196
Occasional Survey
USING THE GENETICS OF INFLUENZA VIRUS TO MAKE LIVE ATTENUATED VACCINES D. A. Division
J. TYRRELL
of Communicable Diseases, Clinical Research Centre, Watford Road, Harrow, Middlesex HA 1 3UJ
Everyone forgets how long and hard the work really important applications become applicable. LEWIS THOMAS. The Lives
must
of a
be before the
Cell; p. 117. 1974.
virus exercises several different functions-gaining entry to the host, replication, overcoming host defences, and damaging cells-which require the use of different parts of its genome. Temperature-sensitive (ts) mutations preventing normal function at the normal murine body-temperature have been introduced at several sites in the genome of a murine influenza virus and in each case the virus became attenuated or avirulent, even though some mutants multiplied to some extent. Presumably each genetic lesion prevented some function necessary for the virus to spread and damage enough cells to make the mouse ill. Introducing a new gene at one of several loci can also attenuate an influenza virus of chickens.2 This finding seems to offer a new approach to making attenuated live virus vaccines. Traditionally viruses which cause disease in man have been adapted by serial passage to grow well in a laboratory animal or tissue culture system which is a convenient source of a vaccine, such as a chick embryo. In many instances the adapted virus is no longer pathogenic for man and can be used as a live vaccine--e.g., yellow fever, measles, or rubella. Adaptation is gradual and seems to result from the selection of mutations which make the virus grow faster in the laboratory but make it less able to produce symptoms in man. The new approach to attenuation is to expose a virus,—e.g., influenza virus-to a mutagen and select from the progeny a ts strain which grows less well in man than in the parent. Workers at the National Institutes of Health have shown that in ts mutants produced by this means the degree of attenuation for man depends on the "cut-off" temperature,-i.e., the degree of temperature sensitivity.3·4 Mutants unable to grow at lower temperatures may be so attenuated that they do not multiply and so are non-immunogenic. Strains with a less reduced cut-off temperature multiply and immunise but cause few symptoms. This type of attenuation can be produced by ts mutation at more than one genetic site. Mutant IE with an intermediate degree of temperature sensitivity, which is immunogenic in adult volunteers and causes negligible symptoms, has been evaluated in some detail. Because the ts gene does not control the structure of haemagglutinin or neuraminidase antigens, the ts property can be transferred to viruses of other serotypes by recombination (gene reassortment). On each occasion the recombinant has been attenuated. However, there are difficulties .5 The viruses are attenuated and immunise when given to adults who have antineuraminidase antibody but cause unaccep-
To
produce disease
a
table symptoms when given to adults or children without such antibodies,3,6 presumably because there is free multiplication and reversion to the virulent wild type. Furthermore, ts mutants are produced in relatively low yield by egg or tissue cultures, and I believe that recombining them with egg-adapted strains to improve this yield would profoundly alter their degree of attenuation for man. Maassab7 has confirmed an earlier Russian finding that attenuated ts mutants appear after viruses are passaged at low temperature (thus avoiding the use of mutagens which troubles some licensing authorities) and there is preliminary evidence that these strains are more genetically stable and do not become virulent so
easily.8 Attenuating viruses by passage in a laboratory host, the approach used in Britain, requires multiple passages be effective as in the case of the PR8 and Okuda strains.9 Recombinants between these and current influenza viruses which grew well in eggs had the antigens of the current viruses, were attenuated, and behaved like live vaccines.l0,11 This approach has yielded a vaccine now marketed in Britain. But not all the recombinants are properly attenuated and attempts have thus been made to "mark" those which are not. Pathogenicity for an animal might indicate pathogenicity for man, but while this may be true for ferrets it is not for mice. 10 The ability to damage human or ferret ciliated epithelium distinguishes virulent recombinants with PR8’2 and ts strains" but not recombinants with others.14 This is no surprise-human and animal cells are different and even if the virus can grow well in human respiratory epithelium it may lack some other ability and so be attenuated. The use of ts strains has the advantage that even a few temperature-resistant particles (those likely to become virulent in the respiratory tract of volunteers) can be detected in the laboratory. to
Viruses attenuated by serial passage recently genetically quite undefined. This
until acceptable in the past,-e.g., with measles and rubella-when it was possible to do large-scale field trials before licensing a new vaccine but not now. Furthermore live influenza vaccine would be most needed in the face of an impending pandemic when there would be no time for large-scale trials. If field testing were replaced by genetic and other in-vitro studies, we could detect recombinants with the same genes as a strain which had been used successfully before. were
was
VIRUS STRUCTURE AS A GENETIC MARKER
The behaviour of a virus in its host reflects the specific peptides it produces. Most of these are found in the virus particle, but not all. It is usually said that only the antigens-i.e., the peptides of the haemagglutinin and neuraminidase-vary from strain to strain, while the other peptides remain the same, but it seems more reasonable to me that differences in characteristics such as host range and attenuation are evidence of subtle differences in the structure of components such as the matrix protein, nucleoprotein, and polymerase which are not detected by studies of the antigens. There is evidence to support this view. The matrix protein of different influenza-A viruses and recombinants may have different aminoacid sequences,15 and blocking tests in a
197 between the matrix proavian and a human virus.16 Hydrolysis and "finger-printing" the peptide fragments has also shown that the matrix protein and nucleoprotein of the attenuated recombinant WRL 105 are derived from the attenuated parent.17,18 Is a recombinant possessing its attenuated parent’s matrix and nucleoprotein itself regularly attenuated for man? I doubt it, but these two peptides might be most important in attenuation. If they are not, it will be difficult to study other peptides which are present in small amounts, such as the P peptides, or those found only in the cell. In the long run it would be better to characterise viruses by their riboncleic acid (R.N.A.)-to identify genes rather than the gene product. About eight complementation groups have been found by genetic analysis of influenza virus’9 and about eight pieces of R.N.A. can now be separated by electrophoresis.2o,21 They are apparently equally represented in the particle nucleic acid and are distinct both by specific hydrolysis with endonucleases and by hybridisation with complementary nucleic acid.22,23 Nucleic-acid pieces from a laboratory strain and a wild virus which can be distinguished by polyacrylamide-gel electrophoresis have each been shown to code for a specific protein. Analysis of the nucleic acid of a recombinant then showed what genetic information it contained and which parent it came from.24,2s The R.N.A. and peptides of ts viruses can sometimes be distinguished by these techniques.24,27 I suggest that we look now for parental strains which have been attenuated by multiple serial passages in acceptable hosts and which have R.N.A. pieces which can be separated and identified by mobility, by the pattern of oligonucleotides which are released enzymically, or by hybridisation. A number of viruses have been well tested in man and these and their recombinants could be fully defined by R.N.A. analysis. The inventory of their genes could then be related to the degree of attenuation and other properties. In time this would show if there were any pieces of nucleic acid always found in attenuated strains. If there are unpredictable interactions, or if the hasmagglutinin or neuraminidase of the wild virus has a major influence on virulence, recombinants with genes from an attenuated parent may not be attenuated. This would still not give a method for detecting a few reverted virulent particles in the presence of an excess of attenuated particles-which can be done with ts strains. On the other hand in recombinants with strains like PR8 it would be less important since a strain in which several genes determine attenuation is unlikely to return rapidly or in one step to virulence. There are already some indications that attenuation may be predicted from the viral R.N.A. Florent et al. have hybridised the R.N.A. of possible recombinant vaccines with complementary R.N.A. derived from the attenuated parent PR8. The recombinants were selected because their haemagglutinin and neuraminidase, which are coded in about 40% of the R.N.A., are different from PR8 and so complete hybridisation is impossible. However, recombinants in which the amount of hybridisable R.N.A. was equivalent to most of,the remaining R.N.A.-i.e., over 50%-were attenuated. This method shows that the recombinant has R.N.A. sequences like those of the attenuated parent, but gives no indication of which piece of R.N.A. the sequences are
radioimmunoassay distinguish
in. It will be
tein of
separated
an
fascinating to study the hybridisation of pieces but this has not yet been done
R.N.A.
with potential vaccines. All this new genetic information does not eliminate the need for volunteer experiments or clinical trials since it is unlikely that the exact degree of attenuation will be predictable. The PR8 recombinants obtained from swine influenza strains were too attenuated to be useful,29 possibly because the swine haemagglutinin and neuraminidase did not contribute to virulence to the same extent as in the epidemic strains used previously. CONCLUSION
Influenza recombinants can be harmless and immunogenic as vaccines. We need to identify them rapidly and certainly, and a combination of clinical assessment with study of the viral genome should make this possible. American workers have made progress using temperature sensitive (ts) mutant genes. Satisfactory attenuated live influenza virus vaccines have also been made with strains passed serially in laboratory hosts such as chick embryos. If such strains are to be used on a large scale, particularly in face of a pandemic, vaccine developers will have to have relatively complete information on the genetic composition of a potential vaccine strain as well as its behaviour in man. To this end it is now possible to identify some of the peptides (gene products) and most of the R.N.A. fragments (genes). It is likely that recombinants with genes derived mainly from the laboratory-adapted attenuated parent will be attenuated too. Work is now needed to establish this and to find out how to tailor strains for use in individuals with and without some basic immunity. REFERENCES
Mackenzie, J. S. Br. med. J. 1969, iii, 757. Scholtissek, C., Rott, R., Orlich, M., Harms, E., Rohde, W. Virology, 1977, 81, 74. 3. Murphy, B. R., Richman, D. D., Springs, S. B., Chanock, R. M. Postgrad. 1. 2.
med. J. 1976, 52, 381. Mills, J., Van Kirk, J., Hill, D. A., Chanock, R. M. Bull. Wld Hlth Org. 1969, 41, 599. 5. Kim, H. W., Arrobio, J. O., Brandt, C. D., Parrott, R. H., Murphy, B. R., Richman, D. D., Chanock, R. M. Pediat. Res. 1976, 10, 238. 6. Murphy, B. R., Markoff, L. J., Chanock, R. M., Douglas, R. G., Betts, R. F., Cate, T. R., Couch, R. B. Presented at I.A.B.S. Symposium on Influenza Immunization, June, 1977. 7. Maassab, H. F. J. Immun. 1969, 102, 728. 8. Spring, S. B., Maassab, H. F., Kendal, A. P., Murphy, B. R., Chanock, R. M. Virology, 1977, 77, 337. 9. Beare, A. S., Hall, T. S. Lancet, 1971, ii, 1271. 10. McCahon, D., Schild, G. C. J. gen. Virol. 1972, 15, 73. 11. McCahon, D., Beare, A. S., Stealey, V. Postgrad. med. J. 1976, 52, 389. 12. Mostow, S. R., Tyrrell, D. A. J. Arch. ges. Virusforsch. 1973, 43, 385. 13. Mostow, S. R., Flatauer, S., Pater, M., Murphy, B. R. J. infect. Dis. 1977, 4.
136, 1.
14. Hara, K., Beare, A. S., Tyrrell, D. A. J. Arch. ges. Virusforsch. 1974, 44, 227. 15. Laver, W. G., Downie, J. C. Virology, 1976, 70, 105. 16. Lecomte, J., Oxford, J. Unpublished. 17. Brand, C. M., Stealey, V. M., Rowe, J. J. gen. Virol. 1977, 36, 385. 18. Dimmock, N. J., Carver, A. S., Kennedy, S. I. T., Lee, M. R., Luscombe, S. ibid. 1977, 36, 503. 19. Sugiura, A. in The Influenza Viruses and Influenza (edited by E. D. Kil-
bourne); p. 171. London, 1975. 20. Palese, P., Schulman, J. L. J. Virol. 1976, 17, 876. 21. McGeoch, D., Fellner, P., Newton, C. Proc. natn. Acad. Sci. 1976, 73, 3045. 22. Stephenson, J. R., Hay, A. J., Skehel, J. J. J. gen. Virol. 1977, 36, 237. 23. Inglis, S. C., McGeoch, D. J., Mahy, B. W. J. Virology, 1977, 78, 522. 24. Ritchey, M. B., Palese, P., Schulman, J. L. J. Virol. 1976, 20, 307. 25. Schulman, J. L., Palese, P. ibid. 1976, 20, 248. 26. Palese, P., Ritchey, M. B. Virology, 1977, 78, 183. 27. Kendal, A. P., Cox, N. J., Murphy, B. R., Spring, S. B., Maassab, H. F. J. gen. Virol. 1977, 37, 145. 28. Florent, G., Lobmann, M., Beare, A. S., Zygraich, N. Archs Virol. 1977, 54, 19. 29. Beare, A. S. Unpublished.
Occasional Survey
USING THE GENETICS OF INFLUENZA VIRUS TO MAKE LIVE ATTENUATED VACCINES D. A. Division
J. TYRRELL
of Communicable Diseases, Clinical Research Centre, Watford Road, Harrow, Middlesex HA 1 3UJ
Everyone forgets how long and hard the work really important applications become applicable. LEWIS THOMAS. The Lives
must
of a
be before the
Cell; p. 117. 1974.
virus exercises several different functions-gaining entry to the host, replication, overcoming host defences, and damaging cells-which require the use of different parts of its genome. Temperature-sensitive (ts) mutations preventing normal function at the normal murine body-temperature have been introduced at several sites in the genome of a murine influenza virus and in each case the virus became attenuated or avirulent, even though some mutants multiplied to some extent. Presumably each genetic lesion prevented some function necessary for the virus to spread and damage enough cells to make the mouse ill. Introducing a new gene at one of several loci can also attenuate an influenza virus of chickens.2 This finding seems to offer a new approach to making attenuated live virus vaccines. Traditionally viruses which cause disease in man have been adapted by serial passage to grow well in a laboratory animal or tissue culture system which is a convenient source of a vaccine, such as a chick embryo. In many instances the adapted virus is no longer pathogenic for man and can be used as a live vaccine--e.g., yellow fever, measles, or rubella. Adaptation is gradual and seems to result from the selection of mutations which make the virus grow faster in the laboratory but make it less able to produce symptoms in man. The new approach to attenuation is to expose a virus,—e.g., influenza virus-to a mutagen and select from the progeny a ts strain which grows less well in man than in the parent. Workers at the National Institutes of Health have shown that in ts mutants produced by this means the degree of attenuation for man depends on the "cut-off" temperature,-i.e., the degree of temperature sensitivity.3·4 Mutants unable to grow at lower temperatures may be so attenuated that they do not multiply and so are non-immunogenic. Strains with a less reduced cut-off temperature multiply and immunise but cause few symptoms. This type of attenuation can be produced by ts mutation at more than one genetic site. Mutant IE with an intermediate degree of temperature sensitivity, which is immunogenic in adult volunteers and causes negligible symptoms, has been evaluated in some detail. Because the ts gene does not control the structure of haemagglutinin or neuraminidase antigens, the ts property can be transferred to viruses of other serotypes by recombination (gene reassortment). On each occasion the recombinant has been attenuated. However, there are difficulties .5 The viruses are attenuated and immunise when given to adults who have antineuraminidase antibody but cause unaccep-
To
produce disease
a
table symptoms when given to adults or children without such antibodies,3,6 presumably because there is free multiplication and reversion to the virulent wild type. Furthermore, ts mutants are produced in relatively low yield by egg or tissue cultures, and I believe that recombining them with egg-adapted strains to improve this yield would profoundly alter their degree of attenuation for man. Maassab7 has confirmed an earlier Russian finding that attenuated ts mutants appear after viruses are passaged at low temperature (thus avoiding the use of mutagens which troubles some licensing authorities) and there is preliminary evidence that these strains are more genetically stable and do not become virulent so
easily.8 Attenuating viruses by passage in a laboratory host, the approach used in Britain, requires multiple passages be effective as in the case of the PR8 and Okuda strains.9 Recombinants between these and current influenza viruses which grew well in eggs had the antigens of the current viruses, were attenuated, and behaved like live vaccines.l0,11 This approach has yielded a vaccine now marketed in Britain. But not all the recombinants are properly attenuated and attempts have thus been made to "mark" those which are not. Pathogenicity for an animal might indicate pathogenicity for man, but while this may be true for ferrets it is not for mice. 10 The ability to damage human or ferret ciliated epithelium distinguishes virulent recombinants with PR8’2 and ts strains" but not recombinants with others.14 This is no surprise-human and animal cells are different and even if the virus can grow well in human respiratory epithelium it may lack some other ability and so be attenuated. The use of ts strains has the advantage that even a few temperature-resistant particles (those likely to become virulent in the respiratory tract of volunteers) can be detected in the laboratory. to
Viruses attenuated by serial passage recently genetically quite undefined. This
until acceptable in the past,-e.g., with measles and rubella-when it was possible to do large-scale field trials before licensing a new vaccine but not now. Furthermore live influenza vaccine would be most needed in the face of an impending pandemic when there would be no time for large-scale trials. If field testing were replaced by genetic and other in-vitro studies, we could detect recombinants with the same genes as a strain which had been used successfully before. were
was
VIRUS STRUCTURE AS A GENETIC MARKER
The behaviour of a virus in its host reflects the specific peptides it produces. Most of these are found in the virus particle, but not all. It is usually said that only the antigens-i.e., the peptides of the haemagglutinin and neuraminidase-vary from strain to strain, while the other peptides remain the same, but it seems more reasonable to me that differences in characteristics such as host range and attenuation are evidence of subtle differences in the structure of components such as the matrix protein, nucleoprotein, and polymerase which are not detected by studies of the antigens. There is evidence to support this view. The matrix protein of different influenza-A viruses and recombinants may have different aminoacid sequences,15 and blocking tests in a
197 between the matrix proavian and a human virus.16 Hydrolysis and "finger-printing" the peptide fragments has also shown that the matrix protein and nucleoprotein of the attenuated recombinant WRL 105 are derived from the attenuated parent.17,18 Is a recombinant possessing its attenuated parent’s matrix and nucleoprotein itself regularly attenuated for man? I doubt it, but these two peptides might be most important in attenuation. If they are not, it will be difficult to study other peptides which are present in small amounts, such as the P peptides, or those found only in the cell. In the long run it would be better to characterise viruses by their riboncleic acid (R.N.A.)-to identify genes rather than the gene product. About eight complementation groups have been found by genetic analysis of influenza virus’9 and about eight pieces of R.N.A. can now be separated by electrophoresis.2o,21 They are apparently equally represented in the particle nucleic acid and are distinct both by specific hydrolysis with endonucleases and by hybridisation with complementary nucleic acid.22,23 Nucleic-acid pieces from a laboratory strain and a wild virus which can be distinguished by polyacrylamide-gel electrophoresis have each been shown to code for a specific protein. Analysis of the nucleic acid of a recombinant then showed what genetic information it contained and which parent it came from.24,2s The R.N.A. and peptides of ts viruses can sometimes be distinguished by these techniques.24,27 I suggest that we look now for parental strains which have been attenuated by multiple serial passages in acceptable hosts and which have R.N.A. pieces which can be separated and identified by mobility, by the pattern of oligonucleotides which are released enzymically, or by hybridisation. A number of viruses have been well tested in man and these and their recombinants could be fully defined by R.N.A. analysis. The inventory of their genes could then be related to the degree of attenuation and other properties. In time this would show if there were any pieces of nucleic acid always found in attenuated strains. If there are unpredictable interactions, or if the hasmagglutinin or neuraminidase of the wild virus has a major influence on virulence, recombinants with genes from an attenuated parent may not be attenuated. This would still not give a method for detecting a few reverted virulent particles in the presence of an excess of attenuated particles-which can be done with ts strains. On the other hand in recombinants with strains like PR8 it would be less important since a strain in which several genes determine attenuation is unlikely to return rapidly or in one step to virulence. There are already some indications that attenuation may be predicted from the viral R.N.A. Florent et al. have hybridised the R.N.A. of possible recombinant vaccines with complementary R.N.A. derived from the attenuated parent PR8. The recombinants were selected because their haemagglutinin and neuraminidase, which are coded in about 40% of the R.N.A., are different from PR8 and so complete hybridisation is impossible. However, recombinants in which the amount of hybridisable R.N.A. was equivalent to most of,the remaining R.N.A.-i.e., over 50%-were attenuated. This method shows that the recombinant has R.N.A. sequences like those of the attenuated parent, but gives no indication of which piece of R.N.A. the sequences are
radioimmunoassay distinguish
in. It will be
tein of
separated
an
fascinating to study the hybridisation of pieces but this has not yet been done
R.N.A.
with potential vaccines. All this new genetic information does not eliminate the need for volunteer experiments or clinical trials since it is unlikely that the exact degree of attenuation will be predictable. The PR8 recombinants obtained from swine influenza strains were too attenuated to be useful,29 possibly because the swine haemagglutinin and neuraminidase did not contribute to virulence to the same extent as in the epidemic strains used previously. CONCLUSION
Influenza recombinants can be harmless and immunogenic as vaccines. We need to identify them rapidly and certainly, and a combination of clinical assessment with study of the viral genome should make this possible. American workers have made progress using temperature sensitive (ts) mutant genes. Satisfactory attenuated live influenza virus vaccines have also been made with strains passed serially in laboratory hosts such as chick embryos. If such strains are to be used on a large scale, particularly in face of a pandemic, vaccine developers will have to have relatively complete information on the genetic composition of a potential vaccine strain as well as its behaviour in man. To this end it is now possible to identify some of the peptides (gene products) and most of the R.N.A. fragments (genes). It is likely that recombinants with genes derived mainly from the laboratory-adapted attenuated parent will be attenuated too. Work is now needed to establish this and to find out how to tailor strains for use in individuals with and without some basic immunity. REFERENCES
Mackenzie, J. S. Br. med. J. 1969, iii, 757. Scholtissek, C., Rott, R., Orlich, M., Harms, E., Rohde, W. Virology, 1977, 81, 74. 3. Murphy, B. R., Richman, D. D., Springs, S. B., Chanock, R. M. Postgrad. 1. 2.
med. J. 1976, 52, 381. Mills, J., Van Kirk, J., Hill, D. A., Chanock, R. M. Bull. Wld Hlth Org. 1969, 41, 599. 5. Kim, H. W., Arrobio, J. O., Brandt, C. D., Parrott, R. H., Murphy, B. R., Richman, D. D., Chanock, R. M. Pediat. Res. 1976, 10, 238. 6. Murphy, B. R., Markoff, L. J., Chanock, R. M., Douglas, R. G., Betts, R. F., Cate, T. R., Couch, R. B. Presented at I.A.B.S. Symposium on Influenza Immunization, June, 1977. 7. Maassab, H. F. J. Immun. 1969, 102, 728. 8. Spring, S. B., Maassab, H. F., Kendal, A. P., Murphy, B. R., Chanock, R. M. Virology, 1977, 77, 337. 9. Beare, A. S., Hall, T. S. Lancet, 1971, ii, 1271. 10. McCahon, D., Schild, G. C. J. gen. Virol. 1972, 15, 73. 11. McCahon, D., Beare, A. S., Stealey, V. Postgrad. med. J. 1976, 52, 389. 12. Mostow, S. R., Tyrrell, D. A. J. Arch. ges. Virusforsch. 1973, 43, 385. 13. Mostow, S. R., Flatauer, S., Pater, M., Murphy, B. R. J. infect. Dis. 1977, 4.
136, 1.
14. Hara, K., Beare, A. S., Tyrrell, D. A. J. Arch. ges. Virusforsch. 1974, 44, 227. 15. Laver, W. G., Downie, J. C. Virology, 1976, 70, 105. 16. Lecomte, J., Oxford, J. Unpublished. 17. Brand, C. M., Stealey, V. M., Rowe, J. J. gen. Virol. 1977, 36, 385. 18. Dimmock, N. J., Carver, A. S., Kennedy, S. I. T., Lee, M. R., Luscombe, S. ibid. 1977, 36, 503. 19. Sugiura, A. in The Influenza Viruses and Influenza (edited by E. D. Kil-
bourne); p. 171. London, 1975. 20. Palese, P., Schulman, J. L. J. Virol. 1976, 17, 876. 21. McGeoch, D., Fellner, P., Newton, C. Proc. natn. Acad. Sci. 1976, 73, 3045. 22. Stephenson, J. R., Hay, A. J., Skehel, J. J. J. gen. Virol. 1977, 36, 237. 23. Inglis, S. C., McGeoch, D. J., Mahy, B. W. J. Virology, 1977, 78, 522. 24. Ritchey, M. B., Palese, P., Schulman, J. L. J. Virol. 1976, 20, 307. 25. Schulman, J. L., Palese, P. ibid. 1976, 20, 248. 26. Palese, P., Ritchey, M. B. Virology, 1977, 78, 183. 27. Kendal, A. P., Cox, N. J., Murphy, B. R., Spring, S. B., Maassab, H. F. J. gen. Virol. 1977, 37, 145. 28. Florent, G., Lobmann, M., Beare, A. S., Zygraich, N. Archs Virol. 1977, 54, 19. 29. Beare, A. S. Unpublished.
