Journal
of Biological
Standardization
1975 3, 341-347
Towards a split influenza virus vaccine* C. J. M. Rondle,? M. J. Walker,? J. B. Krahnt and R. G. Bird?
A simple method is proposed whereby extracts of influenza virus can be injected intraperitoneally in mice and protect them against subsequent challenge by live virus by the intranasal route.
INTRODUCTION It is generally recognized that an attenuated or genetically modified live virus is probably the best vaccine to combat epidemics of influenza which arise from time to time. In some cases, however, live virus is contra-indicated and a ‘dead’ split vaccine would be of advantage. One criterion of such a vaccine is that it should contain those antigens giving rise to protective antibodies and be free from nucleoprotein and other materials not involved in the protective immune process. Influenza virus has been disrupted by a number of agents amongst which may be cited ether (Hoyle, 1950; Hoyle, Horne, & Waterson 1961), the anionic detergents sodium dodecyl sulphate (SDS) and sodium deoxycholate (SDC), (Laver, 1963, 1964), the enzyme bromelain (Brand & Skehel, 1972; Brand, 1973) and the non-ionic detergents Non-idet P40 (Shell) and Triton NlOl (Rohm & Haas Co., Philadelphia). These two compounds are similar if not identical in composition being based on nonylphenoxyethanol. However, Corbel & Rondle (1970) and Corbel, Rondle & Bird (1970) showed that under controlled conditions T NlOl degraded all strains of influenza virus tested in a unique way. Under the action of T NlOl outer ‘spikes’ of neuraminidase (NA) and haemagglutinin (HA) were preferentially stripped off leaving the inner virus particle ‘ghosts’ intact. The resultant mixture had NA activity, did not haemagglutinate normally susceptible chick * Received for publication 10 March 1975. t London School of Hygiene and Tropical WC1 E7HT, England.
Medicine,
Keppel
Street (Gower
Street),
London
341
C. J. M.
RONDLE
ET
AL.
cells but blocked haemagglutination-inhibiting antibody (serum blocking substance activity, SBS). Preliminary experiments showed that these mixtures were antigenic in ferrets and guinea pigs. This paper describes our attempts to separate ‘spikes’ from ‘ghosts’ and to investigate the chemical and serological properties of each fraction. MATERIALS
AND
METHODS
Virus strains used Strains used in this work were X-31 (Kilboume, 1969) said to be a recombinant H2 N2 between AZ/HongKong/68 and AO/PRS; 64b, described by Schild as A/PR8/34A/Eng/959/69 (HON2); and BAP/13. This last strain was most generously provided by Dr G. Appleyard of M.R.E. Porton and he suggested the designation A/BEL/40Singapore/i/57-PR/8/34 (HCN,). X-31 was useful for ferret and guinea-pig experiments, most of which are reported elsewhere. For the present work its low virulence for mice was disadvantageous. Strain 64b was used extensively but in our hands it was difficult to obtain ‘clean’ virus preparations for study. Moreover, its effect on mice was best assessedby lung consolidation and this tended to be subjective. BAP/13 was better for our purposes as it showed 85-95% virulence for mice 5-10 days after intranasal challenge. These present virus strains were chosen because our previous work had been done with ‘conventional’ strains and we wished to test that our methods were of general application. Virus growth and purification Viruses were grown in the allantoic cavity of lo-day-old fertile hen eggs. Allantoic fluids were harvested after 48 h incubation, clarified by centrifugation at 3000 rev/min for 20 min and the precipitate discarded. Virus was recovered by absorption and elution onto and from barium sulphate (Mituzani, 1963). Further purification was effected by sugar density gradient (SDG) centrifugation using 45-60% sucrose concentrations in a ‘Spinco’ SW30 rotor at 20 000 rev/min for 16 h. Virus degradation Virus bands recovered from SDG were suspended in phosphate-buffered saline (PBSA) containing 2% T NlOl and sedimented at 100 000 g in a ‘Spinco’ centrifuge. Supernatants were removed carefully with a syringe and needle and stored at 4 “C. Virus pellets were allowed to drain in the now inverted centrifuge tubes supported on filter pads to absorb excess moisture. Many attempts were made to remove T NlOl from the supernatant fluids. These included partition experiments with Arcton and hexane, SDG centrifugation and SDG in borate buffers (borate chelates with the hydroxy groups of T NlOl which results in a charged molecule) and iso-electric focusing under varied conditions. It was eventually discovered that the slow and careful addition with stirring of 4 vol/vol of cold (4 “C) ethanol to the cold (4 “C) supematant effected precipitation of the material present in the supernatant and left T NlOl in solution. After centrifugation at 800 g for 12 min the supematant was discarded and the precipitate washed with absolute ethanol. By the spectrophotometric method of Corbel & Rondle (1970) less than O*OOOl%T NlOl was present in this ethanol washing. Before study in the electron microscope the ‘supernatant precipitate’ was treated in the same way as the first ‘ghost’ precipitate. 342
SPLIT
INFLUENZA
VIRUS
VACCINE
Electron microscopy
Drained pellets of purified virus and drained pellets of degraded material were kept at 4 “C and processed within 1 h of preparation. The method used for making the negative stained preparations was a slight modification of that given in our earlier publication (Corbel et al., 1970). Each inverted tube was inspected to make sure that no diluent remained on the side of the tube, any present being removed by a strip of filter paper before treatment. To each tube was added 5 drops (v/v) neutralized phosphotungstic acid, using a 5 ml plastic syringe fitted with a 19G2 50/11 stainless steel needle. A fresh sterilized glass pipette was used to suspend each pellet in the negative stain and apply the suspensions to formvar-coated Smethurst new type 400 grids. Excess fluid was removed with strips of filter paper after 15-20 s and the grids allowed to dry in air in a dust-free atmosphere. Preparations were examined within 3 h of preparation using a GEC/AEI 811 electron microscope. Photographs were taken on Ilford N50 film. Polyacrylamide
gel electrophoresis
In an attempt to compare fractions obtained by us with those reported by others some experiments were done using analytical polyacrylamide electrophoresis. In some cases the method of Laver & Valentine (1969) was used employing SDS and dithiothreitol; in most others the technique of Skehel & Schild (1971) using SDS, 2-mercaptoethanol and urea was employed. Chemical analysis
Due to paucity of material little work could be done. In a few casesprotein was estimated by the method of Sutherland, Cori, Haynes & Olsen (1949). More reliance was placed on spectrophotometric observations using an Optica CF4Nl double-beam recording spectrophotometer. Serological tests
NA and HA tests were done as described by Corbel & Rondle (1970). A few SSB tests were done as control experiments and in two cases (X-31) and (64b) gel diffusion tests were done on degraded materials to show that T NlOl was releasing strain-specific materials as had previously been reported. Protection tests
Available mice, usually inbred white, but occasionally unselected hybrids, were used in batches of 10. One batch was inoculated intraperitoneally (i/p) with 0.1 ml saline per mouse. Other batches were inoculated with preparations of ‘spikes’ or ‘ghosts’. Mice were challenged intranasally (i/n) 7-10 days after ‘protection’. Usually 0~05-0*1 ml inoculum was introduced under light anaesthesia using 95% ether 5% chloroform as an anaesthetic. The inoculum was a 10m3dilution of freshly harvested allantoic fluid obtained from eggs infected with the homologous virus.
RESULTS Virus degradation
After treatment with T NlOl (up to 19 h with concentrated suspensions of material) no preparation possessedHA activity within the limits of the test although all preparations 343
C. J. M. RONDLE
ET
AL.
showed slight NA. Inoculation of 0.1 ml samples of mixtures into the allantoic sac of lo-day-old fertile hen eggs failed to produce HA in the allantoic fluid within 48 h indicating that no live virus was present in the mixtures. Separation of components This has been described under ‘Materials and Methods’. It was essential that the differential centrifugation was done in the presence of 2% T NlOl or reaggregation of ‘spikes’ and ‘ghosts’ was liable to occur. It is interesting that reaggregation of disrupted influenza virus was commented upon by Hoyle (1972). Electron microscopy Similar results were obtained for X-31,64b and BAP/13. The best resultswere obtained with BAP/lS and a typical series of electron micrographs is shown in Plate 1. Plate l(a) shows a purified virus preparation. Plate l(c) shows aggregated, amorphous ‘spikes’ with no sign of the structures shown by Laver & Valentine (1969). Plate l(b) shows ‘ghosts’ from which not all ‘spikes’ have been removed. Polyacrylamide gel electrophoresis The results are equivocal and are included only to show that the ‘simple’ electron microscope pictures do not relate directly to the chemical changes apparently taking place. Figure l(a) shows BAP/lS treated with T NlOl and subjected to analysis by the method of Ornstein & Davies (1962). Figure l(b) sh ows BAP/13 treated with T NlOl and then treated as described by Skehel & Schild (1971). Figures l(c) and l(d) show comparable data for ‘spikes’ and ‘ghosts’. All results are taken from readings on a Joyce-Loebl ‘Chromoscan’ using a 1.0 OD wedge, a ‘C’ cam, an aperture of 05803 and a filter and lamp at 620 mp. It would appear that T NlOl does not by itself break virus into very small pieces. Indeed in Figure l(a) most of the material remained at the origin and did not pass into the ‘small pore’ gel A breakdown of T NlOl-treated virus by the method of Skehel Sz Schild (1971) gives results comparable to those obtained by them on otherwise untreated virus. Indeed possible positions of ‘marker’ peptides have been tentatively ascribed to the results shown in Figure l(a). However, if one assumes that ‘spikes’ and ‘ghosts’ are respectively external and internal virus components there is little correlation between our work and that summarized in Table 1 of Skehel & Schild (1971). Chemical properties of fractions Due to the very small amounts of material available little progress was made in this part of the work. The method of Sutherland et al. (1949) detected ‘some’ protein in both ‘spikes’ and ‘ghosts’. Spectrophotometrically no nucleic acid could be detected in ‘spikes’ but traces of absorption at 260 rnp were found in ‘ghosts’ degraded with SDS. Protection tests (a) Experiments with ferrets using X-31 will be reported in detail elsewhere. Suffice it to say that intramuscular injections of virus degraded with T NlOl delayed or abolished the typical diphasic temperature responses of these animals when infected by the i/n route. (b) Experiments with ‘spikes’ and ‘ghosts’ with 64b tended to be subjective. In a few cases subjective assessment was most kindly checked histologically by Dr R. Darnell; 344
3441
SPLIT
INFLUENZA
VIRUS
Fig. 1. Polyacrylamide 0 = origin of run.
VACCINE
gel analysis of BAP/13
and fractions. Explanation in the text;
subjective and objective assessments agreed. A typical experiment with ‘spikes’ is shown in Table 1. Each mouse was examined by three observers and it is clear that some mitigation of symptoms over direct challenge has occurred. TABLE 1. Modification of mouse lung consolidation by 64b by ‘spikes’ obtained from T NlOl treatment. (a) Virus dilution 2 x lo4 HAU/ml; split by T NlOl and ‘spikes’ and ‘ghosts’ obtained. Mice inoculated intraperitoneally with 0.1 ml preparation. (b) Challenged at day 9 intranasally with 800 HAU live virus in 0.1 ml PBSA
Controls
l/3
l/30
++/+++ ++ +++ +++ ++/+++ ++ +++ + ++
-
+ iI
+++ -
++ + -
+ + + +
++ + ++ +++ ++
LIZ -
l/300
(c) Of numerous experiments done with BAP/13 three are listed in Table 2 as similar protocols were followed in each case. Batches of 10 mice were used in each case. Apart from the controls mice were injected i/p with 0.1 ml ‘spikes’ or ‘ghosts’. Before treatment 34.5
C. J. M.
RONDLE
ET
AL.
with T NlOl the virus preparations had a titre of 10’ HAU/ml. After 7-8 days mice were challenged i/n with a 10-s dilution of freshly prepared, infected allantoic fluid. The inoculum was 0.05 ml. TABLE 2. Mouse protection by ‘spikes’ and ‘ghosts’ from BAP/lS treated
with Triton NlOl r 3 Experiment A Controls Spikes Ghosts Experiment B Controls Spikes Ghosts
4 N/T
1 0 0
6 0 0
Experiment C Controls Spikes Ghosts Ten mice were used for each Numbers refer to accumulative
Deaths of day * 5 6 7 8 9 0 1 00 00
6
10
8 0 0
9 0 0
N/T N/T N/T
N/T N/T N/T
N/T N/T N/T
8 0 0
8 9 N/T 0 0 N/T 0 0 N/T 7 8 N/T 0 0 N/T 0 0 N/T
9 0 0
group. deaths per day.
Protective results with BAP/13 ‘spikes’ appear unequivocal. Protection against challenge by ‘ghosts’ is not surprising since as shown in Plate l(b) ‘spikes’ had not been removed completely from the virus surface. One point of interest may be mentioned. Due to the small amounts of material handled it has not been possible to weigh doses of vaccine injected. Skehel & Schild (1971) state that 10s HAU virus was approximately equivalent to 1 mg protein. This figure agrees reasonably well with a value of approximately 3 mg protein per lo6 HAU for 64b. In the experiment given in Table 2 virus at 10’ HAU/ml was used. This was degraded and separated into ‘spikes’ and ‘ghosts’. Parallel experiments showed that this procedure gave an approximately 50-50 split in the fractions. This leads to the conclusion that the mice received approximately 0.5-1.5 mg material per immunizing dose.
DISCUSSION The work presented in this paper suggests that it is possible to obtain a split vaccine from influenza virus which contains those antigens necessary for the production of protective antigen (i.e. against infection) but contains little nucleic acid or other non-essential material. Unlike some of the more recently proposed split vaccines it does not require adjuvant for activity. This is possibly because the liberated ‘spikes’ form a conglomerate which is highly antigenic. The material described does not haemagglutinate susceptible chick cells and is poor in NA activity. It has been shown previously by Tyrrell & Horsfall (1954), Hobson (1966) 346
SPLIT
INFLUENZA
VIRUS
VACCINE
and Larin & Gallimore (1971) that there is no correlation between the production of the appropriate antibody and the presence of HA or NA in the immunizing material. The present work is considered only as a preliminary to a more detailed study but it is not known if such a study can take place in this laboratory. One pressing need is to determine whether or not the protection afforded by ‘spikes’ is strain specific or shows overlap with other virus strains. Acknowledgements The authors wish to thank the Medical Research Council who financed this work and supplied emoluments for M. J. W. and J. B. K. They are also grateful to the Visual Aids Department of the London School of Hygiene and Tropical Medicine for the artwork supplied. REFERENCES Brand, C. M. (1973). Crystalline haemagglutinin antigen from influenza virus. Symposia Series in Immunobiological Standardization 20, 59-70. Brand, C. M. & Skehel ,J. J. (1972). Symposium on 1nfEuenza Vaccines. The Royal Institution, April 1972. Corbel, M. J. & Rondle, C. J. M. (1970). Soluble antigens obtained from influenza virus by treatment with non-ionic detergent. Journal of Hygiene, Cambridge 68, 81-96. Corbel, M. J., Rondle, C. J. M. & Bird, R. G. (1970). Degradation influenza virus by nonionic detergent. Journal of Hygiene, Cambridge 68, 77-80. Hobson, D. (1966). The strain-specific serological activity of a non-haemagglutinating fraction of influenza. BritishJournal of Experimental Pathology 47, 257-265. Hoyle, L. (1950). The multiplication of influenza viruses in the fertile egg. Journal of Hygiene 48, 277-297. Hoyle, L. (1972). Fractionation of the influenza virus with the object of producing subunit vaccine. Symposium on Inj?uenza Virus. The Royal Institution, April 1972. Hoyle, L., Horne, R. W. & Waterson, A. P. (1961). Structure and composition of the myxoviruses. 11. Components released from the virus particle by ether. Virology 13, 448-459. Kilbourne, E. D. (1969). Future influenza vaccines and the use of genetic recombinants. Bulletin of the World Health Organization 41, 643. Larin, N. M. & Gallimore, P. H. (1971). Journal of Hygiene, Cambridge 69, 35-46. Laver, W. G. (1963). The structure of influenza viruses. 3. Disruption of the virus particles and separation of neuraminidase activity. Virology 20, 251-262. Laver, W. G. (1964). Structural studies on the protein subunits from three strains of influenza virus. Journal of Molecular Biology 9, 109-124. Laver, W. G. & Valentine R. C. (1969). Morphology of the isolated haemagglutinin and neuraminidase subunits of influenza virus. Virology 38, 105-119. Mituzani, H. (1963). A simple method for purification of influenza virus. Nature 198, 109. Ornstein, L. & Davis, B. J. (1962). Disc Electrophoresis. Preprinted by Distillation Products Industries, Eastman Kodak Co. Skehel, J. J. & Schild, G. C. (1971). The polypeptide composition of influenza A viruses. Virology 44, 396-408. Sutherland, W. E., Cori, C. F., Haynes, R. & Olsen, A. S. (1949). Purification of the hyperglycaemic-glycogenolytic factor from insulin and from gastric mucosa. Journal of Biological Chemistry 180, 825-837. Tyrrell, D. A. J. & Horsfall, F. L. (1954). Disruption of influenza virus. Properties of degradation products of the virus particle. Journal of Experimental Medicine 99, 321-342.
347
of Biological
Standardization
1975 3, 341-347
Towards a split influenza virus vaccine* C. J. M. Rondle,? M. J. Walker,? J. B. Krahnt and R. G. Bird?
A simple method is proposed whereby extracts of influenza virus can be injected intraperitoneally in mice and protect them against subsequent challenge by live virus by the intranasal route.
INTRODUCTION It is generally recognized that an attenuated or genetically modified live virus is probably the best vaccine to combat epidemics of influenza which arise from time to time. In some cases, however, live virus is contra-indicated and a ‘dead’ split vaccine would be of advantage. One criterion of such a vaccine is that it should contain those antigens giving rise to protective antibodies and be free from nucleoprotein and other materials not involved in the protective immune process. Influenza virus has been disrupted by a number of agents amongst which may be cited ether (Hoyle, 1950; Hoyle, Horne, & Waterson 1961), the anionic detergents sodium dodecyl sulphate (SDS) and sodium deoxycholate (SDC), (Laver, 1963, 1964), the enzyme bromelain (Brand & Skehel, 1972; Brand, 1973) and the non-ionic detergents Non-idet P40 (Shell) and Triton NlOl (Rohm & Haas Co., Philadelphia). These two compounds are similar if not identical in composition being based on nonylphenoxyethanol. However, Corbel & Rondle (1970) and Corbel, Rondle & Bird (1970) showed that under controlled conditions T NlOl degraded all strains of influenza virus tested in a unique way. Under the action of T NlOl outer ‘spikes’ of neuraminidase (NA) and haemagglutinin (HA) were preferentially stripped off leaving the inner virus particle ‘ghosts’ intact. The resultant mixture had NA activity, did not haemagglutinate normally susceptible chick * Received for publication 10 March 1975. t London School of Hygiene and Tropical WC1 E7HT, England.
Medicine,
Keppel
Street (Gower
Street),
London
341
C. J. M.
RONDLE
ET
AL.
cells but blocked haemagglutination-inhibiting antibody (serum blocking substance activity, SBS). Preliminary experiments showed that these mixtures were antigenic in ferrets and guinea pigs. This paper describes our attempts to separate ‘spikes’ from ‘ghosts’ and to investigate the chemical and serological properties of each fraction. MATERIALS
AND
METHODS
Virus strains used Strains used in this work were X-31 (Kilboume, 1969) said to be a recombinant H2 N2 between AZ/HongKong/68 and AO/PRS; 64b, described by Schild as A/PR8/34A/Eng/959/69 (HON2); and BAP/13. This last strain was most generously provided by Dr G. Appleyard of M.R.E. Porton and he suggested the designation A/BEL/40Singapore/i/57-PR/8/34 (HCN,). X-31 was useful for ferret and guinea-pig experiments, most of which are reported elsewhere. For the present work its low virulence for mice was disadvantageous. Strain 64b was used extensively but in our hands it was difficult to obtain ‘clean’ virus preparations for study. Moreover, its effect on mice was best assessedby lung consolidation and this tended to be subjective. BAP/13 was better for our purposes as it showed 85-95% virulence for mice 5-10 days after intranasal challenge. These present virus strains were chosen because our previous work had been done with ‘conventional’ strains and we wished to test that our methods were of general application. Virus growth and purification Viruses were grown in the allantoic cavity of lo-day-old fertile hen eggs. Allantoic fluids were harvested after 48 h incubation, clarified by centrifugation at 3000 rev/min for 20 min and the precipitate discarded. Virus was recovered by absorption and elution onto and from barium sulphate (Mituzani, 1963). Further purification was effected by sugar density gradient (SDG) centrifugation using 45-60% sucrose concentrations in a ‘Spinco’ SW30 rotor at 20 000 rev/min for 16 h. Virus degradation Virus bands recovered from SDG were suspended in phosphate-buffered saline (PBSA) containing 2% T NlOl and sedimented at 100 000 g in a ‘Spinco’ centrifuge. Supernatants were removed carefully with a syringe and needle and stored at 4 “C. Virus pellets were allowed to drain in the now inverted centrifuge tubes supported on filter pads to absorb excess moisture. Many attempts were made to remove T NlOl from the supernatant fluids. These included partition experiments with Arcton and hexane, SDG centrifugation and SDG in borate buffers (borate chelates with the hydroxy groups of T NlOl which results in a charged molecule) and iso-electric focusing under varied conditions. It was eventually discovered that the slow and careful addition with stirring of 4 vol/vol of cold (4 “C) ethanol to the cold (4 “C) supematant effected precipitation of the material present in the supernatant and left T NlOl in solution. After centrifugation at 800 g for 12 min the supematant was discarded and the precipitate washed with absolute ethanol. By the spectrophotometric method of Corbel & Rondle (1970) less than O*OOOl%T NlOl was present in this ethanol washing. Before study in the electron microscope the ‘supernatant precipitate’ was treated in the same way as the first ‘ghost’ precipitate. 342
SPLIT
INFLUENZA
VIRUS
VACCINE
Electron microscopy
Drained pellets of purified virus and drained pellets of degraded material were kept at 4 “C and processed within 1 h of preparation. The method used for making the negative stained preparations was a slight modification of that given in our earlier publication (Corbel et al., 1970). Each inverted tube was inspected to make sure that no diluent remained on the side of the tube, any present being removed by a strip of filter paper before treatment. To each tube was added 5 drops (v/v) neutralized phosphotungstic acid, using a 5 ml plastic syringe fitted with a 19G2 50/11 stainless steel needle. A fresh sterilized glass pipette was used to suspend each pellet in the negative stain and apply the suspensions to formvar-coated Smethurst new type 400 grids. Excess fluid was removed with strips of filter paper after 15-20 s and the grids allowed to dry in air in a dust-free atmosphere. Preparations were examined within 3 h of preparation using a GEC/AEI 811 electron microscope. Photographs were taken on Ilford N50 film. Polyacrylamide
gel electrophoresis
In an attempt to compare fractions obtained by us with those reported by others some experiments were done using analytical polyacrylamide electrophoresis. In some cases the method of Laver & Valentine (1969) was used employing SDS and dithiothreitol; in most others the technique of Skehel & Schild (1971) using SDS, 2-mercaptoethanol and urea was employed. Chemical analysis
Due to paucity of material little work could be done. In a few casesprotein was estimated by the method of Sutherland, Cori, Haynes & Olsen (1949). More reliance was placed on spectrophotometric observations using an Optica CF4Nl double-beam recording spectrophotometer. Serological tests
NA and HA tests were done as described by Corbel & Rondle (1970). A few SSB tests were done as control experiments and in two cases (X-31) and (64b) gel diffusion tests were done on degraded materials to show that T NlOl was releasing strain-specific materials as had previously been reported. Protection tests
Available mice, usually inbred white, but occasionally unselected hybrids, were used in batches of 10. One batch was inoculated intraperitoneally (i/p) with 0.1 ml saline per mouse. Other batches were inoculated with preparations of ‘spikes’ or ‘ghosts’. Mice were challenged intranasally (i/n) 7-10 days after ‘protection’. Usually 0~05-0*1 ml inoculum was introduced under light anaesthesia using 95% ether 5% chloroform as an anaesthetic. The inoculum was a 10m3dilution of freshly harvested allantoic fluid obtained from eggs infected with the homologous virus.
RESULTS Virus degradation
After treatment with T NlOl (up to 19 h with concentrated suspensions of material) no preparation possessedHA activity within the limits of the test although all preparations 343
C. J. M. RONDLE
ET
AL.
showed slight NA. Inoculation of 0.1 ml samples of mixtures into the allantoic sac of lo-day-old fertile hen eggs failed to produce HA in the allantoic fluid within 48 h indicating that no live virus was present in the mixtures. Separation of components This has been described under ‘Materials and Methods’. It was essential that the differential centrifugation was done in the presence of 2% T NlOl or reaggregation of ‘spikes’ and ‘ghosts’ was liable to occur. It is interesting that reaggregation of disrupted influenza virus was commented upon by Hoyle (1972). Electron microscopy Similar results were obtained for X-31,64b and BAP/13. The best resultswere obtained with BAP/lS and a typical series of electron micrographs is shown in Plate 1. Plate l(a) shows a purified virus preparation. Plate l(c) shows aggregated, amorphous ‘spikes’ with no sign of the structures shown by Laver & Valentine (1969). Plate l(b) shows ‘ghosts’ from which not all ‘spikes’ have been removed. Polyacrylamide gel electrophoresis The results are equivocal and are included only to show that the ‘simple’ electron microscope pictures do not relate directly to the chemical changes apparently taking place. Figure l(a) shows BAP/lS treated with T NlOl and subjected to analysis by the method of Ornstein & Davies (1962). Figure l(b) sh ows BAP/13 treated with T NlOl and then treated as described by Skehel & Schild (1971). Figures l(c) and l(d) show comparable data for ‘spikes’ and ‘ghosts’. All results are taken from readings on a Joyce-Loebl ‘Chromoscan’ using a 1.0 OD wedge, a ‘C’ cam, an aperture of 05803 and a filter and lamp at 620 mp. It would appear that T NlOl does not by itself break virus into very small pieces. Indeed in Figure l(a) most of the material remained at the origin and did not pass into the ‘small pore’ gel A breakdown of T NlOl-treated virus by the method of Skehel Sz Schild (1971) gives results comparable to those obtained by them on otherwise untreated virus. Indeed possible positions of ‘marker’ peptides have been tentatively ascribed to the results shown in Figure l(a). However, if one assumes that ‘spikes’ and ‘ghosts’ are respectively external and internal virus components there is little correlation between our work and that summarized in Table 1 of Skehel & Schild (1971). Chemical properties of fractions Due to the very small amounts of material available little progress was made in this part of the work. The method of Sutherland et al. (1949) detected ‘some’ protein in both ‘spikes’ and ‘ghosts’. Spectrophotometrically no nucleic acid could be detected in ‘spikes’ but traces of absorption at 260 rnp were found in ‘ghosts’ degraded with SDS. Protection tests (a) Experiments with ferrets using X-31 will be reported in detail elsewhere. Suffice it to say that intramuscular injections of virus degraded with T NlOl delayed or abolished the typical diphasic temperature responses of these animals when infected by the i/n route. (b) Experiments with ‘spikes’ and ‘ghosts’ with 64b tended to be subjective. In a few cases subjective assessment was most kindly checked histologically by Dr R. Darnell; 344
3441
SPLIT
INFLUENZA
VIRUS
Fig. 1. Polyacrylamide 0 = origin of run.
VACCINE
gel analysis of BAP/13
and fractions. Explanation in the text;
subjective and objective assessments agreed. A typical experiment with ‘spikes’ is shown in Table 1. Each mouse was examined by three observers and it is clear that some mitigation of symptoms over direct challenge has occurred. TABLE 1. Modification of mouse lung consolidation by 64b by ‘spikes’ obtained from T NlOl treatment. (a) Virus dilution 2 x lo4 HAU/ml; split by T NlOl and ‘spikes’ and ‘ghosts’ obtained. Mice inoculated intraperitoneally with 0.1 ml preparation. (b) Challenged at day 9 intranasally with 800 HAU live virus in 0.1 ml PBSA
Controls
l/3
l/30
++/+++ ++ +++ +++ ++/+++ ++ +++ + ++
-
+ iI
+++ -
++ + -
+ + + +
++ + ++ +++ ++
LIZ -
l/300
(c) Of numerous experiments done with BAP/13 three are listed in Table 2 as similar protocols were followed in each case. Batches of 10 mice were used in each case. Apart from the controls mice were injected i/p with 0.1 ml ‘spikes’ or ‘ghosts’. Before treatment 34.5
C. J. M.
RONDLE
ET
AL.
with T NlOl the virus preparations had a titre of 10’ HAU/ml. After 7-8 days mice were challenged i/n with a 10-s dilution of freshly prepared, infected allantoic fluid. The inoculum was 0.05 ml. TABLE 2. Mouse protection by ‘spikes’ and ‘ghosts’ from BAP/lS treated
with Triton NlOl r 3 Experiment A Controls Spikes Ghosts Experiment B Controls Spikes Ghosts
4 N/T
1 0 0
6 0 0
Experiment C Controls Spikes Ghosts Ten mice were used for each Numbers refer to accumulative
Deaths of day * 5 6 7 8 9 0 1 00 00
6
10
8 0 0
9 0 0
N/T N/T N/T
N/T N/T N/T
N/T N/T N/T
8 0 0
8 9 N/T 0 0 N/T 0 0 N/T 7 8 N/T 0 0 N/T 0 0 N/T
9 0 0
group. deaths per day.
Protective results with BAP/13 ‘spikes’ appear unequivocal. Protection against challenge by ‘ghosts’ is not surprising since as shown in Plate l(b) ‘spikes’ had not been removed completely from the virus surface. One point of interest may be mentioned. Due to the small amounts of material handled it has not been possible to weigh doses of vaccine injected. Skehel & Schild (1971) state that 10s HAU virus was approximately equivalent to 1 mg protein. This figure agrees reasonably well with a value of approximately 3 mg protein per lo6 HAU for 64b. In the experiment given in Table 2 virus at 10’ HAU/ml was used. This was degraded and separated into ‘spikes’ and ‘ghosts’. Parallel experiments showed that this procedure gave an approximately 50-50 split in the fractions. This leads to the conclusion that the mice received approximately 0.5-1.5 mg material per immunizing dose.
DISCUSSION The work presented in this paper suggests that it is possible to obtain a split vaccine from influenza virus which contains those antigens necessary for the production of protective antigen (i.e. against infection) but contains little nucleic acid or other non-essential material. Unlike some of the more recently proposed split vaccines it does not require adjuvant for activity. This is possibly because the liberated ‘spikes’ form a conglomerate which is highly antigenic. The material described does not haemagglutinate susceptible chick cells and is poor in NA activity. It has been shown previously by Tyrrell & Horsfall (1954), Hobson (1966) 346
SPLIT
INFLUENZA
VIRUS
VACCINE
and Larin & Gallimore (1971) that there is no correlation between the production of the appropriate antibody and the presence of HA or NA in the immunizing material. The present work is considered only as a preliminary to a more detailed study but it is not known if such a study can take place in this laboratory. One pressing need is to determine whether or not the protection afforded by ‘spikes’ is strain specific or shows overlap with other virus strains. Acknowledgements The authors wish to thank the Medical Research Council who financed this work and supplied emoluments for M. J. W. and J. B. K. They are also grateful to the Visual Aids Department of the London School of Hygiene and Tropical Medicine for the artwork supplied. REFERENCES Brand, C. M. (1973). Crystalline haemagglutinin antigen from influenza virus. Symposia Series in Immunobiological Standardization 20, 59-70. Brand, C. M. & Skehel ,J. J. (1972). Symposium on 1nfEuenza Vaccines. The Royal Institution, April 1972. Corbel, M. J. & Rondle, C. J. M. (1970). Soluble antigens obtained from influenza virus by treatment with non-ionic detergent. Journal of Hygiene, Cambridge 68, 81-96. Corbel, M. J., Rondle, C. J. M. & Bird, R. G. (1970). Degradation influenza virus by nonionic detergent. Journal of Hygiene, Cambridge 68, 77-80. Hobson, D. (1966). The strain-specific serological activity of a non-haemagglutinating fraction of influenza. BritishJournal of Experimental Pathology 47, 257-265. Hoyle, L. (1950). The multiplication of influenza viruses in the fertile egg. Journal of Hygiene 48, 277-297. Hoyle, L. (1972). Fractionation of the influenza virus with the object of producing subunit vaccine. Symposium on Inj?uenza Virus. The Royal Institution, April 1972. Hoyle, L., Horne, R. W. & Waterson, A. P. (1961). Structure and composition of the myxoviruses. 11. Components released from the virus particle by ether. Virology 13, 448-459. Kilbourne, E. D. (1969). Future influenza vaccines and the use of genetic recombinants. Bulletin of the World Health Organization 41, 643. Larin, N. M. & Gallimore, P. H. (1971). Journal of Hygiene, Cambridge 69, 35-46. Laver, W. G. (1963). The structure of influenza viruses. 3. Disruption of the virus particles and separation of neuraminidase activity. Virology 20, 251-262. Laver, W. G. (1964). Structural studies on the protein subunits from three strains of influenza virus. Journal of Molecular Biology 9, 109-124. Laver, W. G. & Valentine R. C. (1969). Morphology of the isolated haemagglutinin and neuraminidase subunits of influenza virus. Virology 38, 105-119. Mituzani, H. (1963). A simple method for purification of influenza virus. Nature 198, 109. Ornstein, L. & Davis, B. J. (1962). Disc Electrophoresis. Preprinted by Distillation Products Industries, Eastman Kodak Co. Skehel, J. J. & Schild, G. C. (1971). The polypeptide composition of influenza A viruses. Virology 44, 396-408. Sutherland, W. E., Cori, C. F., Haynes, R. & Olsen, A. S. (1949). Purification of the hyperglycaemic-glycogenolytic factor from insulin and from gastric mucosa. Journal of Biological Chemistry 180, 825-837. Tyrrell, D. A. J. & Horsfall, F. L. (1954). Disruption of influenza virus. Properties of degradation products of the virus particle. Journal of Experimental Medicine 99, 321-342.
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