influenza viruses, J. Virol. Methods 116 (2004) 71 – 78. [2] A.I. Klimov, N.J. Cox, PCR restriction analysis of genome composition and stability of cold-adapted reassortant live influenza vaccines, J. Virol. Methods 52 (1995) 41 – 49. [3] S.-Y. Feng, et al., Genetic recombination between temperature-sensitive and wild-type influenza A virus strains, Acta Virol. 27 (1983) 21 – 26. [4] R. Youil, et al., Phenotypic and genetic analysis of the heterogeneous population present in the cold-adapted master donor strain: A/Leningrad/134/17/57 (H2N2) Virus Research (2004) in press.
www.ics-elsevier.com
Development and evaluation of live influenza (LIV) cold-adapted reassortant vaccines in cell culture I. Kiseleva a,b, Q. Su a, T. Toner a, C. Szymkowiak a, W.-S. Kwan a, N. Kraiouchkine a, L. Rudenko b, A. Shaw a, R. Youil a,* a
MRL, Merck and Co., Inc., Virus and Cell Biology, 770 Sumneytown Pike, WP44L-206B, West Point, PA 19486, USA b Institute of Experimental Medicine RAMS, St. Petersburg, Russia
Abstract. The Russian influenza A live virus vaccine (LIV) is based on the temperature sensitive (ts) and cold-adapted (ca) A/Leningrad/134/17/57 (H2N2) donor strain (Len/17). Reassortants between current circulating A strains and the ca donor strain are traditionally prepared in eggs. MDCK cells have been used for the commercial manufacture of inactivated whole virus influenza vaccines. There has, as yet, been no transition into cell culture for the development of LIV. In this report we have looked at cell-culture derived reassortants with respect to their ability to maintain their phenotypic and genetic characteristics. D 2004 Elsevier B.V. All rights reserved. Keywords: Influenza; Cold-adapted; Reassortant
1. Introduction Developments made in cell culture will hopefully allow for greater ease in the manufacture of influenza vaccines. Unlike eggs, cell culture is amenable to process modifications allowing for improvements to be made with respect to viral output, as well as purity and clarity in the final vaccine product. The development of reassortants based on the Russian ca influenza donor strain, Len/17, has traditionally taken place in eggs. In this study we have developed type A influenza reassortants between the H2N2 Len/17 and H1N1 A/New Caledonia/20/99 (A/NC). The reassortment was performed entirely in MDCK cells, which were also used to evaluate ts phenotype of isolated reassortants. The Vero cell line was then used to study the growth of two reassortants, which were subsequently genetically characterized.
* Corresponding author. Tel.: +1-215-652-6328; fax: +1-215-993-2096. E-mail address: [email protected] (R. Youil). 0531-5131/ D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2004.02.158
552
I. Kiseleva et al. / International Congress Series 1263 (2004) 551–554
2. Reassortment in MDCK cells between Len/17 donor strain and A/New Caledonia/ 20/99 wild-type virus Len/17 donor strain and wt A/NC influenza virus were co-infected into MDCK cells at equal ratios (5 log TCID50/ml). Virus stocks were obtained from Dr. A. Klimov (CDC, Atlanta, GA). Five rounds of selective propagation were performed in MDCK cells (passage #55– 75) in the presence of anti-H2 serum (goat) and anti-N2 serum (goat). Anti-serum was derived from A/Singapore/1/57 (H2N2) and obtained from the NIAID, Bethesda, MD. Low temperature propagation (26 jC) was used except for P1 (the initial crossing) and passage 5 (P5) which were carried out at 32 jC. Cloning was performed at P4 and P5. Incubation at each passage ranged between 4 and 7 days. 3. Initial screening of type A reassortants Initial screening for suitable reassortants was performed on MDCK cells [1]. 4. Screening of type A reassortants for their genome composition Clones which displayed the ca/ts phenotype were assayed for either presence of amplicon or by RFLP analysis [2]. Reassortants (6:2) were selected. 5. Subcloning of reassortants Each reassortant was subcloned by plaque assay. Plaques were selected from each clone and propagated in MDCK cells (DMEM media containing 20 Ag/ml trypsin). This passage
Fig. 1. Differential plaque assay of the 38E clone and its subclone 38E/1 following its serial passaging in Vero cells at 32 jC. Plaque assays were performed at 32, 37 and 39 jC. No plaques were observed at 39 jC.
I. Kiseleva et al. / International Congress Series 1263 (2004) 551–554
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Fig. 2. Differential plaque assay of subclone 39E/2 following its serial passaging in Vero cells at 32 jC. Plaque assays were performed at 32, 37 and 39 jC. No plaques were observed at 39 jC.
is referred to as P1. Subclones 38E/1 and 39E/2 were selected for further study. Each subclone was subsequently passaged through several rounds in Vero cells at 32 jC. At each passage plaque assay was performed to measure virus growth at 32, 37 and 39 jC. 6. Passaging of virus in Vero cells FOR each subsequent passage 6 cm dishes containing Vero cells at semi-confluence were prepared (passages #145 –160). Media consisted of DMEM containing 2 mM Table 1 The predicted amino acid changes for Vero-passaged reassortants 38E1 and 39E2 Gene segment
38E/1 (P1 Vero)
38E/1 (P5 Vero)
39E/2 (P1 Vero)
39E/2 (P5 Vero)
PB1 PB2 PA NPa
– – – Gln ! Pro (459) –
– ND ND Gln ! Pro (459) –
M1 M2 NS1 NS2 HA NA
– – – – Asn (203)b –
ND
– – – – – – – – – Glu ! Gly (67) Asp (203)b –
– – – – Asp ! Asn (51) Ser ! Arg (287) His ! Tyr (110) – – Glu ! Gly (67) Asp (203)b –
ND Asn (203)b –
Sequence analysis was performed at passage 1 and passage 5. Numbers in parentheses refer to codon number. a Both 38E/1 and 39E/2 reassortants carried the A/Leningrad/134/47/57 (Leu ! Iso) mutation at codon #341 of the NP gene [2]. b Both residues (Asn and Asp) were identified within the original A/New Caledonia/20/99 viral stock.
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I. Kiseleva et al. / International Congress Series 1263 (2004) 551–554
glutamine and 1 Ag/ml trypsin. For propagation, 30 Al of virus supernatant (from P1) was diluted in a total of 1 ml of DMEM and added into each plate. Following a 1 h adsorption at 32 jC, 2 ml of media containing trypsin was added and plates were left to incubate at 32 jC until CPE was reached (48 – 96 h). Trypsin (1 Ag/ml) was added every 24 h. This passage was referred to as P2. A similar procedure was carried out for P3 to P5. MOI for passages P1 –P5 were in the range of 0.1– 1.0. 7. Viral titer determinations Fifty percent egg infectious dose (EID50) and fifty percent tissue culture infectious dose (TCID50) assays were performed as previously described [1]. Plaque assays using MDCK cells were performed as previously described [3]. Plates were incubated at the appropriate temperature with 5% CO2 for 72 h. In this study, 32 jC represents the optimal temperature of viral growth and 37 jC represents the restrictive temperature. 8. Results and discussion Fig. 1 shows results of the differential plaque assay for 38E/1 reassortant following serial passaging at 32 jC in Vero cells. No plaques were observed at 39 jC at any passage. Fig. 2 shows results of the differential plaque assay for subclone 39E/2 following serial passaging in Vero cells at 32 jC. Interestingly, the reduced growth differential observed at passages 4 and 5 could be widened upon re-plaque isolation of the reassortant (data not shown). Table 1 outlines predicted amino acid changes for 38E/1 and 39E/2 at P1 and P5. All mutations described for Len/17 [2] were found to be present. Any changes observed at P1 may be due to the heterogeneous nature of the Len/17 master donor stock [4]. 9. Conclusion MDCK cells provide a good substrate for obtaining reassortments. The Vero cell line is also suitable for their propagation. While the heterogeneous nature of the Len/17 made it difficult to decipher possible cell line-induced changes from variants already existing within the donor stock, genetic characterization confirmed all Len/17 mutations to be present and stable. The ts phenotype was also preserved, as demonstrated by the lack of plaque formation at 39 jC. References [1] I. Kiseleva, et al., Cell based assay for the determination of temperature sensitive and cold adapted phenotype of influenza viruses, J. Virol. Methods 116 (2004) 71 – 78. [2] A.I. Klimov, N.J. Cox, PCR restriction analysis of genome composition and stability of cold-adapted reassortant live influenza vaccines, J. Virol. Methods 52 (1995) 41 – 49. [3] S.-Y. Feng, et al., Genetic recombination between temperature-sensitive and wild-type influenza A virus strains, Acta Virol. 27 (1983) 21 – 26. [4] R. Youil, et al., Phenotypic and genetic analysis of the heterogeneous population present in the cold-adapted master donor strain: A/Leningrad/134/17/57 (H2N2) Virus Research (2004) in press.