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Determination of the viral particle content of influenza vaccines by electron microscopy
Journal
of Biological
Standardization
1975 3, 281-289
Determination of the viral particle content of influenza vaccinesby electron microscopy* R. c. Dunlap,-f E. R. Brown-f and D. W. Barry-f
The virus particle content of influenza vaccines was determined by electron microscopy using a modification of Sharp’s sedimentation technique. Aggregates of 2-100 virus particles were found to a variable extent in all vaccines, but these aggregates could be dispersed by suspending the preparations in buffers of high molality. The mean ratio of viral particles to CCA units was 140 x lo* and the CCA titer of influenza vaccine preparations could be increased if the test employed buffers which dispersed aggregates. The virus particle content of split-product vaccines could not be quantitated with precision because of degradation of the virus produced by the disruption process. The technique of direct particle counting provided additional information concerning the antigen content of influenza vaccines and permitted evaluation not only of the integrity of the viruses in the preparations but also the amount of non-virion particulate contamination.
INTRODUCTION The problems associated with the quantitation of the potency of inactivated influenza vaccine have been well recognized in the past (Perkins, 1973) and have been the subject of intensive investigation both in our laboratories and elsewhere for several years. Because such an inactivated product does not replicate in either in vitro or in vivo test systems, various indirect systems have been employed in an attempt to evaluate the antigenic content of these vaccines. Difficulties associated with all of these methods, however, limit their usefulness or relevancy in clinical or control situations. Techniques which depend on virus-red blood cell (RBC) interactions, such as hemagglutination (HA) or chick cell agglutination (CCA) titrations (Miller & Stanley, 1944), measure only one component of the virus (hemagglutinin), are only moderately reproducible even when a * Received for publication 2.5 November 1974. 7 Division of Virology, Bureau of Biologics, Food and Drug Administration, Bethesda, Maryland 20014, U.S.A.
8800 Rockville
Pike,
281
R. C. DUNLAP,
E. R. BROWN
AXD
D.
W.
BARRY
standard reference is employed (Tauraso, O’Brien & Seligmann, 1969) and may be subject to further variation because of the differing avidity which various strains of influenza virus have for RBC (Fazekas de St Groth, 1949). Antigen extinction tests in animals have been shown to be poorly reproducible (Tauraso et aE., 1969) and their relevance to human protection is unknown (Heller, 1967). The reproducibility, reliability and relcvancy of more recent techniques, such as single radial immunodiffusion (Schild, HenryAymard & Pereira, 1972), remain to be tested. The enumeration of viral particles (VP) by electron microscopic (EM) observation, on the other hand, would appear to have the advantages of being a direct technique not subject to most of the inherent variations of the indirect tests noted above. While the technique of counting VP by EM has been well established (Sharp, 1965) and its theoretical advantages for influenza antigen quantitation have been previously reviewed (Fazekas de St Groth, 1962), certain questions remain concerning its practical applicability to influenza vaccine standardization. The present study describes several of the variables encountered in employing this technique as a method of influenza vaccine antigenic quantitation and compares the results obtained from particle counting to those obtained with the CCA test, currently the only official test of influenza vaccine potency in the United States. MATERIALS
AND
METHODS
Vaccines
Experimental monovalent influenza vaccines prepared from A/Aichi strains were obtained from commercial manufacturers by special contract. Licensed bivalent vaccines containing A/England and B/Mass prototypes and monovalent B/Hong Kong vaccines were obtained from commercial distributors. Two U.S. Reference influenza vaccines, Lot 72 (bivalent A/England and B/Mass) and Lot 73 (monovalent B/Mass), were included inthestudies. Vaccinesfrommanufacturers A, B and C were purified by zonal centrifugation. Vaccine from manufacturer D was chromatographically purified. Vaccines made by manufacturers E and G were ‘split-product’ vaccines prepared by treatment with Tween-80 and either tri-(n)-butyl phosphate (TBP) or ether, respectively. Reagents
Buffers used as vaccine diluents were obtained from the National Institutes of Health Media Unit or were prepared in this laboratory. The following buffers were used: 0.001 M-phosphate (PO,) in water, 0.01 M-PO, in water, 0.01 M-PO, in saline (PBS), 0.1 M-PBS, McIlvaine’s buffer in water (McIlvaine, 1921) and McIlvaine’s buffer made up in saline. The phosphate buffers had a pH of 7.2 and the McIlvaine’s buffers were pH 7.0. Virus particle
coulit
Quantitation of virus was done by the method of Sharp (1960) utilizing the Sorvall SU counting rotor. Two methods of particle count preparation were used : (1) the pseudoreplica method which consisted of sedimenting the diluted vaccine onto agar, applying a film of collodion to the agar and floating the film (to which the virus adheres) onto water ; or (2) spinning the virus suspension directly onto collodion-coated grids which replaced the agar block and were held in place by double-coated Scotch brand tape. In the direct sedimentation method, it was necessary to rinse the grids in distilled water after virus 282
Plate 1. Aggregation
of undiluted U.S. Reference influenza stained with uranyl acetate.
vaccine, Lot 72. Negatively
Plate 2. Viral aggregation in different buffers. U.S. Reference influenza vaccine, Lot 72, diluted in (A) 0.001 M-PO, (H,O); (B) 0.01 M-PO, (H,O); (C) 0.01 M-PO, (saline); (D) McIlvaine’s buffer (H,O). Virus sedimented by centrifugation onto grids. Shadowed with chromium.
Plate 3. Particle count preparations of vaccines manufactured by different procedures. (A) Zonal centrifugation; (B) chromatography; (C) ether extraction; (D) TBP treatment. Preparations are chromium-shadowed.
T’IRUS
PARTICLE
CONTENT
OF
INFLUENZA
VACCINES
deposition in order to remove salt crystals. Total VP counts by this method were divided by 1.2 to compensate for the absence of the 2 mm thick agar block in the rotor cell. All grids were shadow-cast with chromium and examined in an RCA EMU 3 G electron microscope. Aggregation
determinations
Twenty randomly chosen electron microscopic fields of each preparation were photographed. Tabulations were made of numbers of single VP and the total number of particles from which the per cent of the total number of particles occurring singly (% singles) was calculated. Molality
Molality of the buffers was determined by the freezing point depression technique of Shoemaker & Garland (1967). CCA tests
CCA determinations were performed by the method of Miller & Stanley (1944) as modified by Tauraso et al. (1969) to include a standard reference. Vaccine/reference (V/R) ratios thus obtained were multiplied by the assigned value of the Lot 72 Reference (1886 CCA units/ml) and the results expressed as CCA units/ml. The standard diluent that was used in the CCA tests was 0.01 M-PBS unless otherwise specified in the text. RESULTS Particle counts
Preliminary EM inspection of the U.S. Reference influenza vaccine, Lot 72, revealed that the undiluted product was highly aggregated (Plate 1). Particles were tightly clumped in large massesor connected in long chains. Occasionally, groups were joined by slender filaments. Attempts to disperse the virus by sonication at various frequencies and time intervals with a Branson sonifier were not entirely successful, although some improvement was attained. Prolonged sonication (3 min) appeared to degrade the particles and increase aggregation. Choice of diluent, however, proved to have a marked effect on the state of virus aggregation as seen in Plate 2. A dilute phosphate buffer (0.001 M-PO& in water caused massive aggregates to form [Plate 2(A)]. Increasing the molarity to 0.01 I+PO~ in water reduced the size of the aggregates [Plate 2(B)]. A 0.01 M-PO, buffer prepared in normal saline greatly increased dispersion [Plate 2(C)]. Even better dispersion was attained by the use of McIlvaine’s buffer prepared in water [Plate 2(D)]. Consequently, McIlvaine’s buffer was used routinely for particle count preparations. Molality rather than pH (which was 7-O-7.2) or phosphate content appeared to be responsible for this aggregation phenomenon as there was a direct relationship between increasing molality and the percentage of single (non-aggregated) particles (Fig. 1). For example, 0.01 M-PO, in water (b) which had a molality of 0.01 was associated with only 10% single particles whereas the same buffer in saline (d) with a molality of 0.23 was associated with 64% single particles. There was no discernible difference in the tendency of either recombinants or nonrecombinants to form aggregates as shown in Table 1, where four A and four B strains were examined. Of the two preparations most highly aggregated, one was a nonrecombinant A strain (vaccine B) and the other was a recombinant B (vaccine C) strain. 283
R. C. DUNLAP,
E. R. BROWN
AND
D.
W.
BARRY
The remainder of the vaccines tested displayed similar degrees of dispersion regardless of strain or type. At the outset, counts were prepared by the agar-collodion pseudo-replica method. To test whether all the virus was being stripped off the agar, counts also were done by the direct deposition of the virus onto collodion-coated grids as described above. Table 2 contains counts of vaccines done by both methods. There was a considerable increase in particle count when the virus was sedimented directly onto the microscope grid indicating that residual virus remained on the agar block. The direct method was therefore adopted for routine influenza particle counting. The method of manufacture of influenza vaccines had an effect both on the EM appearance of the preparation and on the precision and reliability with which it could be
Mololity hoi/kg)
Fig. 1. Effect of molality of buffer on aggregation.
Molality was calculated from the freezing point depression produced by different buffers. (a) 0901 M-PO& (HsO); (b) 0.01 M-PO, (saline) 1 : 2; (d) 0.01 M-PO, (saline); M-PO, (H,O); (c) 0.001 M-PO, (H,O)+O*Ol (e) McIlvaine’s buffer in saline; (f) McIlvaine’s buffer (HsO); (g) McIlvaine’s (HaO)+ 0.01 M-PO, (saline) 1 : 2; (h) 0.1 M-PO, (saline).
TABLE
1. Measurement of aggregation in vaccines prepared binant or non-recombinant influenza strains Vaccine A* B C D Ref. 73 A B C
Strain
Type Recombinant Non-recombinant Non-recombinant Recombinant Non-recombinant Non-recombinant Non-recombinant Recombinant
Single particles (%) 58 33 65 49 59 60 57 38
Vaccines were diluted in O-01 M-PBS. * Letters A-D refer to commercial manufacturers.
284
from recom-
VIRUS
PARTICLE
CONTENT
OF
INFLUENZA
VACCINES
TABLE 2. Comparison of counts done by pseudo-replication from agar and by direct sedimentation onto coated grids Vaccine Ref. 72
D (monovalent A strain) A (bivalent A and B)
Agar
Grid
Increase (%)
I.80 x lo”* 0.72 x IO”
5.53 x 10” 2.57 x 10” 2.93 x 10”
207 257 150
1.17 x 1011
* = VP/ml.
quantitated by EM enumeration of VP. Vaccines purified by zonal centrifugation contained little extraneous debris and the virus particles were clearly defined [Plate 3(A)], while the chromatographically purified preparation contained more debris and had less clearly defined VP [Plate 3(B)]. Ether-extracted vaccines demonstrated degraded particles and recognition and differentiation were uncertain [Plate 3(C)], while the integrity of the VP in the TBP-treated vaccines was almost completely destroyed and only presumably subviral units were observed [Plate 3(D)]. Particle count and CCA content of vaaines Table 3 contains the particle counts obtained with several different vaccine preparations: Experimental vaccines (monovalent A) and commercial vaccines (bivalent A and B and monovalent B). Only vaccines purified by zonal centrifugation or chromatography are included in the table. Vaccines prepared by other methods could not be counted reliably. It will be noted that while the mean ratio of VP/CCA units was 1.40 x 10s, the range of this ratio was 0.60 x 108 to 2.34 x 10s indicating that there could be as much as a fourfold difference in the number of viral particles in two preparations containing the same number of CCA units. TABLE
Type Monovalent
3. Virus Vaccine
A
particle
content
of influenza
VP/ml
( x 10”)
CCA/rnl
VP/CCA (x lO8) 0.82 2.08 I.34 0.72
A* B C D
1.27 2.70 2.84 1.59
1540 1300 2120 2200
A B C D
I.80 3.03 5.13 4.54
2380 1980 2880 2100
vaccines
Mean = I.24 Bivalent A and B
0.76 1.53 I.78 2.16
Mean= I.56 Monovalent
B
A B C
0.74 I.51 2.69
0.60 I.24 2.34
1240 1220 1150
Mean =I*05 Total
mean
= 1.40
Particle counts are the average of five fields taken from each of eight grids (40 pictures). * Letters A-D refer to manufacturers.
285
R.
C. DUNLAP,
E. R. BROWN
AND
D.
W.
BARRY
Eflect of virus aggregation on the CCA test
To determine whether the aggregation of VP noted on EM examination of the vaccine might affect the CCA results, three vaccine preparations were diluted in 0.001 M-PO, buffer to produce highly aggregated virus and in McIlvaine’s buffer for maximum dispersion of virus. These preparations were compared in the CCA test and aggregation counts were made. In order to prevent lysis of the chicken RBC by these buffers, however, the final dilutions of all preparations used in both the CCA test and the EM enumeration tests were mixed with equal volumes of 0.01 M-PBS. Although the addition of saline reduced the aggregation in the dilute phosphate buffer and increased the aggregation in the McIlvaine’s buffer, a twofold or more difference in the per cent single particles between the two preparations could still be obtained. Figure 2 depicts the relationship 300
!OOO
100 L$ 90 g
80
: .g
70 60
E
50
i
40
500
000
30
E .E 2
500
20 IO 0
1 x
Y
C(monovalent A)
I/
Y
x Ref.72 Vaccines
11
x
Y
0
Ref.73
Fig. 2. Effect of aggregation on CCA values. Solid bar = per cent single particles. X = 0.001 M-PO, (H,O) buffer as diluent. Y = Cross-hatched bar = CCA titer. C refers to the commercial manufacturer. 1 indicates McIlvaine’s buffer (HsO) as diluent. range of values from different tests.
found between aggregation and CCA value for the three vaccines. Increased dispersion raised the CCA value while highly aggregated virus was associated with a reduced CCA value. Calculations of the variability of the CCA test and the particle count procedure were made on repeated tests using the U.S. Reference vaccine, Lot 72. These data are shown in Table 4 and indicate that the two tests are approximately equal in reproducibility. TABLE
CCA Particle counts
4. Reproducibility of CCA test and particle counts
Mean
Number of tests
Standard deviation
1460 238*
161 45
215 34
* Virus particles/EM
286
picture.
950/0Confidence limits 1030-1890 170-306
Coefficient of error (“/6) 15 14
VIRUS
PARTICLE
CONTENT
OF
INFLUENZA
VACCINES
DISCUSSION Direct EM enumeration of VP in influenza vaccines offers several advantages over indirect techniques of antigen quantitation which are subject to variations in the interaction of various strains of virus with either the RBC used in agglutination titrations (Fazekas de St Groth, 1949) or the particular animal species used in antigen extinction titrations (Nakamura, 1965). The technique also provides considerable insight and information concerning the morphology and structural integrity of the virus in such preparations as well as an estimate of the amount of extraneous non-viral material which might be in such vaccines. Several considerations must be borne in mind, however, before direct EM enumeration can be considered as an adjunct to currently employed methods of quantitation of influenza vaccine potency. The first is that the degree of aggregation of VP in the vaccines appears to be variable and unrelated to the type or strain of virus. Although Dalton, Kim & Sharp (1967) and Hirst & Pons (1973) have shown that aggregates of live virus particles are more effective in causing infection in vitro than single particles, the significance of such aggregation of inactivated virus to the immunogenicity of vaccine in humans is unknown. It may be speculated that large aggregates might increase the immunogenicity of influenza vaccines as Davenport (1968) has shown that increasing the size of subunit particles by adsorption to AlPO, markedly increased their immunogenicity in animals and possibly in man (Hennessy & Davenport, 1974). However, comparative studies of the immunogenicity of whole virus preparations adsorbed to AlPO, or in aggregates have not been performed. It is clear, however, that such aggregates affect agglutination titrations such as the CCA test, where the relatively low CCA values associated with highly aggregated preparations could be elevated if the preparations were suspended in a diluent of high molality which dispersed such aggregates. The use of high molality buffers also facilitated the precise quantitation of VP numbers in a preparation not only because aggregation prevents uniform distribution of particles but also because enumeration of the individual viral particles in an aggregate is exceedingly difficult. Donald & Isaacs (1954) noted that the relationship between agglutinating units and VP tends to remain relatively constant regardless of the strain being examined, with as much variation observed between examinations of the same strain on different days as between different strains. In the present investigation we found that a three- to fourfold variation in the ratio could be observed between vaccines of the same strains produced by different methods of manufacture but that the mean ratios of vaccines containing different strains varied only slightly. The mean ratio of 10sl is approximately 1 log,, higher than the value obtained by Donald & Isaacs (1954) and Tyrrell & Valentine (1957) and may be related to the lower absolute values obtained with the CCA test than with the end-point hemagglutination test used by the previous investigators (Tauraso & O’Brien, 1970). Because of the higher CCA values obtained when the vaccines were tested in buffers which increased dispersion (Fig. 2), CCA titers may be more related to the number of separate groups of particles than to total VP because aggregates composed of many VP may behave as a single active unit in virus-RBC interactions. The reproducibility of the direct VP count, nevertheless, was approximately that of the CCA test when a standard reference was employed (Tauraso et al., 1969). The advantages of direct sedimentation of the vaccine suspension onto the grid over previously described techniques of direct influenza virus enumeration are readily apparent. Stripping of the VP from the agar block by the use of collodion, as described by Sharp (1960), appeared to be incomplete, as considerably higher counts were obtained when the 287
R. C. DUNLAP,
E. R. BROWN
AND
D. W. BARRY
agar block was omitted. Spray techniques for distribution of the virus on grids (Luria, Williams & Backus, 1951) have the disadvantage of being subject to variations not only in the distance of the grid from the spray gun but also are affected by the bore of the gun, the force applied to create the spray and uneven distribution secondary to drying of the spray droplets. Enumeration of VP on laked RBC (Dawson & Elford, 1949) does not permit estimation of the contamination of the vaccine with non-virion material. The technique used in our study permitted detection of relatively large amounts of non-virion material in the chromatographically purified vaccine (D) which contained approximately three times as much protein as all other vaccines when tested by the method of Lowry, Rosebrough, Farr & Randall (1951). The technique is not sensitive enough, however, to detect small variations in non-virion protein content which could be determined better by either the Lowry technique or polyacrylamide gel electrophoresis (Perkins, 1973) but would be of use in determining whether or not vaccines high in total protein content had correspondingly large numbers of VP. While the technique also does not permit accurate quantitation of VP in vaccines where the virus has been subjected to disruption by Tween-80 and either TBP or ether, it does allow evaluation of the integrity and size of the particles which may severely affect the primary immunogenic capacity of a vaccine produced by these manufacturing methods (Barry, Staton & Mayner, 1974). It is clear that TBP produced much greater degradation than did ether. Studies are currently in progress in this laboratory to relate VP counts by EM with the immunogenicity in animals and humans in an attempt to expand our knowledge concerning the reliability and relevancy of this technique to the protection of man from influenza infection. Acknowledgement The authors are deeply appreciative of the advice and assistance of Dr Joan May in the freezing point determination experiments.
REFERENCES Barry, D. W., Staton, E. & Mayner, R. E. (1974). Inactivated influenza vaccine efficacy: Diminished antigenicity of split-product vaccines in mice. Infection and Immunity 10, 1329-1336. Dalton, D., Kim, K. S. & Sharp, D. G. (1967). Multiplicity activation of vaccinia virus in L cells. Proceedings of the National Academy of Sciences 58, 1758-1761. Davenport, F. M. (1968). Antigenic enhancement of ether-extracted influenza virus vaccines by AlPOd. Proceedings of the Society for Experimental Biology and Medicine 127, 587490. Dawson, I. M. & Elford, W. J. (1949). The investigation of influenza and related viruses in the electron microscope, by a new technique. Journal of General Microbiology 3, 298-3 11. Donald, H. B. & Isaacs, A. (1954). Counts of influenza virus particles. Journal of General Microbiology 10, 4.57-464. Fazekas de St Groth, S. (1949). Modification of virus receptors by metaperiodate. I. Properties of IO,-treated red cells. AustralianJburnal of Experimental Biological and Medical Science 27, 65-81. Fazekas de St Groth, S. (1962). The neutralization of viruses. Advances in Virus Research 9, l-125. HeIIer, L. (1967). The precision of three different types of response metometers used in mouse protection assays of the relative potencies of inactivated influenza virus vaccines. International Symposium on Biological Assay Methods for Vaccines and Sera 10, 33-42. 288
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CONTENT
OF
INFLUENZA
VACCINES
Hennessy, A. V. & Davenport, F. M. (1974). Studies on vaccination of infants against influenza with influenza hemagglutinin. Proceedings of the Society for Experimental Biology and Medicine 146, 200-204. Hirst, G. K. & Pons, M. W. (1973). Mechanism of influenza recombination. II. Virus aggregation and its effect on plaque formation by so-called non-infective virus. Virology
56,620-631. Lowry, 0. H., Rosebrough, N. J ., Farr, A. L. & Randall, R. J. (195 1). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 26.5-275. Luria, S. E., Williams, R. C. & Backus, R. C. (1951). Electron micrographic counts of bacteriophage particles. Journal of Bacteriology 61, 179-188. McIlvaine, T. C. (1921). A buffer solution for calorimetric comparison. Journal of Biological Chemistry 49, 183-l 86. Miller, G. L. & Stanley, W. M. (1944). Q uantitative aspects of the red blood cell agglutination test of influenza virus. Journal of Experimental Medicine 79, 185-195. Nakamura, K. (1965). Pathogenicity and immunogenicity of various strains of influenza virus for mice. Bihen’sJournal 8, 155-165. Perkins, F. T. (1973). In: Report of an informal meeting of manufacturers and the Control Laboratory on Influenza Vaccine. Journal of Biological Standardization 1, 195-197. Schild, G. C., Henry-Aymard, M. & Pereira, H. G. (1972). A quantitative single radial diffusion test for immunological studies with influenza virus. JournaZ of General Virology
16, 231-236. Sharp, D. G. (1960). Sedimentation counting of particles via electron microscopy. Fourth International Conference on Electron Microscopy, Berlin 1958, pp. 542-548. Berlin: Springer-Verlag. Sharp, D. G. (1965). Quantitative use of the electron microscope in virus research. Methods and recent results of particle counting. Laboratory Investigation 14, 93-125. Shoemaker, D. P. & Garland, C. W. (1967). Experiments in Physical Chemistry, 2nd ed., pp. 141-146. New York: McGraw-Hill. Tauraso, N. M. & O’Brien, T. C. (1970). Comparison of the haemagglutinin production of influenza A2 Hong Kong variant and recombinant strains. Bulletin of the World Health Organization 43, 275-279. Tauraso, N. M., O’Brien, T. C. & Seligmann, E. B. (1969). Problems of influenza vaccine standardization. Bulletin of the World Health Organization 41, 497-506. Tyrrell, D. A. J. & Valentine, R. C. (1957). The assay of influenza virus particles by haemagglutination and electron microscopy. Journal of General Microbiology 16, 668-675.
289
of Biological
Standardization
1975 3, 281-289
Determination of the viral particle content of influenza vaccinesby electron microscopy* R. c. Dunlap,-f E. R. Brown-f and D. W. Barry-f
The virus particle content of influenza vaccines was determined by electron microscopy using a modification of Sharp’s sedimentation technique. Aggregates of 2-100 virus particles were found to a variable extent in all vaccines, but these aggregates could be dispersed by suspending the preparations in buffers of high molality. The mean ratio of viral particles to CCA units was 140 x lo* and the CCA titer of influenza vaccine preparations could be increased if the test employed buffers which dispersed aggregates. The virus particle content of split-product vaccines could not be quantitated with precision because of degradation of the virus produced by the disruption process. The technique of direct particle counting provided additional information concerning the antigen content of influenza vaccines and permitted evaluation not only of the integrity of the viruses in the preparations but also the amount of non-virion particulate contamination.
INTRODUCTION The problems associated with the quantitation of the potency of inactivated influenza vaccine have been well recognized in the past (Perkins, 1973) and have been the subject of intensive investigation both in our laboratories and elsewhere for several years. Because such an inactivated product does not replicate in either in vitro or in vivo test systems, various indirect systems have been employed in an attempt to evaluate the antigenic content of these vaccines. Difficulties associated with all of these methods, however, limit their usefulness or relevancy in clinical or control situations. Techniques which depend on virus-red blood cell (RBC) interactions, such as hemagglutination (HA) or chick cell agglutination (CCA) titrations (Miller & Stanley, 1944), measure only one component of the virus (hemagglutinin), are only moderately reproducible even when a * Received for publication 2.5 November 1974. 7 Division of Virology, Bureau of Biologics, Food and Drug Administration, Bethesda, Maryland 20014, U.S.A.
8800 Rockville
Pike,
281
R. C. DUNLAP,
E. R. BROWN
AXD
D.
W.
BARRY
standard reference is employed (Tauraso, O’Brien & Seligmann, 1969) and may be subject to further variation because of the differing avidity which various strains of influenza virus have for RBC (Fazekas de St Groth, 1949). Antigen extinction tests in animals have been shown to be poorly reproducible (Tauraso et aE., 1969) and their relevance to human protection is unknown (Heller, 1967). The reproducibility, reliability and relcvancy of more recent techniques, such as single radial immunodiffusion (Schild, HenryAymard & Pereira, 1972), remain to be tested. The enumeration of viral particles (VP) by electron microscopic (EM) observation, on the other hand, would appear to have the advantages of being a direct technique not subject to most of the inherent variations of the indirect tests noted above. While the technique of counting VP by EM has been well established (Sharp, 1965) and its theoretical advantages for influenza antigen quantitation have been previously reviewed (Fazekas de St Groth, 1962), certain questions remain concerning its practical applicability to influenza vaccine standardization. The present study describes several of the variables encountered in employing this technique as a method of influenza vaccine antigenic quantitation and compares the results obtained from particle counting to those obtained with the CCA test, currently the only official test of influenza vaccine potency in the United States. MATERIALS
AND
METHODS
Vaccines
Experimental monovalent influenza vaccines prepared from A/Aichi strains were obtained from commercial manufacturers by special contract. Licensed bivalent vaccines containing A/England and B/Mass prototypes and monovalent B/Hong Kong vaccines were obtained from commercial distributors. Two U.S. Reference influenza vaccines, Lot 72 (bivalent A/England and B/Mass) and Lot 73 (monovalent B/Mass), were included inthestudies. Vaccinesfrommanufacturers A, B and C were purified by zonal centrifugation. Vaccine from manufacturer D was chromatographically purified. Vaccines made by manufacturers E and G were ‘split-product’ vaccines prepared by treatment with Tween-80 and either tri-(n)-butyl phosphate (TBP) or ether, respectively. Reagents
Buffers used as vaccine diluents were obtained from the National Institutes of Health Media Unit or were prepared in this laboratory. The following buffers were used: 0.001 M-phosphate (PO,) in water, 0.01 M-PO, in water, 0.01 M-PO, in saline (PBS), 0.1 M-PBS, McIlvaine’s buffer in water (McIlvaine, 1921) and McIlvaine’s buffer made up in saline. The phosphate buffers had a pH of 7.2 and the McIlvaine’s buffers were pH 7.0. Virus particle
coulit
Quantitation of virus was done by the method of Sharp (1960) utilizing the Sorvall SU counting rotor. Two methods of particle count preparation were used : (1) the pseudoreplica method which consisted of sedimenting the diluted vaccine onto agar, applying a film of collodion to the agar and floating the film (to which the virus adheres) onto water ; or (2) spinning the virus suspension directly onto collodion-coated grids which replaced the agar block and were held in place by double-coated Scotch brand tape. In the direct sedimentation method, it was necessary to rinse the grids in distilled water after virus 282
Plate 1. Aggregation
of undiluted U.S. Reference influenza stained with uranyl acetate.
vaccine, Lot 72. Negatively
Plate 2. Viral aggregation in different buffers. U.S. Reference influenza vaccine, Lot 72, diluted in (A) 0.001 M-PO, (H,O); (B) 0.01 M-PO, (H,O); (C) 0.01 M-PO, (saline); (D) McIlvaine’s buffer (H,O). Virus sedimented by centrifugation onto grids. Shadowed with chromium.
Plate 3. Particle count preparations of vaccines manufactured by different procedures. (A) Zonal centrifugation; (B) chromatography; (C) ether extraction; (D) TBP treatment. Preparations are chromium-shadowed.
T’IRUS
PARTICLE
CONTENT
OF
INFLUENZA
VACCINES
deposition in order to remove salt crystals. Total VP counts by this method were divided by 1.2 to compensate for the absence of the 2 mm thick agar block in the rotor cell. All grids were shadow-cast with chromium and examined in an RCA EMU 3 G electron microscope. Aggregation
determinations
Twenty randomly chosen electron microscopic fields of each preparation were photographed. Tabulations were made of numbers of single VP and the total number of particles from which the per cent of the total number of particles occurring singly (% singles) was calculated. Molality
Molality of the buffers was determined by the freezing point depression technique of Shoemaker & Garland (1967). CCA tests
CCA determinations were performed by the method of Miller & Stanley (1944) as modified by Tauraso et al. (1969) to include a standard reference. Vaccine/reference (V/R) ratios thus obtained were multiplied by the assigned value of the Lot 72 Reference (1886 CCA units/ml) and the results expressed as CCA units/ml. The standard diluent that was used in the CCA tests was 0.01 M-PBS unless otherwise specified in the text. RESULTS Particle counts
Preliminary EM inspection of the U.S. Reference influenza vaccine, Lot 72, revealed that the undiluted product was highly aggregated (Plate 1). Particles were tightly clumped in large massesor connected in long chains. Occasionally, groups were joined by slender filaments. Attempts to disperse the virus by sonication at various frequencies and time intervals with a Branson sonifier were not entirely successful, although some improvement was attained. Prolonged sonication (3 min) appeared to degrade the particles and increase aggregation. Choice of diluent, however, proved to have a marked effect on the state of virus aggregation as seen in Plate 2. A dilute phosphate buffer (0.001 M-PO& in water caused massive aggregates to form [Plate 2(A)]. Increasing the molarity to 0.01 I+PO~ in water reduced the size of the aggregates [Plate 2(B)]. A 0.01 M-PO, buffer prepared in normal saline greatly increased dispersion [Plate 2(C)]. Even better dispersion was attained by the use of McIlvaine’s buffer prepared in water [Plate 2(D)]. Consequently, McIlvaine’s buffer was used routinely for particle count preparations. Molality rather than pH (which was 7-O-7.2) or phosphate content appeared to be responsible for this aggregation phenomenon as there was a direct relationship between increasing molality and the percentage of single (non-aggregated) particles (Fig. 1). For example, 0.01 M-PO, in water (b) which had a molality of 0.01 was associated with only 10% single particles whereas the same buffer in saline (d) with a molality of 0.23 was associated with 64% single particles. There was no discernible difference in the tendency of either recombinants or nonrecombinants to form aggregates as shown in Table 1, where four A and four B strains were examined. Of the two preparations most highly aggregated, one was a nonrecombinant A strain (vaccine B) and the other was a recombinant B (vaccine C) strain. 283
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The remainder of the vaccines tested displayed similar degrees of dispersion regardless of strain or type. At the outset, counts were prepared by the agar-collodion pseudo-replica method. To test whether all the virus was being stripped off the agar, counts also were done by the direct deposition of the virus onto collodion-coated grids as described above. Table 2 contains counts of vaccines done by both methods. There was a considerable increase in particle count when the virus was sedimented directly onto the microscope grid indicating that residual virus remained on the agar block. The direct method was therefore adopted for routine influenza particle counting. The method of manufacture of influenza vaccines had an effect both on the EM appearance of the preparation and on the precision and reliability with which it could be
Mololity hoi/kg)
Fig. 1. Effect of molality of buffer on aggregation.
Molality was calculated from the freezing point depression produced by different buffers. (a) 0901 M-PO& (HsO); (b) 0.01 M-PO, (saline) 1 : 2; (d) 0.01 M-PO, (saline); M-PO, (H,O); (c) 0.001 M-PO, (H,O)+O*Ol (e) McIlvaine’s buffer in saline; (f) McIlvaine’s buffer (HsO); (g) McIlvaine’s (HaO)+ 0.01 M-PO, (saline) 1 : 2; (h) 0.1 M-PO, (saline).
TABLE
1. Measurement of aggregation in vaccines prepared binant or non-recombinant influenza strains Vaccine A* B C D Ref. 73 A B C
Strain
Type Recombinant Non-recombinant Non-recombinant Recombinant Non-recombinant Non-recombinant Non-recombinant Recombinant
Single particles (%) 58 33 65 49 59 60 57 38
Vaccines were diluted in O-01 M-PBS. * Letters A-D refer to commercial manufacturers.
284
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TABLE 2. Comparison of counts done by pseudo-replication from agar and by direct sedimentation onto coated grids Vaccine Ref. 72
D (monovalent A strain) A (bivalent A and B)
Agar
Grid
Increase (%)
I.80 x lo”* 0.72 x IO”
5.53 x 10” 2.57 x 10” 2.93 x 10”
207 257 150
1.17 x 1011
* = VP/ml.
quantitated by EM enumeration of VP. Vaccines purified by zonal centrifugation contained little extraneous debris and the virus particles were clearly defined [Plate 3(A)], while the chromatographically purified preparation contained more debris and had less clearly defined VP [Plate 3(B)]. Ether-extracted vaccines demonstrated degraded particles and recognition and differentiation were uncertain [Plate 3(C)], while the integrity of the VP in the TBP-treated vaccines was almost completely destroyed and only presumably subviral units were observed [Plate 3(D)]. Particle count and CCA content of vaaines Table 3 contains the particle counts obtained with several different vaccine preparations: Experimental vaccines (monovalent A) and commercial vaccines (bivalent A and B and monovalent B). Only vaccines purified by zonal centrifugation or chromatography are included in the table. Vaccines prepared by other methods could not be counted reliably. It will be noted that while the mean ratio of VP/CCA units was 1.40 x 10s, the range of this ratio was 0.60 x 108 to 2.34 x 10s indicating that there could be as much as a fourfold difference in the number of viral particles in two preparations containing the same number of CCA units. TABLE
Type Monovalent
3. Virus Vaccine
A
particle
content
of influenza
VP/ml
( x 10”)
CCA/rnl
VP/CCA (x lO8) 0.82 2.08 I.34 0.72
A* B C D
1.27 2.70 2.84 1.59
1540 1300 2120 2200
A B C D
I.80 3.03 5.13 4.54
2380 1980 2880 2100
vaccines
Mean = I.24 Bivalent A and B
0.76 1.53 I.78 2.16
Mean= I.56 Monovalent
B
A B C
0.74 I.51 2.69
0.60 I.24 2.34
1240 1220 1150
Mean =I*05 Total
mean
= 1.40
Particle counts are the average of five fields taken from each of eight grids (40 pictures). * Letters A-D refer to manufacturers.
285
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Eflect of virus aggregation on the CCA test
To determine whether the aggregation of VP noted on EM examination of the vaccine might affect the CCA results, three vaccine preparations were diluted in 0.001 M-PO, buffer to produce highly aggregated virus and in McIlvaine’s buffer for maximum dispersion of virus. These preparations were compared in the CCA test and aggregation counts were made. In order to prevent lysis of the chicken RBC by these buffers, however, the final dilutions of all preparations used in both the CCA test and the EM enumeration tests were mixed with equal volumes of 0.01 M-PBS. Although the addition of saline reduced the aggregation in the dilute phosphate buffer and increased the aggregation in the McIlvaine’s buffer, a twofold or more difference in the per cent single particles between the two preparations could still be obtained. Figure 2 depicts the relationship 300
!OOO
100 L$ 90 g
80
: .g
70 60
E
50
i
40
500
000
30
E .E 2
500
20 IO 0
1 x
Y
C(monovalent A)
I/
Y
x Ref.72 Vaccines
11
x
Y
0
Ref.73
Fig. 2. Effect of aggregation on CCA values. Solid bar = per cent single particles. X = 0.001 M-PO, (H,O) buffer as diluent. Y = Cross-hatched bar = CCA titer. C refers to the commercial manufacturer. 1 indicates McIlvaine’s buffer (HsO) as diluent. range of values from different tests.
found between aggregation and CCA value for the three vaccines. Increased dispersion raised the CCA value while highly aggregated virus was associated with a reduced CCA value. Calculations of the variability of the CCA test and the particle count procedure were made on repeated tests using the U.S. Reference vaccine, Lot 72. These data are shown in Table 4 and indicate that the two tests are approximately equal in reproducibility. TABLE
CCA Particle counts
4. Reproducibility of CCA test and particle counts
Mean
Number of tests
Standard deviation
1460 238*
161 45
215 34
* Virus particles/EM
286
picture.
950/0Confidence limits 1030-1890 170-306
Coefficient of error (“/6) 15 14
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DISCUSSION Direct EM enumeration of VP in influenza vaccines offers several advantages over indirect techniques of antigen quantitation which are subject to variations in the interaction of various strains of virus with either the RBC used in agglutination titrations (Fazekas de St Groth, 1949) or the particular animal species used in antigen extinction titrations (Nakamura, 1965). The technique also provides considerable insight and information concerning the morphology and structural integrity of the virus in such preparations as well as an estimate of the amount of extraneous non-viral material which might be in such vaccines. Several considerations must be borne in mind, however, before direct EM enumeration can be considered as an adjunct to currently employed methods of quantitation of influenza vaccine potency. The first is that the degree of aggregation of VP in the vaccines appears to be variable and unrelated to the type or strain of virus. Although Dalton, Kim & Sharp (1967) and Hirst & Pons (1973) have shown that aggregates of live virus particles are more effective in causing infection in vitro than single particles, the significance of such aggregation of inactivated virus to the immunogenicity of vaccine in humans is unknown. It may be speculated that large aggregates might increase the immunogenicity of influenza vaccines as Davenport (1968) has shown that increasing the size of subunit particles by adsorption to AlPO, markedly increased their immunogenicity in animals and possibly in man (Hennessy & Davenport, 1974). However, comparative studies of the immunogenicity of whole virus preparations adsorbed to AlPO, or in aggregates have not been performed. It is clear, however, that such aggregates affect agglutination titrations such as the CCA test, where the relatively low CCA values associated with highly aggregated preparations could be elevated if the preparations were suspended in a diluent of high molality which dispersed such aggregates. The use of high molality buffers also facilitated the precise quantitation of VP numbers in a preparation not only because aggregation prevents uniform distribution of particles but also because enumeration of the individual viral particles in an aggregate is exceedingly difficult. Donald & Isaacs (1954) noted that the relationship between agglutinating units and VP tends to remain relatively constant regardless of the strain being examined, with as much variation observed between examinations of the same strain on different days as between different strains. In the present investigation we found that a three- to fourfold variation in the ratio could be observed between vaccines of the same strains produced by different methods of manufacture but that the mean ratios of vaccines containing different strains varied only slightly. The mean ratio of 10sl is approximately 1 log,, higher than the value obtained by Donald & Isaacs (1954) and Tyrrell & Valentine (1957) and may be related to the lower absolute values obtained with the CCA test than with the end-point hemagglutination test used by the previous investigators (Tauraso & O’Brien, 1970). Because of the higher CCA values obtained when the vaccines were tested in buffers which increased dispersion (Fig. 2), CCA titers may be more related to the number of separate groups of particles than to total VP because aggregates composed of many VP may behave as a single active unit in virus-RBC interactions. The reproducibility of the direct VP count, nevertheless, was approximately that of the CCA test when a standard reference was employed (Tauraso et al., 1969). The advantages of direct sedimentation of the vaccine suspension onto the grid over previously described techniques of direct influenza virus enumeration are readily apparent. Stripping of the VP from the agar block by the use of collodion, as described by Sharp (1960), appeared to be incomplete, as considerably higher counts were obtained when the 287
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agar block was omitted. Spray techniques for distribution of the virus on grids (Luria, Williams & Backus, 1951) have the disadvantage of being subject to variations not only in the distance of the grid from the spray gun but also are affected by the bore of the gun, the force applied to create the spray and uneven distribution secondary to drying of the spray droplets. Enumeration of VP on laked RBC (Dawson & Elford, 1949) does not permit estimation of the contamination of the vaccine with non-virion material. The technique used in our study permitted detection of relatively large amounts of non-virion material in the chromatographically purified vaccine (D) which contained approximately three times as much protein as all other vaccines when tested by the method of Lowry, Rosebrough, Farr & Randall (1951). The technique is not sensitive enough, however, to detect small variations in non-virion protein content which could be determined better by either the Lowry technique or polyacrylamide gel electrophoresis (Perkins, 1973) but would be of use in determining whether or not vaccines high in total protein content had correspondingly large numbers of VP. While the technique also does not permit accurate quantitation of VP in vaccines where the virus has been subjected to disruption by Tween-80 and either TBP or ether, it does allow evaluation of the integrity and size of the particles which may severely affect the primary immunogenic capacity of a vaccine produced by these manufacturing methods (Barry, Staton & Mayner, 1974). It is clear that TBP produced much greater degradation than did ether. Studies are currently in progress in this laboratory to relate VP counts by EM with the immunogenicity in animals and humans in an attempt to expand our knowledge concerning the reliability and relevancy of this technique to the protection of man from influenza infection. Acknowledgement The authors are deeply appreciative of the advice and assistance of Dr Joan May in the freezing point determination experiments.
REFERENCES Barry, D. W., Staton, E. & Mayner, R. E. (1974). Inactivated influenza vaccine efficacy: Diminished antigenicity of split-product vaccines in mice. Infection and Immunity 10, 1329-1336. Dalton, D., Kim, K. S. & Sharp, D. G. (1967). Multiplicity activation of vaccinia virus in L cells. Proceedings of the National Academy of Sciences 58, 1758-1761. Davenport, F. M. (1968). Antigenic enhancement of ether-extracted influenza virus vaccines by AlPOd. Proceedings of the Society for Experimental Biology and Medicine 127, 587490. Dawson, I. M. & Elford, W. J. (1949). The investigation of influenza and related viruses in the electron microscope, by a new technique. Journal of General Microbiology 3, 298-3 11. Donald, H. B. & Isaacs, A. (1954). Counts of influenza virus particles. Journal of General Microbiology 10, 4.57-464. Fazekas de St Groth, S. (1949). Modification of virus receptors by metaperiodate. I. Properties of IO,-treated red cells. AustralianJburnal of Experimental Biological and Medical Science 27, 65-81. Fazekas de St Groth, S. (1962). The neutralization of viruses. Advances in Virus Research 9, l-125. HeIIer, L. (1967). The precision of three different types of response metometers used in mouse protection assays of the relative potencies of inactivated influenza virus vaccines. International Symposium on Biological Assay Methods for Vaccines and Sera 10, 33-42. 288
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Hennessy, A. V. & Davenport, F. M. (1974). Studies on vaccination of infants against influenza with influenza hemagglutinin. Proceedings of the Society for Experimental Biology and Medicine 146, 200-204. Hirst, G. K. & Pons, M. W. (1973). Mechanism of influenza recombination. II. Virus aggregation and its effect on plaque formation by so-called non-infective virus. Virology
56,620-631. Lowry, 0. H., Rosebrough, N. J ., Farr, A. L. & Randall, R. J. (195 1). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 26.5-275. Luria, S. E., Williams, R. C. & Backus, R. C. (1951). Electron micrographic counts of bacteriophage particles. Journal of Bacteriology 61, 179-188. McIlvaine, T. C. (1921). A buffer solution for calorimetric comparison. Journal of Biological Chemistry 49, 183-l 86. Miller, G. L. & Stanley, W. M. (1944). Q uantitative aspects of the red blood cell agglutination test of influenza virus. Journal of Experimental Medicine 79, 185-195. Nakamura, K. (1965). Pathogenicity and immunogenicity of various strains of influenza virus for mice. Bihen’sJournal 8, 155-165. Perkins, F. T. (1973). In: Report of an informal meeting of manufacturers and the Control Laboratory on Influenza Vaccine. Journal of Biological Standardization 1, 195-197. Schild, G. C., Henry-Aymard, M. & Pereira, H. G. (1972). A quantitative single radial diffusion test for immunological studies with influenza virus. JournaZ of General Virology
16, 231-236. Sharp, D. G. (1960). Sedimentation counting of particles via electron microscopy. Fourth International Conference on Electron Microscopy, Berlin 1958, pp. 542-548. Berlin: Springer-Verlag. Sharp, D. G. (1965). Quantitative use of the electron microscope in virus research. Methods and recent results of particle counting. Laboratory Investigation 14, 93-125. Shoemaker, D. P. & Garland, C. W. (1967). Experiments in Physical Chemistry, 2nd ed., pp. 141-146. New York: McGraw-Hill. Tauraso, N. M. & O’Brien, T. C. (1970). Comparison of the haemagglutinin production of influenza A2 Hong Kong variant and recombinant strains. Bulletin of the World Health Organization 43, 275-279. Tauraso, N. M., O’Brien, T. C. & Seligmann, E. B. (1969). Problems of influenza vaccine standardization. Bulletin of the World Health Organization 41, 497-506. Tyrrell, D. A. J. & Valentine, R. C. (1957). The assay of influenza virus particles by haemagglutination and electron microscopy. Journal of General Microbiology 16, 668-675.
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