Biologicals
(1990)
18, 39-44
Free Haemagglutinin in Inactivated Influenza Vaccines L. N. Kastrikina,
V. I. Minaev,
N. I. Lonskaya
Whole Virus
and G. 1. Bizhanovt
The Tarassevich State Research Institute for Standardization and Control of Medical Biological Preparations, 4 1 Sivtsev Vrazhek, 121002 Moscow, U.S. S.R.
Abstract.
While studying the haemagglutinin content of whole virus inactivated influenza vaccines by the single radial diffusion test and quantitative electron microscopy, it was found that not all haemagglutinin measured by single radial diffusion was bound to virions, a part of it being in a free state. The influence of unbound haemagglutinin on the immunogenicity of whole virus inactivated influenza vaccine is discussed. In addition. the use of single radial diffusion for the assessment of unbound haemagglutinin is suggested.
introduction Until now the vaccinal prophylaxis of influenza remains the protection of first choice against this disease and thus the evaluation of influenza vaccine quality and the methods of control are of great importance. According to the experimental data that are available, the optimal type of influenza viral vaccine (we deal here only with inactivated influenza vaccines: IIV) should include only those antigens necessary to evoke an efficient protective immunity and should be free from immunogenically inefficient components of the virus that may cause unwanted side-effects. Subunit vaccines (SU) containing membrane proteins of influenza virus, haemagglutinin (HA) and/or neuraminidase (NA), are most satisfactory in this respect. However, there are many reports of the relatively poor immunogenicity of these vaccine preparations1-3 relative to that of the whole virus (WV) vaccines although some data of SU and WV vaccines having similar immunogenicity are also available.4-7 To enhance the immunogenic response to SU vaccines, repeated administration of such preparations or adjuvants has been used.all The latter, along with the detergents used for SU vaccine production, are among the causes of reactogenicity and postvaccinal complications. On the contrary, the high immunogenicity of WV vaccines ensures that one dose only of such preparations is sufficient for immunization. When free from contamination with chick embryo proteins (which is possible, for example, by purification by the eluate-chromatographic method with subsequent immunosorbtion,i2J3 WV vaccines have a 1045%1056/90/010039+06
$03.00/O
real advantage at present over SU vaccines. In future, the most probable candidates for ideal vaccines appear to be synthetic antigens; however as yet these are only at the beginning of their development. In this study, data on the HA content of some experimental lots of WV vaccines assessed by the single radial diffusion (SRD) test and by quantitative electron microscopy are presented. Materials
and methods
Influenza vaccines
Experimental density gradient phy15 (bivalent 77(H3N2) and 385/80(H3N2),
lots of IIV were purified by sucrose centrifugationi4 and chromatogravaccines: Nib-G(HlNl), A/Texas/l/ monovalent vaccines: A/Leningrad respectively).
tiA content of vaccines
The SRD test? was used for the estimation HA content of the vaccines.
of the
Vu-ion concentration
Sedimentation method with electron microscopy17 was used for the assessment of virion concentration. Protein content of virions
The protein content of influenza lated according to the formula:18 cg = @ 1990 The
International
virions was calcu-
N x 0.6 x M x lo3
A Association
of Biological
9 Standardization
40
L. N. Kastrikina
where Cs = concentration of virus protein in mg/ml, N = concentration of virus particles per ml, M = influenza virus molecular mass (28 x 107D), A = Avogadro’s number (6 = 1023), and O-6 = the part of influenza virus molecular mass consisting of protein. HA content
of virions
The HA content (pg/ml) of the virus particles was calculated from the protein content of the influenza virions on the assumption that the former accounts for 30% of the latter. Electron microscopy
of k-ions
The integrity of virions in IIV was studied by the electron microscopy method of negative staining.lg Statistical
analysis
Standard errors of the means were calculated Student’s t-test.
by
Results The HA content of centrifuged viral preparations evaluated by the SRD test and electron microscopy Table 1. Haemagglutinin centrifugation Electron
Vaccine no. 1
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 M+m
Virion concentration x 1o1O (particles/ml)
5-63 + 1.29 5.43 + o-53 9.77 f 0.23 3.54 f 0.31 10.87 f 1.79 7.53 zk l-61 8-20 f 2.10 6.02 + O-66 6.05 + 1.05 11.01 + 0.85 11.56 f 1.41 9.15 + 1.62
11.50 2 l-31 7.35 + 0.99 10.58 f 1.65 9.83 + 0.37 5-35 f 0.59 7.24 + 0.91 5.24 3~O-27 7-99 AZ0.57
content
et al.
analysis was as shown in Table 1. According to the results obtained the HA content calculated from the concentration of viral particles and virus protein (electron microscopy) was 7.99 + 0.57 pg/ml while SRD test revealed a tenfold greater HA concentration (69.57 f 0.68 pg/ml). The reason for these differences obtained by the two methods is apparently a difference between methods that are used for HA content estimation. The SRD test evaluates the total HA, whereas electron microscopy reveals only HA that is bound to viral particles. In other words, the difference between quantity of HA associated with viral particles and the total HA of vaccines (the latter being about 10 times as great as the former) suggests that, in IIV preparations, the HA of virions (bound HA) is only a tenth of the total HA detected by SRD and that most of the HA is separated from the virions and dispersed in the solution (free HA). Consequently it is possible to suggest that the integrity of the surface glycoprotein layer in the majority of virions is disrupted. The justification for this assumption was obtained in the course of electron microscopy analysis of the prepar-
of bivalent
microscopy Viral protein concentration (pglml) 16
15 27 10 30 21 23 17 17 31
32 26 32 21 30 28 15 20 15 22.42 + 1.59
influenza
vaccines
purified
Single radial diffusion HA concentration calculated from viral protein concentration @g/ml)
Vaccine HA (HINI + H3N2) concentration (pglml)
5.28 4.95 8.91 3.30 9.90 6.93 7.59 5.61 5.61 10.23 10.56 8-58 10.56 6.93 9.90 9-24 4.95 6.60 4.95 7.40 f 1.53
68-O 72.7 64.6 74-9 69-8 64.3 67.3 72.7 70.2 71.7 68.4 71-4 71-l 71.2 66.2 69.8 67.3 67.2 73.1 68.57 rt 0.68
by
Free
41
haemagglutinin
greater quantity of bound HA than centrifuged vaccines. Moreover, there was a good coincidence of SRD and electron microscopy data relating to bound HA (columns 3 and 5 in Table 2) independently of per cent the bound HA makes up.
ations studied. In addition to virions with an intact surface antigen layer, it was possible to observe (Fig. 1) virions with a locally abnormal density of the glycoprotein layer explicable by spontaneous partial dissociation of HA from virions. The HA content of some experimental lots of chromatographic monovaccines was also evaluated. In this case both total and free HA were determined by SRD (the latter being determined in vaccine supernatants obtained after precipitation of the viral particles). By subtracting free HA from the total we obtained the bound HA. The results were presented in Table 2. Chromatographic vaccines contained a
Discussion Influenza virus HA is known to be the most important and strongest antigen of influenza virus but when separated from the virions it evokes a poor immune response. 20*21 As a reason for the poor immunogenicity of unbound HA (and in particular of SU vaccines),
Figure 1. Electron micrograph of influenza virus particles in inactivated stained with 3% phosphotungstic acid (X 160 000).
Table 2. Haemagglutinin graphy Electron Virion concentration Vaccine no. 1 2 3 4 5
x 1010
(particles/ml) 3-65 f O-17 o-94 f 0.19
o-95 2 0.18 O-80 + 0.10 0.59 2 0.11
content of monovalent
influenza
microscopy
(pghnl) k + IL + f
negatively
vaccines purified by chromato-
Single radial diffusion
Bound HA content 32-7 8-6 8.7 7.33 5.45
vaccine (centrifuged)
1x5 2.7 1.6 o-9 l-06
Total HA content Q&d) 29.32 14-19 17.85 11.56 8.16
k 5.18 f 2.51 f 3-15 zk 2-04 z!I l-44
Bound HA content (k&d) 29.32 10.11 9.35 6.54 5.61
* _t f + k
5.18 1.79 l-65 1.16 0.99
Bound HA in % of total HA content 100 71 52 57 69
42
L. N. Kastrikina
some authors suggest that the small size of the antigen evokes a narrow antibody spectrum compared to that induced by the administration of the proteinpolysacchrid+lipid complex of WV vaccines.22 A more important factor in HA immunogenicity may be its bond with the lipid bilayer which provides the membrane proteins with a spatial configuration optimal for antigenic activity. To potentiate the immune response against SU vaccines, different adjuvants (for increasing antigen size) and imitators of lipid bilayer (for reconstitution of the HA configuration similar to that in a native viral particle) have been used. It has been shown that the more a model structure of proteins and lipids (natural or synthetic) mimicks the spatial configuration of membrane proteins in viral particles the greater the immunogenicity.23-26 On the basis of these data it was logical to suppose that the immunogenicity of a vaccine containing predominantly free HA would be less than that of a vaccine with an intact surface glycoprotein layer. On the other hand, an increase of SU vaccine immunogenicity up to the level of WV vaccine was demonstrated after the addition of 5-10% of the whole virus.2740 The presence in our vaccine preparations of 10% and > 10% whole virions in centrifuged and chromatographic vaccines, respectively, consequently gives grounds for the suggestion that the immunogenicity of the preparations under study should be sufficiently high although this needs to be verified experimentally, especially in the light of a recent report of Jennings and coworkers31 that the administration of a mixture of detergent-disrupted influenza virus and inactivated whole virus vaccine induced lower serum antibody titres than equivalent or lower doses of WV vaccine alone. Thus, on studying the HA content of WV IIV by two methods in parallel (SRD and electron microscopy) it was established that not all HA measured by SRD was bound to viral particles, a part being in a free state. To our knowledge, the question of free HA in WV vaccines has not been touched upon in the literature. In our opinion, it is worthy of discussion as being of principal importance. First, the presence of free HA interferes with the standard characteristics of WV vaccines as preparations of a defined type. Second the free HA may influence the immunogenicity of WV vaccine; SRD, however, is recommended for the control of the HA content in IIV, that is of total HA: bound plus free, if the latter is present. Thus a situation is possible in which a vaccine preparation might contain the required quantity of HA according to SRD
et a/.
but its quality (provided free HA is present) might not meet the standard requirements. In this respect the SRD method has apparent limitations. The data obtained do not allow us to make broad generalizations as the investigation was carried out during the elaboration of a technological procedure for vaccine production with the aim of ascertaining the information relating to different methods of vaccine quality evaluation. Nevertheless, one can suggest that for a correct evaluation of WV vaccine immune potency, not only should the SRD test be used but other methods should also be used that control both the quantity and quality of HA. This implies the characterization of the structural integrity of the HA molecule and the strength of its bond with the virion. Among the criteria additional to SRD of HA quality evaluation, electron microscopy may secure one of the main places because it provides some quantitative characterization of influenza virus. In a comparative study of vaccines produced by different firms by electron microscopy and agglutination32 (CCA test) or immunoelectrophoresis and agglutination,33 a fourfold difference between viral particles number32 or HA quantity33 and the HA titre of vaccines equivalent in CCA was found. The agreement between electron microscopy and immunoelectrophoresis data allows us to conclude that, in respect of accuracy, both methods are identical. The good correlation of both SRD and electron microscopy data in the estimation of bound HA in chromatographic IIV studied also testifies to the high accuracy of the electron microscopy method. In addition to the calculation of viral particles using electron microscopy, we have made a further step in obtaining the antigenic characteristics of vaccines: the calculation of quantities of viral protein and HA. Moreover it has been shown that the possibilities of the SRD method might be extended by evaluating not only total but also free HA. References 1. Barry DW, Staton E, Mayner RE. Inactivated influenza vaccine efficacy: diminished antigenicity of split-product vaccines in mice. Infect Immun 1974; 10: 1329-1336. 2. Jennings R, Potter CW, McLaren C, Brady M. A new, surface antigen-adsorbed inf-Iuenza virus vaccine. I. Studies on immunogenicity in hamsters. J Hyg 1975; 75: 341-352. 3. Feery BJ, Gallichio HA, Rodda SJ, Hampson AW. Antibody responses to influenza vaccines containing A/uSSR/90/77. Aust J Exp Biol Med Sci 1979; 57: 335-344.
Free
43
haemagglutinin
4. Barry DW, Mayner RE, Staton E, Dunlap RC, Rastogi SC, Hannah JE, Blackburn RJ, Nortman DF, Graze PR. Comparative trial of influenza vaccines. I. Immunogenicity of whole virus and split product vaccines in man. Am J Epidemiol 1976; 104: 34-46. 5. Couch RB, Webster RG, Kasel JA, Cate TR. Efficacy of purified influenza subunit vaccines and relation to the major antigenic determinants on the hemagglutinin molecule. J Infect Dis 1979; 140: 553-559. 6. Kasel JA, Couch RB, Six HR, Knight V. Antigenicity of licensed whole virion and subvirion influenza vaccines in ‘high risk” persons. Proc Sot Exp Biol Med 1976; 151: 742-747. 7. Webster RG, Kasel JA, Couch RB, Laver WG. Influenza virus subunit vaccines. 2. Immunogenicity and original antigenic sin in humans. J Infect Dis 1976; 134: 48-58. 8. Allison AC. Model of action of immunological acljuvants. J Reticuloendothel Sot 1979; 26: 619-630. 9. Arnon R, Sela M, Parant M, Chedid L. Antiviral response elicited by a completely synthetic antigen with built-in acljuvanticity. Proc Nat1 Acad Sci USA 1980; 77: 6769-6772. 10. Gross PA, Ennis FA. Influenza vaccine: split product versus whole-virus types-how do they differ? New Engl J Med 1977; 296: 567-568. 11. Eastwood LM, Jennings R, Milner RDG, Potter CW. Reactogenicity and immunogenicity of a surfaceantigen-adsorbed influenza virus vaccine in children. J Clin Path01 1979; 32: 534-537. 12. Bichurina MA, Corunova VV, Chubarova NI, Zhebrun AB, Noscov FS, Lonskaya NI, Fridman EA. Comparative study of different methods for ovalbumin assessment in purified influenza virus preparations. Voprosy Virusologiy 1981; 2: 228-230. 13. Zhebrun AB, Polyanaskaya NYU, Noscov FS, Fridman EA. Host antigens in purified influenza virus preparations. Etiology and specific prophylaxis of influenza. Trans Pasteur Instit Sci Papers, Leningrad 1982; 59: 70-81. 14. Igamberdiev VM, Yakovleva NV, Sokolov NN. Securing conditions for biological activity preservation of influenza virus during its purification and concentration by centrifugical methods. Inactivated influenza vaccine. Trans Pasteur Inst Sci Papers, Leningrad 1976; 47: 65-70. 15. Bresler SE, Kolikov VM, Molodkin VM, Mchedlishvili BV, Katushkina NV, Anistchenko LM, Malafeeva LK, Bespalova GI, Fridman EA, Peradze TV, Zheleznova NV. Chromatographic purification and concentration of influenza virus allantoic culture (strain A2136 Victoriai72). Respiratory viral infections. Trans Pasteur Instit Sci Papers, Leningrad 1973; 42: 22-25. 16. Schild GC, Wood JM, Newman RW. A single-radialimmunodiffusion technique for the assay of influenza haemagglutinin antigen. Bull WHO 1975; 52: 223-231. 17. Minaev VI. Modification of chambers for sedimentation method of viral particle calculation in electron microscope. Standards, . strains and viral ._- and bacterial preparation control methods. ‘I’rans Tarassevich Instit Sci Papers, Moscow 1981; 108-112. 18. Potokin IL, Bresler SE, Katushkina NV, Kolikov VM, Vinogradskaya GR. Purity control of influenza virus
vaccinal chromatographic preparations. Inactivated influenza vaccine. Trans Pasteur Instit Sci Papers, Leningrad 1976; 47:49-53. 19. Brenner S, Horne RW. A negative staining method for high resolution electron microscopy of viruses. Biophys Biochem Acta 1959; 34: 103-110. 20. Jennings R, Brand CM, McLaren C, Shephard L, Potter CW. The immune response of hamsters to purified haemagglutinins and whole influenza virus vaccines following live influenza virus infection. Med Microbial Imunol 1974; 160: 295-309. 21. Kuo YC, Oxford JS, Schild GC. Immunological studies with the HA1 and HA2 polypeptides of influenza A virus haemagglutinin. Exp Cell Biol 1978; 46: 338354. 22. Fridman EA, Peradze TV. The main directions of influenza research in Pasteur Institute. Etiology and specific prophylaxis of influenza. Trans Pasteur Instit Sci Papers, Leningrad 1982; 59: 3-6. 23. Morein B, Simons K. Subunit vaccines against enveloped viruses: virosomes, micelles and other protein complexes. Vaccine 1985; 3: 83-93. 24. Oxford JS, Hockley DJ, Heath TD, Patterson S. The interaction of influenza virus haemagglutinin with phospholipicl vesicles-morphological and immunological studies. J Gen Virol 1981; 52: 329-343. 25. Thibodeau L, Naud P, Boudreault A. An influenza immunosome: its structure and antigenic properties. A model for a new type of vaccine. In: Nayak, DP, ea., Genetic variation among influenza viruses. New York, London: Academic Press, 1981: 587-600. 26. Almeida JD, Brand CM, Edwards DC, Heath TD. Formation of virosomes from influenza subunits and liposomes. Lancet 1975; ii: 899-901. 27. Frank AL, Webster RG, Glezen WP, Cate TR. Immunogenicity of influenza A/USSR(HINI) subunit vaccine in unprimecl young adults. J Med Virol 1981; 7: 135142. 28. Glezen WP, Kasel JA, Webster RG, Taber LH. Alternative approaches to immunization of children with inactivated influenza virus vaccines. J Infect Dis 1977; 136: S677-S682. 29. McLaren C, Verbonitz MW, Daniel S, Grubb SE, Ennis FA. Effect of priming infection on serologic response to whole and subunit influenza virus vaccine in animals. J Infect Dis 1977; 136s: S706S711. 30. Webster RG, Glezen WP, Hannoun C, Laver WG. Potentiation of immune response to influenza virus subunit vaccines. J Immunol 1977; 119: 2073-2077. 31. Jennings R, Pemberton RM, Smith TL, Amin T, Potter CW. Demonstration of an immunosuppressive action of detergent-disrupted influenza virus on the antibody response to inactivated whole virus vaccine. J Gen Virol 1987; 68: 441-450. 32. Dunlap RC, Brown ER, Barry DW. Determination of the viral particle content of influenza vaccines by electron microscopy. J Biol Stand 1975; 3: 281-289. 33. Mayner RE, Blackburn RJ, Barry DW. Quantitation of influenza vaccine hemagglutinin by immunoelectrophoresis. Dev Biol Stand 1977; 39: 169-178. Received for publication 16 November accepted 18 July 1989.
1988;
(1990)
18, 39-44
Free Haemagglutinin in Inactivated Influenza Vaccines L. N. Kastrikina,
V. I. Minaev,
N. I. Lonskaya
Whole Virus
and G. 1. Bizhanovt
The Tarassevich State Research Institute for Standardization and Control of Medical Biological Preparations, 4 1 Sivtsev Vrazhek, 121002 Moscow, U.S. S.R.
Abstract.
While studying the haemagglutinin content of whole virus inactivated influenza vaccines by the single radial diffusion test and quantitative electron microscopy, it was found that not all haemagglutinin measured by single radial diffusion was bound to virions, a part of it being in a free state. The influence of unbound haemagglutinin on the immunogenicity of whole virus inactivated influenza vaccine is discussed. In addition. the use of single radial diffusion for the assessment of unbound haemagglutinin is suggested.
introduction Until now the vaccinal prophylaxis of influenza remains the protection of first choice against this disease and thus the evaluation of influenza vaccine quality and the methods of control are of great importance. According to the experimental data that are available, the optimal type of influenza viral vaccine (we deal here only with inactivated influenza vaccines: IIV) should include only those antigens necessary to evoke an efficient protective immunity and should be free from immunogenically inefficient components of the virus that may cause unwanted side-effects. Subunit vaccines (SU) containing membrane proteins of influenza virus, haemagglutinin (HA) and/or neuraminidase (NA), are most satisfactory in this respect. However, there are many reports of the relatively poor immunogenicity of these vaccine preparations1-3 relative to that of the whole virus (WV) vaccines although some data of SU and WV vaccines having similar immunogenicity are also available.4-7 To enhance the immunogenic response to SU vaccines, repeated administration of such preparations or adjuvants has been used.all The latter, along with the detergents used for SU vaccine production, are among the causes of reactogenicity and postvaccinal complications. On the contrary, the high immunogenicity of WV vaccines ensures that one dose only of such preparations is sufficient for immunization. When free from contamination with chick embryo proteins (which is possible, for example, by purification by the eluate-chromatographic method with subsequent immunosorbtion,i2J3 WV vaccines have a 1045%1056/90/010039+06
$03.00/O
real advantage at present over SU vaccines. In future, the most probable candidates for ideal vaccines appear to be synthetic antigens; however as yet these are only at the beginning of their development. In this study, data on the HA content of some experimental lots of WV vaccines assessed by the single radial diffusion (SRD) test and by quantitative electron microscopy are presented. Materials
and methods
Influenza vaccines
Experimental density gradient phy15 (bivalent 77(H3N2) and 385/80(H3N2),
lots of IIV were purified by sucrose centrifugationi4 and chromatogravaccines: Nib-G(HlNl), A/Texas/l/ monovalent vaccines: A/Leningrad respectively).
tiA content of vaccines
The SRD test? was used for the estimation HA content of the vaccines.
of the
Vu-ion concentration
Sedimentation method with electron microscopy17 was used for the assessment of virion concentration. Protein content of virions
The protein content of influenza lated according to the formula:18 cg = @ 1990 The
International
virions was calcu-
N x 0.6 x M x lo3
A Association
of Biological
9 Standardization
40
L. N. Kastrikina
where Cs = concentration of virus protein in mg/ml, N = concentration of virus particles per ml, M = influenza virus molecular mass (28 x 107D), A = Avogadro’s number (6 = 1023), and O-6 = the part of influenza virus molecular mass consisting of protein. HA content
of virions
The HA content (pg/ml) of the virus particles was calculated from the protein content of the influenza virions on the assumption that the former accounts for 30% of the latter. Electron microscopy
of k-ions
The integrity of virions in IIV was studied by the electron microscopy method of negative staining.lg Statistical
analysis
Standard errors of the means were calculated Student’s t-test.
by
Results The HA content of centrifuged viral preparations evaluated by the SRD test and electron microscopy Table 1. Haemagglutinin centrifugation Electron
Vaccine no. 1
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 M+m
Virion concentration x 1o1O (particles/ml)
5-63 + 1.29 5.43 + o-53 9.77 f 0.23 3.54 f 0.31 10.87 f 1.79 7.53 zk l-61 8-20 f 2.10 6.02 + O-66 6.05 + 1.05 11.01 + 0.85 11.56 f 1.41 9.15 + 1.62
11.50 2 l-31 7.35 + 0.99 10.58 f 1.65 9.83 + 0.37 5-35 f 0.59 7.24 + 0.91 5.24 3~O-27 7-99 AZ0.57
content
et al.
analysis was as shown in Table 1. According to the results obtained the HA content calculated from the concentration of viral particles and virus protein (electron microscopy) was 7.99 + 0.57 pg/ml while SRD test revealed a tenfold greater HA concentration (69.57 f 0.68 pg/ml). The reason for these differences obtained by the two methods is apparently a difference between methods that are used for HA content estimation. The SRD test evaluates the total HA, whereas electron microscopy reveals only HA that is bound to viral particles. In other words, the difference between quantity of HA associated with viral particles and the total HA of vaccines (the latter being about 10 times as great as the former) suggests that, in IIV preparations, the HA of virions (bound HA) is only a tenth of the total HA detected by SRD and that most of the HA is separated from the virions and dispersed in the solution (free HA). Consequently it is possible to suggest that the integrity of the surface glycoprotein layer in the majority of virions is disrupted. The justification for this assumption was obtained in the course of electron microscopy analysis of the prepar-
of bivalent
microscopy Viral protein concentration (pglml) 16
15 27 10 30 21 23 17 17 31
32 26 32 21 30 28 15 20 15 22.42 + 1.59
influenza
vaccines
purified
Single radial diffusion HA concentration calculated from viral protein concentration @g/ml)
Vaccine HA (HINI + H3N2) concentration (pglml)
5.28 4.95 8.91 3.30 9.90 6.93 7.59 5.61 5.61 10.23 10.56 8-58 10.56 6.93 9.90 9-24 4.95 6.60 4.95 7.40 f 1.53
68-O 72.7 64.6 74-9 69-8 64.3 67.3 72.7 70.2 71.7 68.4 71-4 71-l 71.2 66.2 69.8 67.3 67.2 73.1 68.57 rt 0.68
by
Free
41
haemagglutinin
greater quantity of bound HA than centrifuged vaccines. Moreover, there was a good coincidence of SRD and electron microscopy data relating to bound HA (columns 3 and 5 in Table 2) independently of per cent the bound HA makes up.
ations studied. In addition to virions with an intact surface antigen layer, it was possible to observe (Fig. 1) virions with a locally abnormal density of the glycoprotein layer explicable by spontaneous partial dissociation of HA from virions. The HA content of some experimental lots of chromatographic monovaccines was also evaluated. In this case both total and free HA were determined by SRD (the latter being determined in vaccine supernatants obtained after precipitation of the viral particles). By subtracting free HA from the total we obtained the bound HA. The results were presented in Table 2. Chromatographic vaccines contained a
Discussion Influenza virus HA is known to be the most important and strongest antigen of influenza virus but when separated from the virions it evokes a poor immune response. 20*21 As a reason for the poor immunogenicity of unbound HA (and in particular of SU vaccines),
Figure 1. Electron micrograph of influenza virus particles in inactivated stained with 3% phosphotungstic acid (X 160 000).
Table 2. Haemagglutinin graphy Electron Virion concentration Vaccine no. 1 2 3 4 5
x 1010
(particles/ml) 3-65 f O-17 o-94 f 0.19
o-95 2 0.18 O-80 + 0.10 0.59 2 0.11
content of monovalent
influenza
microscopy
(pghnl) k + IL + f
negatively
vaccines purified by chromato-
Single radial diffusion
Bound HA content 32-7 8-6 8.7 7.33 5.45
vaccine (centrifuged)
1x5 2.7 1.6 o-9 l-06
Total HA content Q&d) 29.32 14-19 17.85 11.56 8.16
k 5.18 f 2.51 f 3-15 zk 2-04 z!I l-44
Bound HA content (k&d) 29.32 10.11 9.35 6.54 5.61
* _t f + k
5.18 1.79 l-65 1.16 0.99
Bound HA in % of total HA content 100 71 52 57 69
42
L. N. Kastrikina
some authors suggest that the small size of the antigen evokes a narrow antibody spectrum compared to that induced by the administration of the proteinpolysacchrid+lipid complex of WV vaccines.22 A more important factor in HA immunogenicity may be its bond with the lipid bilayer which provides the membrane proteins with a spatial configuration optimal for antigenic activity. To potentiate the immune response against SU vaccines, different adjuvants (for increasing antigen size) and imitators of lipid bilayer (for reconstitution of the HA configuration similar to that in a native viral particle) have been used. It has been shown that the more a model structure of proteins and lipids (natural or synthetic) mimicks the spatial configuration of membrane proteins in viral particles the greater the immunogenicity.23-26 On the basis of these data it was logical to suppose that the immunogenicity of a vaccine containing predominantly free HA would be less than that of a vaccine with an intact surface glycoprotein layer. On the other hand, an increase of SU vaccine immunogenicity up to the level of WV vaccine was demonstrated after the addition of 5-10% of the whole virus.2740 The presence in our vaccine preparations of 10% and > 10% whole virions in centrifuged and chromatographic vaccines, respectively, consequently gives grounds for the suggestion that the immunogenicity of the preparations under study should be sufficiently high although this needs to be verified experimentally, especially in the light of a recent report of Jennings and coworkers31 that the administration of a mixture of detergent-disrupted influenza virus and inactivated whole virus vaccine induced lower serum antibody titres than equivalent or lower doses of WV vaccine alone. Thus, on studying the HA content of WV IIV by two methods in parallel (SRD and electron microscopy) it was established that not all HA measured by SRD was bound to viral particles, a part being in a free state. To our knowledge, the question of free HA in WV vaccines has not been touched upon in the literature. In our opinion, it is worthy of discussion as being of principal importance. First, the presence of free HA interferes with the standard characteristics of WV vaccines as preparations of a defined type. Second the free HA may influence the immunogenicity of WV vaccine; SRD, however, is recommended for the control of the HA content in IIV, that is of total HA: bound plus free, if the latter is present. Thus a situation is possible in which a vaccine preparation might contain the required quantity of HA according to SRD
et a/.
but its quality (provided free HA is present) might not meet the standard requirements. In this respect the SRD method has apparent limitations. The data obtained do not allow us to make broad generalizations as the investigation was carried out during the elaboration of a technological procedure for vaccine production with the aim of ascertaining the information relating to different methods of vaccine quality evaluation. Nevertheless, one can suggest that for a correct evaluation of WV vaccine immune potency, not only should the SRD test be used but other methods should also be used that control both the quantity and quality of HA. This implies the characterization of the structural integrity of the HA molecule and the strength of its bond with the virion. Among the criteria additional to SRD of HA quality evaluation, electron microscopy may secure one of the main places because it provides some quantitative characterization of influenza virus. In a comparative study of vaccines produced by different firms by electron microscopy and agglutination32 (CCA test) or immunoelectrophoresis and agglutination,33 a fourfold difference between viral particles number32 or HA quantity33 and the HA titre of vaccines equivalent in CCA was found. The agreement between electron microscopy and immunoelectrophoresis data allows us to conclude that, in respect of accuracy, both methods are identical. The good correlation of both SRD and electron microscopy data in the estimation of bound HA in chromatographic IIV studied also testifies to the high accuracy of the electron microscopy method. In addition to the calculation of viral particles using electron microscopy, we have made a further step in obtaining the antigenic characteristics of vaccines: the calculation of quantities of viral protein and HA. Moreover it has been shown that the possibilities of the SRD method might be extended by evaluating not only total but also free HA. References 1. Barry DW, Staton E, Mayner RE. Inactivated influenza vaccine efficacy: diminished antigenicity of split-product vaccines in mice. Infect Immun 1974; 10: 1329-1336. 2. Jennings R, Potter CW, McLaren C, Brady M. A new, surface antigen-adsorbed inf-Iuenza virus vaccine. I. Studies on immunogenicity in hamsters. J Hyg 1975; 75: 341-352. 3. Feery BJ, Gallichio HA, Rodda SJ, Hampson AW. Antibody responses to influenza vaccines containing A/uSSR/90/77. Aust J Exp Biol Med Sci 1979; 57: 335-344.
Free
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haemagglutinin
4. Barry DW, Mayner RE, Staton E, Dunlap RC, Rastogi SC, Hannah JE, Blackburn RJ, Nortman DF, Graze PR. Comparative trial of influenza vaccines. I. Immunogenicity of whole virus and split product vaccines in man. Am J Epidemiol 1976; 104: 34-46. 5. Couch RB, Webster RG, Kasel JA, Cate TR. Efficacy of purified influenza subunit vaccines and relation to the major antigenic determinants on the hemagglutinin molecule. J Infect Dis 1979; 140: 553-559. 6. Kasel JA, Couch RB, Six HR, Knight V. Antigenicity of licensed whole virion and subvirion influenza vaccines in ‘high risk” persons. Proc Sot Exp Biol Med 1976; 151: 742-747. 7. Webster RG, Kasel JA, Couch RB, Laver WG. Influenza virus subunit vaccines. 2. Immunogenicity and original antigenic sin in humans. J Infect Dis 1976; 134: 48-58. 8. Allison AC. Model of action of immunological acljuvants. J Reticuloendothel Sot 1979; 26: 619-630. 9. Arnon R, Sela M, Parant M, Chedid L. Antiviral response elicited by a completely synthetic antigen with built-in acljuvanticity. Proc Nat1 Acad Sci USA 1980; 77: 6769-6772. 10. Gross PA, Ennis FA. Influenza vaccine: split product versus whole-virus types-how do they differ? New Engl J Med 1977; 296: 567-568. 11. Eastwood LM, Jennings R, Milner RDG, Potter CW. Reactogenicity and immunogenicity of a surfaceantigen-adsorbed influenza virus vaccine in children. J Clin Path01 1979; 32: 534-537. 12. Bichurina MA, Corunova VV, Chubarova NI, Zhebrun AB, Noscov FS, Lonskaya NI, Fridman EA. Comparative study of different methods for ovalbumin assessment in purified influenza virus preparations. Voprosy Virusologiy 1981; 2: 228-230. 13. Zhebrun AB, Polyanaskaya NYU, Noscov FS, Fridman EA. Host antigens in purified influenza virus preparations. Etiology and specific prophylaxis of influenza. Trans Pasteur Instit Sci Papers, Leningrad 1982; 59: 70-81. 14. Igamberdiev VM, Yakovleva NV, Sokolov NN. Securing conditions for biological activity preservation of influenza virus during its purification and concentration by centrifugical methods. Inactivated influenza vaccine. Trans Pasteur Inst Sci Papers, Leningrad 1976; 47: 65-70. 15. Bresler SE, Kolikov VM, Molodkin VM, Mchedlishvili BV, Katushkina NV, Anistchenko LM, Malafeeva LK, Bespalova GI, Fridman EA, Peradze TV, Zheleznova NV. Chromatographic purification and concentration of influenza virus allantoic culture (strain A2136 Victoriai72). Respiratory viral infections. Trans Pasteur Instit Sci Papers, Leningrad 1973; 42: 22-25. 16. Schild GC, Wood JM, Newman RW. A single-radialimmunodiffusion technique for the assay of influenza haemagglutinin antigen. Bull WHO 1975; 52: 223-231. 17. Minaev VI. Modification of chambers for sedimentation method of viral particle calculation in electron microscope. Standards, . strains and viral ._- and bacterial preparation control methods. ‘I’rans Tarassevich Instit Sci Papers, Moscow 1981; 108-112. 18. Potokin IL, Bresler SE, Katushkina NV, Kolikov VM, Vinogradskaya GR. Purity control of influenza virus
vaccinal chromatographic preparations. Inactivated influenza vaccine. Trans Pasteur Instit Sci Papers, Leningrad 1976; 47:49-53. 19. Brenner S, Horne RW. A negative staining method for high resolution electron microscopy of viruses. Biophys Biochem Acta 1959; 34: 103-110. 20. Jennings R, Brand CM, McLaren C, Shephard L, Potter CW. The immune response of hamsters to purified haemagglutinins and whole influenza virus vaccines following live influenza virus infection. Med Microbial Imunol 1974; 160: 295-309. 21. Kuo YC, Oxford JS, Schild GC. Immunological studies with the HA1 and HA2 polypeptides of influenza A virus haemagglutinin. Exp Cell Biol 1978; 46: 338354. 22. Fridman EA, Peradze TV. The main directions of influenza research in Pasteur Institute. Etiology and specific prophylaxis of influenza. Trans Pasteur Instit Sci Papers, Leningrad 1982; 59: 3-6. 23. Morein B, Simons K. Subunit vaccines against enveloped viruses: virosomes, micelles and other protein complexes. Vaccine 1985; 3: 83-93. 24. Oxford JS, Hockley DJ, Heath TD, Patterson S. The interaction of influenza virus haemagglutinin with phospholipicl vesicles-morphological and immunological studies. J Gen Virol 1981; 52: 329-343. 25. Thibodeau L, Naud P, Boudreault A. An influenza immunosome: its structure and antigenic properties. A model for a new type of vaccine. In: Nayak, DP, ea., Genetic variation among influenza viruses. New York, London: Academic Press, 1981: 587-600. 26. Almeida JD, Brand CM, Edwards DC, Heath TD. Formation of virosomes from influenza subunits and liposomes. Lancet 1975; ii: 899-901. 27. Frank AL, Webster RG, Glezen WP, Cate TR. Immunogenicity of influenza A/USSR(HINI) subunit vaccine in unprimecl young adults. J Med Virol 1981; 7: 135142. 28. Glezen WP, Kasel JA, Webster RG, Taber LH. Alternative approaches to immunization of children with inactivated influenza virus vaccines. J Infect Dis 1977; 136: S677-S682. 29. McLaren C, Verbonitz MW, Daniel S, Grubb SE, Ennis FA. Effect of priming infection on serologic response to whole and subunit influenza virus vaccine in animals. J Infect Dis 1977; 136s: S706S711. 30. Webster RG, Glezen WP, Hannoun C, Laver WG. Potentiation of immune response to influenza virus subunit vaccines. J Immunol 1977; 119: 2073-2077. 31. Jennings R, Pemberton RM, Smith TL, Amin T, Potter CW. Demonstration of an immunosuppressive action of detergent-disrupted influenza virus on the antibody response to inactivated whole virus vaccine. J Gen Virol 1987; 68: 441-450. 32. Dunlap RC, Brown ER, Barry DW. Determination of the viral particle content of influenza vaccines by electron microscopy. J Biol Stand 1975; 3: 281-289. 33. Mayner RE, Blackburn RJ, Barry DW. Quantitation of influenza vaccine hemagglutinin by immunoelectrophoresis. Dev Biol Stand 1977; 39: 169-178. Received for publication 16 November accepted 18 July 1989.
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