Antigenicity of Influenza Vaccine from Bovine Cell Cultures

APPLIED MICROBIOLOGY, Oct. 1969, p. 596-600 Copyright 1969 American Society for Microbiology Vol. 18, No. 4 Printed in U.S.A. Antigenicity of Influenza Vaccine from Bovine Cell Cultures EDUARDO LEIDERMAN1 AND WILLIAM J. MOGABGAB Department of Medicine, Tulane University School of Medicine, New Orleans, Louisiana 70112 Received for publication 26 June 1969 An experimental vaccine prepared from influenza virus strains propagated in bovine kidney cell cultures, purified by zonal centrifugation, and further treated with ether was studied in man for the incidence of clinical reactions and hemagglutination-inhibition antibody levels induced. The results were equivalent to those obtained in a simultaneous study made with a commercially licensed influenza vaccine derived from viruses propagated in the embryonated egg and also purified by zonal centrifugation, but not treated with ether. Comparison of the macromethod and the micromethod for determination of hemagglutination-inhibition antibody titers revealed that lower initial titers and lesser increments in antibody levels following vaccination were obtained by the microtechnique. Influenza vaccines have been conventionally prepared with viruses propagated in embryonated chicken eggs. Although the allantoic sac has provided a suitable medium for growth of all influenza A and B viruses in quantities sufficient for vaccine production, there are certain residual problems that suggest the desirability of additional or substitute methods. Allergy to chickens or their eggs, though uncommon, precludes the use of all presently available vaccines. Some of these egg-allergic persons need annual vaccination because they are among the "high risk group," and all allergic individuals eventually will be in this vulnerable state as they become older (6). Furthermore, there are still technical difficulties present in harvesting allantoic fluids and maintaining sterility. Safety of embryonated chicken egg vaccines is generally accepted, but there are questions regarding the potential of contaminating avian leukosis virus even in inactivated vaccines. Production of purified vaccines has reduced the intensity of local and systemic reactions markedly and provided more uniform concentrations of antigen (1, 8). Described in this report are the local and systemic reactions and antibody responses to two purified influenza vaccines, one derived from bovine kidney cell culture-propagated viruses and the other from the chorioallantoic fluids of the chicken embryo. It will be shown that vaccines derived from viruses propagated in bovine kidney cell cultures is equivalent to vaccine prepared from chicken embryos. I W. K. Kellogg Foundation Fellow. 596 MATERIALS AND METHODS Vaccines. Bovine kidney cell culture vaccine was provided by the Infectious Disease Research Division, Abbott Laboratories, North Chicago, Ill. Viruses for this vaccine were propagated in primary kidney cell cultures from newborn calves. Culture fluids were concentrated and purified by density gradient centrifugation in sucrose, by use of zonal centrifuge equipment. The purified concentrates were subjected to ether treatment and inactivated with formaldehyde (J. L. Gerin et al., Fed. Proc., p. 365, 1968). The vaccine contained: influenza A2/Japan 170/62, 150 chicken cell agglutinating (CCA) units; influenza A2/Taiwan 1/64, 150 CCA units; and influenza B/ Massachusetts 3/66, 300 CCA units, in 0.5 ml of aqueous suspension (4). The vaccine had been safety tested by following the requirements of the Division of Biologics Standards, National Institutes of Health, Bethesda, Md. Chicken embryo vaccine (Zonomune, Eli Lilly and Co.) was a licensed, commercially obtained lot that had been prepared with chicken embryo-propagated viruses and purified by zonal centrifugation (8). It contained the same concentrations and types of viruses as noted for the bovine kidney cell vaccine described above. Vaccinations, observations for reactions, and bleeding plan. A total of 257 volunteers, comprising Tulane University students and staff, aged 20 to 73 years with a median age of 27 years, were given alternately 0.5 ml each of either bovine cell or chicken embryo vaccine. Injection was subcutaneous in the deltoid area. Prior to administering vaccine, oral temperatures were recorded and subjects were questioned concerning allergy to chicken eggs, beef, or any biological products. All individuals were examined and questioned at 24 and 48 hr after vaccination for evidence of local or systemic reactions, and oral temperatures were again

VOL. 18, 1969 ANTIGENICITY OF INFLUENZA VACCINE recorded. Blood specimens were obtained immediately prior to vaccination and 3 weeks later. These procedures were completed during the fall of 1968, prior to the appearance of influenza-like illnesses in this population or the detection of influenza A2 (Hong Kong) in the community. Antibody determinations. All sera were treated with an equal volume of receptor-destroying enzyme (RDE) that had been prepared as a broth culture filtrate of Vibrio cholerae. These sera were then incubated at 37 C overnight and heated at 56 C for 30 min. Viruses propagated in the chicken embryo included those contained in the vaccines and the influenza A2/Aichi 2/68 (Hong Kong) strain (6). For hemagglutinationinhibition antibody determinations, four hemagglutinating units of virus were added to twofold dilutions of sera, and the mixture was incubated at room temperature for 30 min. Inhibition titers were determined at 4 C with human type 0 erythrocytes at a final concentration of 0.25%. The final total volume was 1 ml in tubes with hemispherical bottoms with an internal diameter of 10 mm (macrotechnique). Titers were recorded as the greatest initial dilution of antiserum that inhibited hemagglutination. Microserology. Hemagglutination-inhibition antibody titers were also determined by a microtechnique (10). Takatsy "tulip type" loops (0.025 ml; Cooke Engineering Co., Alexandria, Va.) and disposable plastic trays, with 96 V-shaped wells of 0.3 ml capacity each (No. IS-MVC-96, Linbro Chemical Co., New Haven, Conn.), were used. Diluent was distributed in the trays with 0.025-ml calibrated dropper pipettes (Cooke Engineering Co.), and sera were added and manually diluted with the loops in groups of eight. RESULTS Incidence of local and systemic reactions. Comparison of reactions to bovine cell and chicken embryo vaccines is shown in Table 1. Systemic manifestations were few and mild after either vaccine. Only three persons in each group had a temperature elevation. Other symptoms, such as headache, malaise, and chills, were minimal and infrequent and had usually disappeared by 48 hr. Pain at the site of injection was absent or mild in most subjects. Some degree of erythema was observed in about half of all subjects. Edema and induration in the inoculation area was found in 20% of the individuals. Antigenicity of bovine kidney cell vaccine. The distributions of hemagglutination-inhibition antibody titers to the viruses contained in the bovine cell vaccine are shown in Table 2. Relatively high prevaccination antibody titers were detected by all three antigens, and increases in titer were not of great magnitude. However, final mean antibody titers were high. Lower levels of antibody were observed with influenza A2/Aichi 2/68, and some response occurred even though this virus was not included in the vaccine. The data were analyzed to determine the rela- 597 TABLE 1. Incidence of reactions to bovine cell and chicken embryo influenza vaccines Per cent with reaction after vaccination with a Reaction Degree of Bovine cell Chicken emvaccine bryo vaccine 24 hr 48 hr 24 hr 48 hr Systemic Tempera- >99-2.3 0 2.4 2.4 ture ele- 100.6 F vation Headache 6.8 0.8 1.8 2.4 Malaise 5.2 2.4 5.7 6.7 Chills 3.8 0.8 0 0 Local Pain Severe 2.4 0.8 0 0 Mild 35.4 15.5 54.9 21.2 Calor 52.6 27.2 62.1 30.1 Erythema 1-5 cm 40.6 '34.1 34.9 26.0 6-10 3.8 4.7 11.5 3.2 Swelling 1-5 cm 10.6 8.5 21.8 7.4 6-10 0 1.6 0 1.6 Induration 1-5 cm 8.3 12.4 16.2 9.0 6-10 0 0.8 0 2.4 a The number of subjects observed following bovine cell vaccine was 133 at 24 hr and 129 at 48 hr; the number observed following chicken embryo vaccine was 124 at 24 hr and 122 at 48 hr. tionship of the antibody levels before vaccination to those after vaccination (Table 3). Antibody titers against all four antigens were increased to a greater degree in those with lower preimmunization levels. Antigenicity of chicken embryo vaccine. Results similar to those described in the preceding section were obtained with chicken embryo vaccine (Table 4). Again, relatively high levels of antibody were present prior to vaccination, but mean antibody titers were several-fold greater afterwards. These data were also analyzed according to the antibody levels observed before vaccination (Table 5). Increments in mean antibody titers were greater in those persons with lower levels of prevaccination antibody. Thus, it appears that some of the individuals who received either chicken embryo or bovine cell vaccine did not have much response because they already had relatively high antibody levels. Antibody titers determined by microtechnique. Hemagglutination-inhibition antibody titers were determined by a microprocedure with sera obtained before and after administration of chicken embryo vaccine (Table 6). Prevaccination and postvaccination antibody levels were lower than those obtained by the macrotechnique described

t ~~~~~~~~~~00 598 LEIDERMAN AND MOGABGAB APPL. MICROBIOL. TABLE 2. Distribution of hemagglutination-inhibition antibody titers before and after bovine cell influenza vaccine; macrotechnique Antigen Seruma 8 Total Median Meanb bmean- fold.dq Go ~~~~~~increase V so e4;s00sus 0 00%OeO V0000 4s oc A A2/Japan Pre 1 20 26 46 14 14 2 1 124 1,024 929.0 170/62 Post 2 9 25 20 41 16 8 3 124 4,096 2,858.0 3.1 A2/Taiwan Pre 2 20 35 29 25 1 9 3 124 512 469.0 1/64 Post 2 12 11 28 13 27 18 9 4 124 4,096 2,164.0 4.6 A2/Aichi Pre 1 3 14 28 32 23 13 8 1 123 32 33.2 2/68 Post 1 2 10 32 34 23 15 4 2 123 64 68.0 2.0 B/Mass Pre 1 10 38 23 22 13 13 3 1 124 128 163.0 3/66 Post 1 5 16 15 17 24 15 23 5 3 124 1,024 853.0 5.2 a Pre=before vaccination; Post=3 weeks after vaccination. b Geometric means with titers of less than 1-4 were calculated as 0. TABLE 3. Influence of initial hemagglutination-inhibition antibody titer on response to bovine cell influenza vaccine; macrotechnique Geometric reciprocal Prevaccne, recprocalmean titersa8emti Antigen No. of sera Prevaccre reciprocale mean-fold A2/Japan 170/62 93 128-1024 611.0 2,300.0 3.7 31 2,048->8,192 3,265.0 5,470.0 1.6 A2/Taiwan 1/64 86 64-512. 264.6 1,516.0 5.7 38 1,024->8,192 1,700.0 4,525.0 2.6 A2/Aichi 2/68 46 <4-16 11.1 34.2 3.1 77 32-1,024 63.2 101.8 1.6 B/Ma ss 3/66 94 16-256 95.9 646.0 6.7 30 512->8,192 857.0 2,250.0 2.6 apre = before vaccination; Post = after vaccination. TABLE 4. Distribution of hemagglutination-inhibition antibody titers before and after chicken embryo influenza vaccine; macrotechnique Antigen Serum! Total Median Mean afold 0 4 O o > ^It i8 - increase _5'le4100 '01+1- N u1 4 AI I A2/Japan Pre 5 23 27 15 3 5 1 79 512 544.0 170/62 Post 1 3 17 23 8 15 6 5 1 79 1,024 1,658.0 3.0 A2/Taiwan Pre 3 13 20 15 2 12 11 1 77 1,024 1,089.0 1/64 Post 2 13 14 4 15 18 8 4 77 4,096 3,420.0 3.4 A2/Aichi2/68 Pre 2 16 30 21 9 1 79 32 39.5 Post 3 16 21 23 11 1 3 1 79 128 93.2 2.3 B/Mass 3/66 Pre 1 1 2 11 22 20 13 3 4 2 79 128 105.2 Post 2 3 13 20 8 13 9 3 7 1 79 512 558.0 5.3 a See footnote a, Table 2. Pre Post in the previous section. Mean-fold increases in titer were also smaller. Analysis of the results (Table 7) showed similarities to those found with the macroprocedure in that increments in mean antibody titers were greater in the group that showed lower initial levels of antibody. However, the increments were not as large as were those obtained by the macrotechnique.

VOL. 18, 1969 ANTIGENICITY OF INFLUENZA VACCINE 599 TABLE 5. Influence of initial hemagglutination-inhibition antibody titer on response to chicken embryo influenza vaccine; macrotechnique Geometric reciprocal Antigen Antigen N. No. of sera Prevaccine reciprocal mean titersa Geometric titer mean-fold range -increase Pre Post A2/Japan 170/62 55 128-512 338.0 1,205.0 3.5 24 1,024->8,192 1,624.0 3,555.0 2.2 A2/Taiwan 1/64 36 128-512 355.0 1,218.0 3.7 41 1,024->8,192 2,800.0 8,470.0 3.0 A2/Aichi 2/68 48 <4-32 23.9 67.7 2.8 31 64-2,048 87.4 152.0 1.7 B/Mass 3/66 37 <4-64 37.0 240.0 6.4 42 128->8,192 277.0 1,188.0 4.2 a See footnote a, Table 3. TABLE 6. Distribution of hemagglutination-inhibition antibody titers before and after chicken embryo influenza vaccine; microtechnique Antigen Serum-a Total Median Mean increase 4 8 16 32 64 128 A2/Japan 170/62 Pre 1 7 46 20 5 79 16 19.2 Post 9 56 22 2 79 32 32.1 1.6 A2/Taiwan 1/64 Pre 9 46 23 1 79 16 19.2 Post 18 44 17 79 32 33.1 1.7 A2/Aichi 2/68 Pre 9 42 23 3 1 78 8 9.5 Post 1 16 38 18 3 76 16 16.1 1.6 B/Mass 3/66 Pre 1 15 47 15 1 79 16 15.9 Post 2 10 38 26 3 79 32 37.4 2.3 a See footnote a, Table 2. TABLE 7. Influence of initial hemagglutinationinhibition antibody titer on response to chicken embryo influenza vaccine; microtechnique Geometric No. Prevaccine reciprocal Geometric Antigen of reciprocal mean titersa mean-fold sera titer range increase Pre Post A2/Japan 54 4-16 14.2 33.6 2.3 170/62 25 32-128 36.7 53.7 1.4 A2/Taiwan 55 8-16 14.2 28.5 2.0 1/64 24 32-64 32.9 46.5 1.4 A2/Aichi 51 4-8 7.5 14.7 1.9 2/68 28 16-64 16.5 19.0 1.1 B/Mass 16 4-8 7.6 23.6 3.1 3/66 63 16-128 19.3 42.1 2.1 a See footnote a, Table 3. In an effort to determine the reason for these differences, simultaneous macro- and microtitrations were done with sera from a single subject immunized with influenza A2/Aichi 2/68 monovalent vaccine. Hemagglutination-inhibi- z CD z t-z 16-8 - 4-2 - I - 0.5-,, v MACRO -I -2-3 -4 * o MCRO * L MICRO (cilution with bopu) LOG SERUM DILUTION FIG. 1. Linear relationship between the quantity of virus and the concentration ofprevaccination and postvaccination sera as determined by macro- and microtechniques of hemagglutination-inhibition.

600 LEIDERMAN AND MOGABGAB APPL. MICROBIOL. tion antibody titers were determined separately to 16, 8, 4, 2, 1, and 0.5 hemagglutinating units of virus. Sera and virus were diluted with pipettes in tubes, and samples were distributed in tubes (macro) and plastic trays (micro). For the latter, 0.025-ml amounts were dispersed with calibrated disposable pipettes. The same determinations were also made by the usual microtechnique, diluting the sera with Takatsy loops. The procedures were repeated five times and mean titers were calculated and plotted (Fig. 1). The slopes determined by these results show that, whereas dilution with pipettes produced very similar and parallel results in tubes and trays, dilution with Takatsy loops yielded lower and unpredictable values. DISCUSSION The minimal nature of the local and systemic reactions to the bovine cell and chicken embryo vaccines described herein demonstrates the desirability of refined influenza vaccines (1, 8; J. L. Gerin et al., Fed. Proc., p. 365, 1968). Whether purification and ether treatment reduced potential toxic effects of calf kidney cell-propagated virus vaccine is not known. However, procedures of this type reduce the amount of extraneous substances and certainly provide a preferable product for injection into man. It was of interest that the chicken embryo vaccine prepared with influenza viruses purified by zonal centrifugation, but not ether-treated, caused no more reactions than did the bovine cell vaccine that had been purified by zonal centrifugation and ether-treated as well. However, the two vaccines were derived from viruses propagated in different host systems, and comparisons of the procedures utilized for purification may not be justified. Comparable antibody responses were expected from the bovine cell and chicken embryo vaccines on the basis of their hemagglutinin contents. Whether the host cell used for propagation of the virus altered antigenicity sufficiently to affect protection induced by the vaccine has not been determined (5). Nevertheless, it is likely that both of these vaccines would have provided protection from natural infection by virus strains related to those included in the vaccines (2, 9). The introduction of a new host system for vaccine production raises questions as to the presence of undesirable substances, allergens, or contaminating viruses. However, many types of biologicals, including smallpox vaccine, have been derived from bovine sources without obvious untoward effects in man. Although chicken egg-sensitive individuals are not numerous, the "high risk" persons of this type need an influenza vaccine prepared in cells other than those of the chicken embryo. To establish efficacy of an influenza vaccine, large-scale field studies are required to determine protective effects. Whether many thousands of individuals should be given bovine kidney cell influenza vaccine until this can be determined, or whether the vaccine should be made available on a limited basis for these special persons, is uncertain. Degree of protection is predictable on the basis of antigenicity studies in man and animals. The lower hemagglutination-inhibition antibody titers and, particularly, the smaller increments in titer obtained by the microtechnique present significant problems that must be considered in evaluating influenza vaccines by this procedure. These results most probably were caused by the diluting method. Variations in volume due to presence of microbubbles in the loops, contact of loops with the well walls, and other factors such as those described by Hirata et al. (3) might have been responsible. Thus, the savings gained from the microtechnique do not justify its use instead of the standardized and wellknown macroprocedure for measurement of hemagglutination-inhibition antibody levels, especially for evaluation of influenza vaccine. ACKNOWLEDGMENTS This investigation was supported by a grant from Abbott Laboratories, Scientific Divisions, North Chicago, Ill. The contributions of John 0. McLain are acknowledged. LITERATURE CITED 1. Davenport, F. M., A. V. Hennessy, F. M. Brandon, R. G. Webster, C. D. Barrett, and G. 0. Lease. 1964. Comparisons of serologic and febrile responses in humans to vaccination with influenza A viruses or their hemagglutinins. J. Lab. Clin. Med. 63:5-13. 2. Francis, T., and H. F. Maasab. 1965. Influenza viruses, p. 689-740. In F. L. Horsfall and I. Tamm (ed.), Viral and rickettsial infections in man, 4th ed. J. B. Lippincott Co., Philadelphia. 3. Hirata, A. A., D. S. Grant, and L. R. Draper. 1969. Factors affecting the passive hemagglutination titration: dilution loops, titration trays, vibration, diluents. Appl. Microbiol 17:563-567. 4. Miller, G. L., and W. M. Stanley. 1944. Quantitative aspects of the red blood cell agglutination test for influenza virus. J. Exp. Med. 79:185-195. 5. Mogabgab, W. J. 1961. Influenza A and B viruses in monkey kidney cultures. II. Immunogenicity and characteristics of the neutralization reaction as compared to those of chick embryo-propagated viruses. J. Infec. Dis. 109:136-146. 6. National Communicable Disease Center. 1962. Influenza surveillance report no. 72, 31 May 1962. National Cornmunicable Disease Center, Public Health Service, Atlanta, Ga. 7. National Communicable Disease Center. 1968. Morbidity and mortality, vol. 17, no. 33-39. National Communicable Disease Center, Public Health Service, Atlanta, Ga. 8. Peck, F. B. 1968. Purified influenza virus vaccine. A study of viral reactivity and antigenicity. J. Amer. Med. Ass., 206: 2277-2282. 9. Salk, J. E., W. J. Menke, and T. Francis. 1945. A clinical, epidemiological and immunological evaluation of vaccination against epidemic influenza. Amer. J. Hyg. 42:57-93. 10. Sever, J. L. 1962. Application of a microtechnique to vira serological investigations. J. Immunol. 88:320-329.