JGV Papers in Press. Published December 12, 2014 as doi:10.1099/jgv.0.000025
Journal of General Virology Chimeric NA and mutant PB1 gene constellation improves growth and yield of H5N1 vaccine candidate virus --Manuscript Draft-Manuscript Number:
JGV-D-14-00166R1
Full Title:
Chimeric NA and mutant PB1 gene constellation improves growth and yield of H5N1 vaccine candidate virus
Short Title:
Improving Influenza Vaccine Virus Yield
Article Type:
Short Communication
Section/Category:
Animal - Negative-strand RNA Viruses
Corresponding Author:
Zhiping Ye, M.D., Ph.D. US Food and Drug Administration UNITED STATES
First Author:
Ewan P. Plant, Ph.D.
Order of Authors:
Ewan P. Plant, Ph.D. Zhiping Ye, M.D., Ph.D.
Abstract:
We previously showed that a mutated PB1 gene improved the growth kinetics of a H3N2 influenza reassortant. Here we show that the same mutations improve the growth kinetics of a virus containing the A/VietNam/1203/2004 (H5N1) HA and NA. Total protein yield and neuraminidase activity were increased when a chimeric NA was included. These increases indicate that the synergistic effect is due to the gene constellation containing both the altered PB1 gene and the chimeric NA gene.
Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation
Manuscript Including References (Word document) Click here to download Manuscript Including References (Word document): Plant & Ye JGV revised2.doc
1
Chimeric NA and mutant PB1 gene constellation improves growth and yield of
2
H5N1 vaccine candidate virus
3 4
Running title: Improving Influenza Vaccine Virus Yield
5 6
Contents Category: Animal, RNA viruses
7 8
Ewan P. Plant* and Zhiping Ye*
9 10
Laboratory of Respiratory Viral Diseases, Division of Viral Products, Office of Vaccine
11
Research and Review, United States Food and Drug Administration, Silver Spring,
12
Maryland 20993, United States of America
13 14
* Corresponding authors. Tel.: +1 240 402 6733, Fax: +1 301 595 1460
15
Email addresses: [email protected]; [email protected]
16 17 18
Word Count (summary & main text): 1810
19
Summary
20
We previously showed that a mutated PB1 gene improved the growth kinetics of a H3N2
21
influenza reassortant. Here we show that the same mutations improve the growth kinetics
22
of a virus containing the A/VietNam/1203/2004 (H5N1) HA and NA. Total protein yield
23
and neuraminidase activity were increased when a chimeric NA was included. These
24
increases indicate that the synergistic effect is due to the gene constellation containing
25
both the altered PB1 gene and the chimeric NA gene.
26 27
Keywords
28
Influenza, vaccine, polymerase, protein yield, antigenic shift
29 30
2
31
The object of this work is to improve the genomic backbone segments used in
32
vaccine candidate viruses while avoiding detrimental gene constellation effects. It is not
33
known what attributes from each segment affect the gene constellation or reassortment
34
but it is desirable that vaccine seed strains containing contemporary HA and NA
35
segments in the context of known backbone segments can be recovered. It is also
36
desirable that those strains replicate well and that satisfactory protein yields can be
37
harvested. We have previously shown that five mutations to the PB1 gene of the high
38
growth backbone virus PR/8 improved the growth kinetics of a reassortant containing the
39
HA and NA segments from an H3N2 virus (Plant et al., 2012). These mutations (G180E,
40
S216G, S361R, Q621R and N654S) were not selected based on any structural or
41
functional information. The five mutations made to the PB1 gene (PB1m5) were based on
42
sequence variations found in vaccine strain reassortants. We have also described the
43
improved growth and protein yield of a reassortant containing the H5N1
44
A/Vietnam/1203/2004 (A/VN/1203) HA segment and a chimeric NA segment comprised
45
of the carboxy terminus (non-coding region and first 72 amino acids) from the
46
A/VN/1203 strain with the amino terminus from PR/8 (Adamo et al., 2009). The stalk
47
region of the NA protein differs between different influenza virus strains in both length
48
and amino acid identity and has been shown to affect growth, protein yield and
49
pathogenesis (Adamo et al., 2009;Baigent & McCauley, 2001;Zhou et al., 2009). Here
50
we continue these studies by creating reassortants containing PB1wt or PB1m5 and the
51
A/VN/1203 HA with either the wild-type (NAwt) or chimeric (NACh) NA from
52
A/VN/1203.
3
53
Viruses were generated using the eight plasmid reverse genetic system (Hoffmann
54
et al., 2000). The polybasic cleavage site of the A/VN/1203 HA had previously been
55
removed (Adamo et al., 2009). Co-cultured 293T and Madin-Darby canine kidney
56
(MDCK) cells were transfected using the method of Bourret et al., (Bourret et al., 2012).
57
Culture supernatant was used to infect eggs and viruses containing either the wild-type
58
(PB1wt) or mutant (PB1m5) PB1, in conjunction with either the wild-type (NAwt) or
59
chimeric (NACh) NA from A/VN/1203, were recovered. The HA, NA and PB1 genes
60
were sequenced and did not contain any additional mutations. Hemagglutination titers
61
were comparable between the viruses (Table 1) and all the viruses produced large plaques
62
on MDCK cells (Fig. 1a). The longer stalk region of the H5N1 neuraminidase has been
63
shown to contribute to pathogenicity (Zhou et al., 2009). Here we confirm that removing
64
this region reduces pathogenicity, with or without the PB1M5 allele, by determining mean
65
death time (MDT). 106 virus particles were inoculated into the allantoic fluid of eight
66
eggs which were then incubated at 35°C and candled at 12 hour intervals. Eggs were
67
scored as alive or dead and the time of death recorded. MDT was obtained by
68
multiplying the number of embryos dead at each interval post inoculation by the hours
69
post inoculation and dividing by the total number of embryos inoculated. The MDT range
70
was similar for all viruses (Table 1).
71
Optimal polymerase activity is required for efficient replication (Giesendorf et al.,
72
1986;Li et al., 2009;Ngai et al., 2013). PB1 from a seasonal strain or with targeted
73
mutations has been shown to improve growth or HA yield of H3 reassortants (Cobbin et
74
al., 2013;Plant et al., 2012), however mixed results have been observed for H5
75
reassortants (Abt et al., 2011;Rudneva et al., 2007;Wanitchang et al., 2010). All three
4
76
polymerase segments reassort together more frequently in experimental settings than
77
natural reassortment suggesting gene constellation effects (Macken et al., 2006;Varich et
78
al., 2008;Wille et al., 2013). Here we examined the growth of H5 reassortants with the
79
PB1m5 allele with or without a chimeric neuraminidase. MDCK cells infected at a
80
multiplicity of infection of 0.01 were monitored by plaque assay (Fig. 1b). Plaque assays
81
were performed on MDCK cells incubated for 72 hours at 33°C with an EMEM, 0.8%
82
agarose overlay and 1 µg/ml of TPCK trypsin. The Pm5Nwt and Pm5NCh viruses reached
83
higher peak titers than analogous viruses containing the PB1wt allele. The presence of the
84
chimeric NA and the PB1m5 allele together resulted in the highest titers among the four
85
viruses (Fig. 1b).
86
The amount of HA produced from vaccine strains is important because it is the
87
main antigenic component of inactivated vaccines. The protein profiles of each of the
88
strains were assessed. Virus stocks were quantified using the Virus Counter and eggs
89
were inoculated with 104 virus particles. After 48 hours 36mL of allantoic fluid was
90
harvested and virus was pelleted at 30 000 rpm for 90 minutes in a Beckman SW32Ti
91
rotor. The washed pellets were then purified through a sucrose cushion, pelleted and
92
resuspended in 0.5mL PBS. The purified virus was subjected to deglycosylation and the
93
resultant HA1 and HA2 fragments separated by SDS-PAGE under reducing conditions
94
(Fig. 2a). Deglycosylation has previously been shown to improve the quantification of
95
HA (Harvey et al., 2012). The protein bands were stained and the density of the bands
96
used to determine the percentage of total protein. The viral bands were quantified and the
97
proportion of HA calculated. The viruses containing the chimeric NA (PwtNCh and
5
98
Pm5NCh) had a slightly higher proportion of HA than the viruses with the wild-type NA
99
(Fig. 2b).
100
Total protein content for each of the reassortants was determined using a Pierce
101
BCA Protein Assay Kit (Catalog #23225, Thermo Fisher Scientific, Rockford, IL).
102
Measurements from three independent virus amplifications resulted in similar yields of
103
approximately 200 µg mL-1 for the virus with the A/VN/1203 surface proteins in a PR/8
104
background (PwtNwt) and the viruses with either the chimeric NA (PwtNCh) or the PB1m5
105
allele (Pm5Nwt). The yield of total proteins for the virus containing both the chimeric NA
106
and PB1m5 alleles (Pm5NCh) was significantly higher at 1200 µg/mL (Fig. 2c).
107
Modulation of hemagglutinin and neuraminidase activities has been shown to be
108
important for viral growth (Mitnaul et al., 2000;Wagner et al., 2000) and protein yield
109
(Adamo et al., 2009;Jing et al., 2012). We previously showed that viruses with wild-type
110
or chimeric A/VN/1203 NA (JV20 and JV24 respectively) had similar neuraminidase
111
activity (Adamo et al., 2009). We assessed the analogous viruses produced in this work
112
(PwtNwt and PwtNCh) along with the viruses containing the PB1M5 allele (Pm5Nwt and
113
Pm5NCh). Three independent sucrose purified preparations for each virus were serially
114
diluted and assayed for neuraminidase activity as previously described (Sultana et al.,
115
2011). Vibrio cholerae neuraminidase (Sigma) was used to generate a standard curve for
116
each assay from which activity was calculated. Neuraminidase activity for the PwtNwt and
117
PwtNCh viruses was similar (Fig. 2d) as previously observed for the analogous JV20 and
118
JV24 viruses (Adamo et al., 2009). The average neuraminidase activity was higher for
119
the Pm5NCh virus containing both the chimeric NA segment and the PB1m5. It has been
120
shown that replacement of the non-coding region of the NA segment of another H5N1
6
121
strain with a PR/8 backbone also improved growth and HA content through increased
122
packaging of the NA segment (Pan et al., 2012).
123
Here we increase protein yield in eggs by changing the PB1 gene and NA gene of
124
a reassortant H5N1 vaccine candidate strain. This work builds on previous work
125
improving the backbone segments of donor viruses used for vaccine candidate strain
126
production. A small number of donor viruses with high protein yield and good growth
127
characteristics are used to produce vaccine candidate strains. However, introduction of
128
HA and NA segments from contemporary strains sometimes results in reduced growth
129
and protein yield. Amino acids common to PB1 segments from circulating strains that
130
had reassorted with the HA and NA into vaccine candidate strains have previously been
131
shown to improve the growth of an H3N2 reassortant when they were introduced to the
132
backbone PB1 segment from PR/8 (Plant et al., 2012). Here we show that these
133
mutations also enhance growth of a reassortant containing HA and NA segments from an
134
H5N1 strain. We also recapitulate the introduction of an altered stalk region in the
135
neuraminidase. This by itself resulted in a slight increase in the proportion of HA
136
produced from each virus. However when the chimeric HA was present in combination
137
with the PB1 mutations an increase in both neuraminidase activity and total protein
138
content was observed. Because neither neuraminidase activity nor protein content
139
increased when only one of the mutant segments was present we suggest that this result is
140
due to a beneficial gene constellation effect. It will be of interest to examine the
141
mechanism behind this effect and determine if it is at the nucleotide or protein level.
142
Regardless of the mechanism this particular gene constellation effect can be harnessed to
143
improve virus growth and protein yield in vaccine manufacture.
7
144 145 146
Acknowledgments
147
We would like to thank ViroCyt personnel for providing technical assistance with the
148
Virus Counter, and Laura Couzens and Maryna Eichelberger for assistance and reagents
149
for the neuraminidase assay. This work was supported in part by the Biomedical
150
Advanced Research and Development Authority, Department of Health and Human
151
Services.
152
8
153 154 155 156 157 158
Reference List Abt, M., de, J. J., Laue, M. & Wolff, T. (2011).Improvement of H5N1 influenza vaccine viruses: influence of internal gene segments of avian and human origin on production and hemagglutinin content. Vaccine 29, 5153-5162.
159 160 161
Adamo, J. E., Liu, T., Schmeisser, F. & Ye, Z. (2009).Optimizing viral protein yield of influenza virus strain A/Vietnam/1203/2004 by modification of the neuraminidase gene. J Virol 83, 4023-4029.
162 163 164
Baigent, S. J. & McCauley, J. W. (2001).Glycosylation of haemagglutinin and stalklength of neuraminidase combine to regulate the growth of avian influenza viruses in tissue culture. Virus Res 79, 177-185.
165 166 167
Bourret, V., Lyall, J., Ducatez, M. F., Guerin, J. L. & Tiley, L. (2012).Development of an improved polykaryon-based influenza virus rescue system. BMC Biotechnol 12, 69.
168 169 170 171
Cobbin, J. C., Verity, E. E., Gilbertson, B. P., Rockman, S. P. & Brown, L. E. (2013).The source of the PB1 gene in influenza vaccine reassortants selectively alters the hemagglutinin content of the resulting seed virus. J Virol 87, 55775585.
172 173 174
Giesendorf, B., Bosch, F. X., Orlich, M., Scholtissek, C. & Rott, R. (1986).Studies on the temperature sensitivity of influenza A virus reassortants nonpathogenic for chicken. Virus Res 5, 27-42.
175 176 177 178
Harvey, R., Hamill, M., Robertson, J. S., Minor, P. D., Vodeiko, G. M., Weir, J. P., Takahashi, H., Harada, Y., Itamura, S., Bamford, P., Dalla, P. T. & Engelhardt, O. G. (2012).Application of deglycosylation to SDS PAGE analysis improves calibration of influenza antigen standards. Biologicals 40, 96-99.
179 180 181
Hoffmann, E., Neumann, G., Kawaoka, Y., Hobom, G. & Webster, R. G. (2000).A DNA transfection system for generation of influenza A virus from eight plasmids. Proc Natl Acad Sci U S A 97, 6108-6113.
182 183 184 185
Jing, X., Phy, K., Li, X. & Ye, Z. (2012).Increased hemagglutinin content in a reassortant 2009 pandemic H1N1 influenza virus with chimeric neuraminidase containing donor A/Puerto Rico/8/34 virus transmembrane and stalk domains. Vaccine 30, 4144-4152.
186 187 188 189
Li, O. T., Chan, M. C., Leung, C. S., Chan, R. W., Guan, Y., Nicholls, J. M. & Poon, L. L. (2009).Full factorial analysis of mammalian and avian influenza polymerase subunits suggests a role of an efficient polymerase for virus adaptation. PLoS One 4, e5658.
9
190 191 192
Macken, C. A., Webby, R. J. & Bruno, W. J. (2006).Genotype turnover by reassortment of replication complex genes from avian influenza A virus. J Gen Virol 87, 2803-2815.
193 194 195 196
Mitnaul, L. J., Matrosovich, M. N., Castrucci, M. R., Tuzikov, A. B., Bovin, N. V., Kobasa, D. & Kawaoka, Y. (2000).Balanced hemagglutinin and neuraminidase activities are critical for efficient replication of influenza A virus. J Virol 74, 6015-6020.
197 198 199
Ngai, K. L., Chan, M. C. & Chan, P. K. (2013).Replication and transcription activities of ribonucleoprotein complexes reconstituted from avian H5N1, H1N1pdm09 and H3N2 influenza A viruses. PLoS One 8, e65038.
200 201 202 203
Pan, W., Dong, Z., Meng, W., Zhang, W., Li, T., Li, C., Zhang, B. & Chen, L. (2012).Improvement of influenza vaccine strain A/Vietnam/1194/2004 (H5N1) growth with the neuraminidase packaging sequence from A/Puerto Rico/8/34. Hum Vaccin Immunother 8, 252-259.
204 205 206
Plant, E. P., Liu, T. M., Xie, H. & Ye, Z. (2012).Mutations to A/Puerto Rico/8/34 PB1 gene improves seasonal reassortant influenza A virus growth kinetics. Vaccine 31, 207-212.
207 208 209 210 211
Rudneva, I. A., Timofeeva, T. A., Shilov, A. A., Kochergin-Nikitsky, K. S., Varich, N. L., Ilyushina, N. A., Gambaryan, A. S., Krylov, P. S. & Kaverin, N. V. (2007).Effect of gene constellation and postreassortment amino acid change on the phenotypic features of H5 influenza virus reassortants. Arch Virol 152, 11391145.
212 213 214
Sultana, I., Gao, J., Markoff, L. & Eichelberger, M. C. (2011).Influenza neuraminidase-inhibiting antibodies are induced in the presence of zanamivir. Vaccine 29, 2601-2606.
215 216 217 218
Varich, N. L., Gitelman, A. K., Shilov, A. A., Smirnov, Y. A. & Kaverin, N. V. (2008).Deviation from the random distribution pattern of influenza A virus gene segments in reassortants produced under non-selective conditions. Arch Virol 153, 1149-1154.
219 220 221 222
Wagner, R., Wolff, T., Herwig, A., Pleschka, S. & Klenk, H. D. (2000).Interdependence of hemagglutinin glycosylation and neuraminidase as regulators of influenza virus growth: a study by reverse genetics. J Virol 74, 6316-6323.
223 224 225
Wanitchang, A., Kramyu, J. & Jongkaewwattana, A. (2010).Enhancement of reverse genetics-derived swine-origin H1N1 influenza virus seed vaccine growth by inclusion of indigenous polymerase PB1 protein. Virus Res 147, 145-148.
10
226 227 228
Wille, M., Tolf, C., Avril, A., Latorre-Margalef, N., Wallerstrom, S., Olsen, B. & Waldenstrom, J. (2013).Frequency and patterns of reassortment in natural influenza A virus infection in a reservoir host. Virology 443, 150-160.
229 230 231 232 233 234
Zhou, H., Yu, Z., Hu, Y., Tu, J., Zou, W., Peng, Y., Zhu, J., Li, Y., Zhang, A., Yu, Z., Ye, Z., Chen, H. & Jin, M. (2009).The special neuraminidase stalk-motif responsible for increased virulence and pathogenesis of H5N1 influenza A virus. PLoS One 4, e6277.
235
11
236
Figure Legends
237 238
Fig. 1. Virus Characterization. (a) Virus Plaques on MDCK cells. All viruses have a
239
PR/8 backbone with the A/VN/1203 HA. Viruses with the PB1wt and PB1m5 alleles and
240
the A/VN/1203 NA are labelled PwtNwt and Pm5Nwt respectively. Viruses with the PB1wt
241
and PB1m5 alleles and the chimeric PR/8- A/VN/1203 NA are labelled PwtNCh and Pm5NCh
242
respectively (see text for details). (b) Multi-step growth curves of viruses on MDCK
243
cells. Virus titers from three independent experiments measured by plaque assay are
244
expressed as an average with the standard deviation.
245 246
Fig. 2. Virus Protein Yield and Enzyme Activity. (a) Total protein yield from virus
247
grown in allantoic fluid for 48 hours and banded on a sucrose cushion. Virus samples
248
treated with the deglycosylation enzyme PNGaseF were analyzed on SDS-PAGE. (b)
249
Virus protein content determined by micro-BCA assay. The results are the average with
250
standard error from three independent purifications. (c) The amount of HA as a
251
percentage of total protein is shown as the average with standard deviation from three
252
independent virus purifications. (d) Neuraminidase activity of viruses. The values
253
shown are the average and standard error measurements of three independent virus
254
preparations purified by ultracentrifugation through a sucrose cushion. Viruses are
255
labelled as in Figure 1.
12
256
Table 1. Virus Characteristics. The virus names and corresponding gene segments are
257
indicated. The HA titer of the virus stock is shown. The average mean death time in
258
hours (with the range) is shown.
259 Virus Name
PB1
NA
HA Titer
MDT
V8
PB1WT
PR8
512
90 (84-96)
V1
PB1M5
PR8
1024
84 (72-96)
PV8
PB1WT
PR8/VN1203
512
87 (60-96)
PV1
PB1M5
PR8/VN1203
1024
81 (72-96)
260 261
13
Figure 1 Click here to download high resolution image
Figure 2 Click here to download high resolution image
Journal of General Virology Chimeric NA and mutant PB1 gene constellation improves growth and yield of H5N1 vaccine candidate virus --Manuscript Draft-Manuscript Number:
JGV-D-14-00166R1
Full Title:
Chimeric NA and mutant PB1 gene constellation improves growth and yield of H5N1 vaccine candidate virus
Short Title:
Improving Influenza Vaccine Virus Yield
Article Type:
Short Communication
Section/Category:
Animal - Negative-strand RNA Viruses
Corresponding Author:
Zhiping Ye, M.D., Ph.D. US Food and Drug Administration UNITED STATES
First Author:
Ewan P. Plant, Ph.D.
Order of Authors:
Ewan P. Plant, Ph.D. Zhiping Ye, M.D., Ph.D.
Abstract:
We previously showed that a mutated PB1 gene improved the growth kinetics of a H3N2 influenza reassortant. Here we show that the same mutations improve the growth kinetics of a virus containing the A/VietNam/1203/2004 (H5N1) HA and NA. Total protein yield and neuraminidase activity were increased when a chimeric NA was included. These increases indicate that the synergistic effect is due to the gene constellation containing both the altered PB1 gene and the chimeric NA gene.
Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation
Manuscript Including References (Word document) Click here to download Manuscript Including References (Word document): Plant & Ye JGV revised2.doc
1
Chimeric NA and mutant PB1 gene constellation improves growth and yield of
2
H5N1 vaccine candidate virus
3 4
Running title: Improving Influenza Vaccine Virus Yield
5 6
Contents Category: Animal, RNA viruses
7 8
Ewan P. Plant* and Zhiping Ye*
9 10
Laboratory of Respiratory Viral Diseases, Division of Viral Products, Office of Vaccine
11
Research and Review, United States Food and Drug Administration, Silver Spring,
12
Maryland 20993, United States of America
13 14
* Corresponding authors. Tel.: +1 240 402 6733, Fax: +1 301 595 1460
15
Email addresses: [email protected]; [email protected]
16 17 18
Word Count (summary & main text): 1810
19
Summary
20
We previously showed that a mutated PB1 gene improved the growth kinetics of a H3N2
21
influenza reassortant. Here we show that the same mutations improve the growth kinetics
22
of a virus containing the A/VietNam/1203/2004 (H5N1) HA and NA. Total protein yield
23
and neuraminidase activity were increased when a chimeric NA was included. These
24
increases indicate that the synergistic effect is due to the gene constellation containing
25
both the altered PB1 gene and the chimeric NA gene.
26 27
Keywords
28
Influenza, vaccine, polymerase, protein yield, antigenic shift
29 30
2
31
The object of this work is to improve the genomic backbone segments used in
32
vaccine candidate viruses while avoiding detrimental gene constellation effects. It is not
33
known what attributes from each segment affect the gene constellation or reassortment
34
but it is desirable that vaccine seed strains containing contemporary HA and NA
35
segments in the context of known backbone segments can be recovered. It is also
36
desirable that those strains replicate well and that satisfactory protein yields can be
37
harvested. We have previously shown that five mutations to the PB1 gene of the high
38
growth backbone virus PR/8 improved the growth kinetics of a reassortant containing the
39
HA and NA segments from an H3N2 virus (Plant et al., 2012). These mutations (G180E,
40
S216G, S361R, Q621R and N654S) were not selected based on any structural or
41
functional information. The five mutations made to the PB1 gene (PB1m5) were based on
42
sequence variations found in vaccine strain reassortants. We have also described the
43
improved growth and protein yield of a reassortant containing the H5N1
44
A/Vietnam/1203/2004 (A/VN/1203) HA segment and a chimeric NA segment comprised
45
of the carboxy terminus (non-coding region and first 72 amino acids) from the
46
A/VN/1203 strain with the amino terminus from PR/8 (Adamo et al., 2009). The stalk
47
region of the NA protein differs between different influenza virus strains in both length
48
and amino acid identity and has been shown to affect growth, protein yield and
49
pathogenesis (Adamo et al., 2009;Baigent & McCauley, 2001;Zhou et al., 2009). Here
50
we continue these studies by creating reassortants containing PB1wt or PB1m5 and the
51
A/VN/1203 HA with either the wild-type (NAwt) or chimeric (NACh) NA from
52
A/VN/1203.
3
53
Viruses were generated using the eight plasmid reverse genetic system (Hoffmann
54
et al., 2000). The polybasic cleavage site of the A/VN/1203 HA had previously been
55
removed (Adamo et al., 2009). Co-cultured 293T and Madin-Darby canine kidney
56
(MDCK) cells were transfected using the method of Bourret et al., (Bourret et al., 2012).
57
Culture supernatant was used to infect eggs and viruses containing either the wild-type
58
(PB1wt) or mutant (PB1m5) PB1, in conjunction with either the wild-type (NAwt) or
59
chimeric (NACh) NA from A/VN/1203, were recovered. The HA, NA and PB1 genes
60
were sequenced and did not contain any additional mutations. Hemagglutination titers
61
were comparable between the viruses (Table 1) and all the viruses produced large plaques
62
on MDCK cells (Fig. 1a). The longer stalk region of the H5N1 neuraminidase has been
63
shown to contribute to pathogenicity (Zhou et al., 2009). Here we confirm that removing
64
this region reduces pathogenicity, with or without the PB1M5 allele, by determining mean
65
death time (MDT). 106 virus particles were inoculated into the allantoic fluid of eight
66
eggs which were then incubated at 35°C and candled at 12 hour intervals. Eggs were
67
scored as alive or dead and the time of death recorded. MDT was obtained by
68
multiplying the number of embryos dead at each interval post inoculation by the hours
69
post inoculation and dividing by the total number of embryos inoculated. The MDT range
70
was similar for all viruses (Table 1).
71
Optimal polymerase activity is required for efficient replication (Giesendorf et al.,
72
1986;Li et al., 2009;Ngai et al., 2013). PB1 from a seasonal strain or with targeted
73
mutations has been shown to improve growth or HA yield of H3 reassortants (Cobbin et
74
al., 2013;Plant et al., 2012), however mixed results have been observed for H5
75
reassortants (Abt et al., 2011;Rudneva et al., 2007;Wanitchang et al., 2010). All three
4
76
polymerase segments reassort together more frequently in experimental settings than
77
natural reassortment suggesting gene constellation effects (Macken et al., 2006;Varich et
78
al., 2008;Wille et al., 2013). Here we examined the growth of H5 reassortants with the
79
PB1m5 allele with or without a chimeric neuraminidase. MDCK cells infected at a
80
multiplicity of infection of 0.01 were monitored by plaque assay (Fig. 1b). Plaque assays
81
were performed on MDCK cells incubated for 72 hours at 33°C with an EMEM, 0.8%
82
agarose overlay and 1 µg/ml of TPCK trypsin. The Pm5Nwt and Pm5NCh viruses reached
83
higher peak titers than analogous viruses containing the PB1wt allele. The presence of the
84
chimeric NA and the PB1m5 allele together resulted in the highest titers among the four
85
viruses (Fig. 1b).
86
The amount of HA produced from vaccine strains is important because it is the
87
main antigenic component of inactivated vaccines. The protein profiles of each of the
88
strains were assessed. Virus stocks were quantified using the Virus Counter and eggs
89
were inoculated with 104 virus particles. After 48 hours 36mL of allantoic fluid was
90
harvested and virus was pelleted at 30 000 rpm for 90 minutes in a Beckman SW32Ti
91
rotor. The washed pellets were then purified through a sucrose cushion, pelleted and
92
resuspended in 0.5mL PBS. The purified virus was subjected to deglycosylation and the
93
resultant HA1 and HA2 fragments separated by SDS-PAGE under reducing conditions
94
(Fig. 2a). Deglycosylation has previously been shown to improve the quantification of
95
HA (Harvey et al., 2012). The protein bands were stained and the density of the bands
96
used to determine the percentage of total protein. The viral bands were quantified and the
97
proportion of HA calculated. The viruses containing the chimeric NA (PwtNCh and
5
98
Pm5NCh) had a slightly higher proportion of HA than the viruses with the wild-type NA
99
(Fig. 2b).
100
Total protein content for each of the reassortants was determined using a Pierce
101
BCA Protein Assay Kit (Catalog #23225, Thermo Fisher Scientific, Rockford, IL).
102
Measurements from three independent virus amplifications resulted in similar yields of
103
approximately 200 µg mL-1 for the virus with the A/VN/1203 surface proteins in a PR/8
104
background (PwtNwt) and the viruses with either the chimeric NA (PwtNCh) or the PB1m5
105
allele (Pm5Nwt). The yield of total proteins for the virus containing both the chimeric NA
106
and PB1m5 alleles (Pm5NCh) was significantly higher at 1200 µg/mL (Fig. 2c).
107
Modulation of hemagglutinin and neuraminidase activities has been shown to be
108
important for viral growth (Mitnaul et al., 2000;Wagner et al., 2000) and protein yield
109
(Adamo et al., 2009;Jing et al., 2012). We previously showed that viruses with wild-type
110
or chimeric A/VN/1203 NA (JV20 and JV24 respectively) had similar neuraminidase
111
activity (Adamo et al., 2009). We assessed the analogous viruses produced in this work
112
(PwtNwt and PwtNCh) along with the viruses containing the PB1M5 allele (Pm5Nwt and
113
Pm5NCh). Three independent sucrose purified preparations for each virus were serially
114
diluted and assayed for neuraminidase activity as previously described (Sultana et al.,
115
2011). Vibrio cholerae neuraminidase (Sigma) was used to generate a standard curve for
116
each assay from which activity was calculated. Neuraminidase activity for the PwtNwt and
117
PwtNCh viruses was similar (Fig. 2d) as previously observed for the analogous JV20 and
118
JV24 viruses (Adamo et al., 2009). The average neuraminidase activity was higher for
119
the Pm5NCh virus containing both the chimeric NA segment and the PB1m5. It has been
120
shown that replacement of the non-coding region of the NA segment of another H5N1
6
121
strain with a PR/8 backbone also improved growth and HA content through increased
122
packaging of the NA segment (Pan et al., 2012).
123
Here we increase protein yield in eggs by changing the PB1 gene and NA gene of
124
a reassortant H5N1 vaccine candidate strain. This work builds on previous work
125
improving the backbone segments of donor viruses used for vaccine candidate strain
126
production. A small number of donor viruses with high protein yield and good growth
127
characteristics are used to produce vaccine candidate strains. However, introduction of
128
HA and NA segments from contemporary strains sometimes results in reduced growth
129
and protein yield. Amino acids common to PB1 segments from circulating strains that
130
had reassorted with the HA and NA into vaccine candidate strains have previously been
131
shown to improve the growth of an H3N2 reassortant when they were introduced to the
132
backbone PB1 segment from PR/8 (Plant et al., 2012). Here we show that these
133
mutations also enhance growth of a reassortant containing HA and NA segments from an
134
H5N1 strain. We also recapitulate the introduction of an altered stalk region in the
135
neuraminidase. This by itself resulted in a slight increase in the proportion of HA
136
produced from each virus. However when the chimeric HA was present in combination
137
with the PB1 mutations an increase in both neuraminidase activity and total protein
138
content was observed. Because neither neuraminidase activity nor protein content
139
increased when only one of the mutant segments was present we suggest that this result is
140
due to a beneficial gene constellation effect. It will be of interest to examine the
141
mechanism behind this effect and determine if it is at the nucleotide or protein level.
142
Regardless of the mechanism this particular gene constellation effect can be harnessed to
143
improve virus growth and protein yield in vaccine manufacture.
7
144 145 146
Acknowledgments
147
We would like to thank ViroCyt personnel for providing technical assistance with the
148
Virus Counter, and Laura Couzens and Maryna Eichelberger for assistance and reagents
149
for the neuraminidase assay. This work was supported in part by the Biomedical
150
Advanced Research and Development Authority, Department of Health and Human
151
Services.
152
8
153 154 155 156 157 158
Reference List Abt, M., de, J. J., Laue, M. & Wolff, T. (2011).Improvement of H5N1 influenza vaccine viruses: influence of internal gene segments of avian and human origin on production and hemagglutinin content. Vaccine 29, 5153-5162.
159 160 161
Adamo, J. E., Liu, T., Schmeisser, F. & Ye, Z. (2009).Optimizing viral protein yield of influenza virus strain A/Vietnam/1203/2004 by modification of the neuraminidase gene. J Virol 83, 4023-4029.
162 163 164
Baigent, S. J. & McCauley, J. W. (2001).Glycosylation of haemagglutinin and stalklength of neuraminidase combine to regulate the growth of avian influenza viruses in tissue culture. Virus Res 79, 177-185.
165 166 167
Bourret, V., Lyall, J., Ducatez, M. F., Guerin, J. L. & Tiley, L. (2012).Development of an improved polykaryon-based influenza virus rescue system. BMC Biotechnol 12, 69.
168 169 170 171
Cobbin, J. C., Verity, E. E., Gilbertson, B. P., Rockman, S. P. & Brown, L. E. (2013).The source of the PB1 gene in influenza vaccine reassortants selectively alters the hemagglutinin content of the resulting seed virus. J Virol 87, 55775585.
172 173 174
Giesendorf, B., Bosch, F. X., Orlich, M., Scholtissek, C. & Rott, R. (1986).Studies on the temperature sensitivity of influenza A virus reassortants nonpathogenic for chicken. Virus Res 5, 27-42.
175 176 177 178
Harvey, R., Hamill, M., Robertson, J. S., Minor, P. D., Vodeiko, G. M., Weir, J. P., Takahashi, H., Harada, Y., Itamura, S., Bamford, P., Dalla, P. T. & Engelhardt, O. G. (2012).Application of deglycosylation to SDS PAGE analysis improves calibration of influenza antigen standards. Biologicals 40, 96-99.
179 180 181
Hoffmann, E., Neumann, G., Kawaoka, Y., Hobom, G. & Webster, R. G. (2000).A DNA transfection system for generation of influenza A virus from eight plasmids. Proc Natl Acad Sci U S A 97, 6108-6113.
182 183 184 185
Jing, X., Phy, K., Li, X. & Ye, Z. (2012).Increased hemagglutinin content in a reassortant 2009 pandemic H1N1 influenza virus with chimeric neuraminidase containing donor A/Puerto Rico/8/34 virus transmembrane and stalk domains. Vaccine 30, 4144-4152.
186 187 188 189
Li, O. T., Chan, M. C., Leung, C. S., Chan, R. W., Guan, Y., Nicholls, J. M. & Poon, L. L. (2009).Full factorial analysis of mammalian and avian influenza polymerase subunits suggests a role of an efficient polymerase for virus adaptation. PLoS One 4, e5658.
9
190 191 192
Macken, C. A., Webby, R. J. & Bruno, W. J. (2006).Genotype turnover by reassortment of replication complex genes from avian influenza A virus. J Gen Virol 87, 2803-2815.
193 194 195 196
Mitnaul, L. J., Matrosovich, M. N., Castrucci, M. R., Tuzikov, A. B., Bovin, N. V., Kobasa, D. & Kawaoka, Y. (2000).Balanced hemagglutinin and neuraminidase activities are critical for efficient replication of influenza A virus. J Virol 74, 6015-6020.
197 198 199
Ngai, K. L., Chan, M. C. & Chan, P. K. (2013).Replication and transcription activities of ribonucleoprotein complexes reconstituted from avian H5N1, H1N1pdm09 and H3N2 influenza A viruses. PLoS One 8, e65038.
200 201 202 203
Pan, W., Dong, Z., Meng, W., Zhang, W., Li, T., Li, C., Zhang, B. & Chen, L. (2012).Improvement of influenza vaccine strain A/Vietnam/1194/2004 (H5N1) growth with the neuraminidase packaging sequence from A/Puerto Rico/8/34. Hum Vaccin Immunother 8, 252-259.
204 205 206
Plant, E. P., Liu, T. M., Xie, H. & Ye, Z. (2012).Mutations to A/Puerto Rico/8/34 PB1 gene improves seasonal reassortant influenza A virus growth kinetics. Vaccine 31, 207-212.
207 208 209 210 211
Rudneva, I. A., Timofeeva, T. A., Shilov, A. A., Kochergin-Nikitsky, K. S., Varich, N. L., Ilyushina, N. A., Gambaryan, A. S., Krylov, P. S. & Kaverin, N. V. (2007).Effect of gene constellation and postreassortment amino acid change on the phenotypic features of H5 influenza virus reassortants. Arch Virol 152, 11391145.
212 213 214
Sultana, I., Gao, J., Markoff, L. & Eichelberger, M. C. (2011).Influenza neuraminidase-inhibiting antibodies are induced in the presence of zanamivir. Vaccine 29, 2601-2606.
215 216 217 218
Varich, N. L., Gitelman, A. K., Shilov, A. A., Smirnov, Y. A. & Kaverin, N. V. (2008).Deviation from the random distribution pattern of influenza A virus gene segments in reassortants produced under non-selective conditions. Arch Virol 153, 1149-1154.
219 220 221 222
Wagner, R., Wolff, T., Herwig, A., Pleschka, S. & Klenk, H. D. (2000).Interdependence of hemagglutinin glycosylation and neuraminidase as regulators of influenza virus growth: a study by reverse genetics. J Virol 74, 6316-6323.
223 224 225
Wanitchang, A., Kramyu, J. & Jongkaewwattana, A. (2010).Enhancement of reverse genetics-derived swine-origin H1N1 influenza virus seed vaccine growth by inclusion of indigenous polymerase PB1 protein. Virus Res 147, 145-148.
10
226 227 228
Wille, M., Tolf, C., Avril, A., Latorre-Margalef, N., Wallerstrom, S., Olsen, B. & Waldenstrom, J. (2013).Frequency and patterns of reassortment in natural influenza A virus infection in a reservoir host. Virology 443, 150-160.
229 230 231 232 233 234
Zhou, H., Yu, Z., Hu, Y., Tu, J., Zou, W., Peng, Y., Zhu, J., Li, Y., Zhang, A., Yu, Z., Ye, Z., Chen, H. & Jin, M. (2009).The special neuraminidase stalk-motif responsible for increased virulence and pathogenesis of H5N1 influenza A virus. PLoS One 4, e6277.
235
11
236
Figure Legends
237 238
Fig. 1. Virus Characterization. (a) Virus Plaques on MDCK cells. All viruses have a
239
PR/8 backbone with the A/VN/1203 HA. Viruses with the PB1wt and PB1m5 alleles and
240
the A/VN/1203 NA are labelled PwtNwt and Pm5Nwt respectively. Viruses with the PB1wt
241
and PB1m5 alleles and the chimeric PR/8- A/VN/1203 NA are labelled PwtNCh and Pm5NCh
242
respectively (see text for details). (b) Multi-step growth curves of viruses on MDCK
243
cells. Virus titers from three independent experiments measured by plaque assay are
244
expressed as an average with the standard deviation.
245 246
Fig. 2. Virus Protein Yield and Enzyme Activity. (a) Total protein yield from virus
247
grown in allantoic fluid for 48 hours and banded on a sucrose cushion. Virus samples
248
treated with the deglycosylation enzyme PNGaseF were analyzed on SDS-PAGE. (b)
249
Virus protein content determined by micro-BCA assay. The results are the average with
250
standard error from three independent purifications. (c) The amount of HA as a
251
percentage of total protein is shown as the average with standard deviation from three
252
independent virus purifications. (d) Neuraminidase activity of viruses. The values
253
shown are the average and standard error measurements of three independent virus
254
preparations purified by ultracentrifugation through a sucrose cushion. Viruses are
255
labelled as in Figure 1.
12
256
Table 1. Virus Characteristics. The virus names and corresponding gene segments are
257
indicated. The HA titer of the virus stock is shown. The average mean death time in
258
hours (with the range) is shown.
259 Virus Name
PB1
NA
HA Titer
MDT
V8
PB1WT
PR8
512
90 (84-96)
V1
PB1M5
PR8
1024
84 (72-96)
PV8
PB1WT
PR8/VN1203
512
87 (60-96)
PV1
PB1M5
PR8/VN1203
1024
81 (72-96)
260 261
13
Figure 1 Click here to download high resolution image
Figure 2 Click here to download high resolution image
