THE JOUR:\AL OF INFECTIOUS DISEASE. VOL. 136, © 1977 by the University of Chicago. All rights reserved.
SUPPLE:\IE~T
• DECE:\IBER 1977
Evaluation and Control of Vaccines for the National Influenza Immunization Program David W. Barry, Ronald E. Mayner, Jules M. Meisler, and Edward B. Seligmann, Jr.
From the Division of Virology, the Division of Compliance, and the Division of Control Activities, Bureau of Biologics, Food and Drug Administration, Bethesda, Maryland
The safety, purity, and potency of monovalent influenza A/New Jersey and bivalent A/New Jersey and A/Victoria virus vaccines are controlled by licensing procedures for manufacturers of influenza vaccine and by lot-by-lot testing of these vaccines by the manufacturers and the Bureau of Biologics (BOB) of the Food and Drug Administration. The composition and immunogenicity of experimental influenza A/New Jersey and A/Victoria vaccines were the subjects of extensive clinical trials during the spring, summer, and fall of 1976. The experimental vaccines may now be compared with licensed influenza vaccines released for use in the National Influenza Immunization Program (NIIP). The large number of licensed lots produced for the 1976-1977 influenza season and the magnitude of testing by the manufacturers and the BOB have permitted accurate determinations of the various components of influenza vaccines and We thank Ms. Eva Brown for performance of the chick cell-agglutination tests, Dr. H. D. Hochstein for endotoxin analysis, and Dr. Joan C. May for the thimerosal and formaldehyde assays. Please address requests for reprints to Dr. David W. Barry, Division of Virology, Bureau of Biologics, Food and Drug Administration, Bethesda, Maryland 20014.
have allowed us to examine the validity of our current procedures for the testing and evaluation of these vaccines. In addition, we have been able to gain experience with a newer method of potency testing, which may be a more accurate and reliable in vitro correlate of influenza vaccine immunogenicity. The NIIP of 1976 has also provided us with precise information concerning the maximal production capability of manufacturers of influenza vaccines in the United Sates as well as the capacity of the government to evaluate and distribute these vaccines. Since the availability and administration of sufficient quantities of an effective vaccine are of prime importance in the prevention of deaths during influenza epidemics, a careful review of the time course of vaccine production, testing, and distribution this year may provide data that will guide us in preparing for future pandemics. Materials and Methods
Vaccines. Influenza vaccine manufacturers participating in the NIIP were Merck Sharp and Dohme (MSD; West Point, Pa.), Merrell-National Laboratories (MN; Cincinnati, Ohio),
S407
Downloaded from http://jid.oxfordjournals.org/ at Florida Atlantic University on August 29, 2015
The National Influenza Immunization Program of 1976 offered an ideal opportunity to test the capability of the system in the United States for production and distribution of maximal amounts of inactivated influenza virus vaccine of carefully regulated quality. The four licensed manufacturers were able to produce and distribute> 10 million doses of vaccine per week over a 14-week period. Assays showed that the quality of these vaccines was comparable to or exceeded that of vaccines produced in recent years under less stressful circumstances. Because of the extensive clinical trials conducted as part of the program, it was possible to make an unprecedented evaluation of the significance of various laboratory tests of vaccines in relation to their pertinence in prediction of immunogenicity and reactivity for humans. This experience demonstrated the superiority of immunodiffusion methods as compared with the standard chick cell-agglutination method for assay of vaccine potency. Qualitative differences in immunogenicity between whole-virus and disruptedvirus vaccines were recognized that are not measured by in vitro potency tests. The results also indicated that influenza viral components are responsible for most febrile reactions to the vaccine.
5408
min (Sigma Chemical Co., St. Louis, Mo.) as a standard. Because thimerosal increases the amount of protein detected by this method at a rate of 0.74 JLg of protein / JLg of thimerosal (R. E. Mayner, unpublished observations), this amount was calculated and subtracted from the absolute protein values. Limulus amebocyte lysate assays. Assays for endotoxin were performed according to methods previously described [7] with use of a single lot of lysate no. 2 (BOB), Reference Endotoxin lot no. IB, (BOB), and Reference Influenza Vaccine for Endotoxin Assay lot no. E-I (BOB). This vaccine contained 1.043 JLg of endotoxinjtl.li-ml dose with use of the present lysate lot no. 2 (mean of 186 tests). Four replicate tests were performed on each vaccine and on each of the references. The concentration of endotoxin in the test vaccine relative to that in the reference vaccine was obtained by dividing the geometric mean of the test vaccine by that of the reference vaccine. In addi tion, the concentration of endotoxin in the test vaccine was expressed in JLg per 0.5-ml dose of vaccine by means of parallel titration of the reference endotoxin. Formaldehyde assays. Quantitation of free formaldehyde was performed according to the colorimetric method of Nash with use of Hantzsch reagent [8]. Thimerosal. The thimerosal content of vaccines was determined as mercury by the cold vapor atomic adsorption method [9, 10] with use of a Coleman MAS-50 mercury analyzer (PerkinElmer Co., Norwalk, Conn.). Vaccine samples were cold-digested in a mixture of 5% potassium permanganate and concentrated sulfuric acid prior to analysis. Other tests. Tests for sterility and general safety in guinea pigs and mice were performed as prescribed in the Code of Federal Regulations
[11]. Production time figures. Manufacturers were contacted to determine the week during which an individual lot had been shipped from its factory, warehouse, or distribution center to a location designated by the Center for Disease Control (CDC; Atlanta, Ga.). Vaccination rates were obtained from the CDC weekly assessment report on influenza immunization. The mean time required for production, testing, and distribution
Downloaded from http://jid.oxfordjournals.org/ at Florida Atlantic University on August 29, 2015
Parke, Davis and Company (PD; Detroit, Mich.), and Wyeth Laboratories (Philadelphia, Pa.). MSD and MN produce zonally purified, wholevirus vaccines, and PD manufactures a zonally purified, polysorbate 80- and ether-disrupted virus vaccine. Wyeth disrupts the virus with polysorbate 80 and tri-N-butylphosphate after chromatographic purification. Sam ples of bulk lots were shipped or delivered to the BOB as part of normal testing and evaluation-for-approval procedures. Only tests that had been completed by December 16, 1976 are included in the present report. Chick cell-agglutination (CCA) potency testing. CCA determinations were performed as previously described [1] and were modified [2] to include a standard reference (Reference Influenza Vaccine for CCA Assay, lot no. 1975; BOB, Bethesda, Md.). Ratios of test vaccine to reference vaccine thus obtained were multiplied by the assigned value of reference vaccine (2,400 CCA units/rnl), and the results were expressed as CCA units per adult dose (0.5 ml). Laurell rocket immunoelectrophoresis test. Quantitative immunoelectrophoresis was performed according to the method of Laurell [3] as modified for hemagglutinin quantitation [4]. Briefly, 1% agarose slabs containing hyperimmune goat antisera to bromelain-purified influenza A/New Jersey /76 hemagglutinin [5] were cast in plexiglass templates. Vaccines were treated with Triton X-IOO® (final concentration, 0.4%; Fisher Scientific Co., Silver Spring, Md.) for 30 min at room temperature (about 24 C), and 10-JLI aliquots were dispensed into wells (diameter, 3 mm) punched into immunoelectrophoresis slabs. Duplicate samples were electrophoresed for 8 hr at 350 V, 40 rnA at 0.1 microfarad with use of an Ortec" power supply (Ortec Inc., Oak Ridge, Tenn.). Plates were fixed and stained for 15 min in 0.1% amido black in 2.5e;to acetic acid and 35% methanol. Gels were destained in tap water, and rocket-shaped zones of antigen-antibody complexes were measured. Estimates of sample hemagglutinin content were calculated by least-squares regression based on four dilutions of a standardized reference vaccine. Total protein. Protein. determinations were performed according to the method of Lowry et al. [6] with use of crystallized bovine serum albu-
Barry et al.
Vaccine Evaluation for National Immunization
5409
was quantitated by comparison of the cumulative number of doses in each stage at weekly intervals. Results
Table 1. Tests performed on bulk lots of influenza virus vaccine by the manufacturer and by the Bureau of Biologics (BOB), Food and Drug Administration. Performed by Manufacturer Test Sterility (7) * Guinea pig safety (7) Mouse safety (7) Identity Virus inactivation (6) Potency (CCA)+ Formaldehyde Endotoxin (LAL) § Proteinll Thimerosal Barium# Polysorbate 80# Tri-N-butylphosphate # Immunoelectrophoresisll
Bulk lot Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No
Final BOB container (bulk lot) Yes Yes Yes Yes No No No No No No No No No No
Yest Yest Yest Yest Yes Yest Yest Yest Yest Yes Yes No No Yes
*Numbers in parentheses indicate minimal number of days required to perform the test. Tests without numbers required one day. tTest applied to all lots tested by the BOB. tCCA = chick cell agglutination. § LAL = limulus amebocyte lysate test. IINot required by regulation. #Not applicable to all manufacturers.
Downloaded from http://jid.oxfordjournals.org/ at Florida Atlantic University on August 29, 2015
Tests performed on each lot of influenza vaccine by the manufacturer and the BOB are indicated in table 1. The manufacturer performed all tests on each bulk lot and on samples of final container material where applicable. All lots were tested by the BOB for sterility, guinea pig and mouse safety, identity, potency, and content of formaldehyde, endotoxin, and protein. Random analyses of thimerosal and virus inactivation were also performed by the BOB. Analyses of barium, polysorbate 80, and tri-N-butylphosphate were performed by the manufacturer only when these materials had been used in vaccine production. Some of the tests, such as for sterility, virus inactivation, and guinea pig and mouse safety, require six to seven days for completion, whereas it was possible to complete the other tests in one day. All lots submitted to the BOB passed the tests for sterility,
guinea pig and mouse safety, identity, and inactivation. The quantity of various components in the influenza vaccines are shown in table 2. Protein determinations showed a wide diversity of values ranging from a low mean value of 36.9 fLg per dose for the MN monovalent influenza A/New Jersey virus vaccine to a high of 193.1 /-Lg per dose for the Wyeth monovalent vaccine. A similar range was noted for the bivalent influenza AI New J ersey-AjVictoria virus vaccines. Mean formaldehyde concentrations ranged between 2.1 and 15.6 fLg per dose and were consistent with the individual manufacturer's license protocol for the production of influenza vaccine. Thimerosal values were all within the required range of 50 ± 10 /-LgjO.5-ml dose. Endotoxin levels were generally lower than those in vaccines studied previously [12] with mean levels of 0.007-0.082 fLg per dose. Results of the potency testing by CCA techniques showed that all monovalent vaccines had a mean somewhat greater than the minimal limit of 200 CCA units/0.5-ml adult dose. The range was rather narrow with a low of 208.8 CCA units for MSD and a high of 231.6 CCA units for Wyeth vaccines. Likewise, all of the bivalent vaccines contained somewhat more than the minimal requirement of 400 CCA units. Since CCA testing is the most common source of differences in results between the manufacturers and the BOB and the most common cause of rejection of a lot, and since it has been relied upon as the measure of vaccine potency, a more detailed analysis of CCA testing is presented in table 3. Results of tests performed by the BOB differed significantly from those of tests performed by MSD and MN on their monovalent vaccines and those by MN on their bivalent vaccine. No significant differences between PD and the BOB were found in the testing of monovalent or bivalent vaccines; however, wide differences were noted between the results of the BO B and those of Wyeth. In table 4 data are presented indicating the amount of influenza A/New Jersey hemagglutinin in the monovalent and bivalent vaccines produced by the various manufacturers. As was true for the vaccines used in the clinical trials, MSD vaccines contained significantly more and Wyeth significantly less hemagglutinin than vaccines
5410
Barry et al.
Table 2. Quantitation of components of commercial influenza vaccines.
Vaccine, manufacturer *
Total.protein (I-Lg)
Thimerosal (I-Lg)
Chick cellagglutinating units
Endotoxin (I-Lg)
67.1 36.9 43.7 193.1
±9.7 (42) ± 7.7(73) ± 11.2 (3) ± 37.6·(8)
3.2 2.1 10.5 15.6
(42) (71) (2) (8)
49 51 45 51
(10) (12) (1) (5)
0.023 0.040 0.082 0.007
(40) (72) (3) (8)
208.8 219.6 226.8 231.6
± 10.8 (47) ± 18.0 (78) ± 1.2 (5) ± 34.8 (10)
157.4 91.6 51.0 242.3
± 19.0 (10) ± 14.9 (10) ± 4.6 (28) ± 22.7 (16)
3.9 5.2 11.2 15.1
(9) (10) (26) (16)
51 51 49 46
(2) (1) (7) (5)
0.042 0.020 0.058 0.009
(9) (10) (28) (16)
430.8 426.0 436.8 448.8
± 26.4 ± 21.6 ± 32.4 ± 61.2
(10) (10) (29) (16)
NOTE. Data are given as means or means ± so per 0.5-ml adult dose of vaccine. Numbers in parentheses indicate the number of lots tested by the Bureau of Biologics. *MSD = Merck Sharp and Dohrne, MN = Merrell-National Laboratories, and PD = Parke, Davis and Company.
produced by the other manufacturers. Values for each individual manufacturer showed little variation as indicated by the relatively small SE values. The number of vaccine lots submitted to the BOB and the number voluntarily withdrawn by the manufacturer from consideration for release as of December 16, 1976 are shown in table 5. These voluntary withdrawals were based on the finding by the BOB of lots that were found to contain below the minimal requirement of 200 CCA units for the monovalent or 400 CCA units for the bivalent vaccine. No lots were withdrawn for failure to meet requirements other than number of CCA units. The 11.6% withdrawal figure is consistent with rates of withdrawal Table 3.
occurring during previous years. These withdrawn lots represent the equivalent of --17 million doses; however, most of the lots withdrawn were subsequently fortified with virus concentrates and then resubmitted to the BOB as new lots. Therefore, the withdrawal of lots because of low potency did not appear to diminish substantially the total equivalent number of doses (150,034, 580) submitted to the BOB by December 16, 1976. As mentioned previously, tests for safety, sterility, and inactivation require six to seven days to complete and were performed by both the manufacturer and the BOB on the bulk lots. The manufacturer also tested final container mater-
Results of chick cell-agglutination (CCA) tests on influenza virus vaccines. Bureau of Biologics
Manufacturer No. of Vaccine, manufacturer * samples Monovalent MSD MN PD Wyeth Bivalent MSD MN PD Wyeth
Mean ±sot
Ranget
No. of samples
Mean ± sot
Ranget
p*
49 78 5 10
213.6 236.4 222.0 264.0
± 9.6 ± 18.0 ± 12.0 ±44.4
201.6-240.0 200.4-280.8 202.8-229.2 217.2-328.8
47 78 5 10
208.8 219.6 226.8 231.6
± 10.8 ± 18.0 ± 1.2 ± 34.8
199.2-228.0 199.2-279.6 205.2-273.6 199.2-291.6
;> 40 I 30
" ffl ! II "l / vff
20/ 10
9110
9/24
10/8 10/22 11/5 11119 12/3 12/17 12/31
WEEK ENDING:
Figure 1. Rates of production, testing, shipment, and distribution of influenza vaccines during the National Influenza Immunization Program. BoB = Bureau of Biologics.
probability and quantity of bacterial contamination during influenza vaccine production. The finding of low levels of endotoxin in present influenza vaccines gives evidence of the success of their efforts. Although the Reference Influenza Vaccine for Endotoxin Assay contains 1.043 /Lg of endotoxin per 1,200 CCA units of virus and therefore might be expected to have considerably higher values of endotoxin than present vaccines which contain only 200-400 CCA units of virus, calculations of the nanograms of endotoxin per CCA unit will indicate that this is not the sole explanation for the near absence of endotoxin in present vaccines. The reference endo-
toxin vaccine has 0.87 ng of endotoxinjCCA unit, whereas the present vaccines contain 0.030.36 ngjCCA unit and therefore only about onethird to less than one-thirtieth of the proportionate amount of endotoxin as vaccines used in the recent past . With the introduction of a standard reference into the CCA test, this measure of potency became more reliable [2], but difficulties in obtaining equivalent results between laboratories have nevertheless occasionally occurred. The present data confirm this finding and indicate that significant differences sometimes occurred between the CCA results of the BOB and those obtained by MSD and MN. However, the actual difference in means between the BOB's results and those of MN in the testing of their monovalent vaccine, for example, is only 7% of the total CCA content of the vaccine, well within the coefficient of error of the test [20]. On the other hand, the large SD value obtained both by the BOB and Wyeth during the testing of representative numbers of both monovalent and bivalent vaccines indicated difficulties on the part of both parties in the precise and consistent determination of potency of this manufacturer's vaccine. In spite of the generally good correlation between CCA results of the BOB and the manufacturers, the test nevertheless remains an indirect one, dependent in part on the affinity of a particular strain of influenza virus for chicken red blood cells. The number of viral particles in preparations containing the same number of CCA units has consequently been found to vary widely [21]. A direct measure of the viral hemagglutinin content of inactivated influenza vaccine, as suggested by Schild et al. [22], would enable us to circumvent these difficulties. We have found that the adaptation of the "rocket" electrophoresis test of Laurell [4] to the quantitation of influenza hemagglutinin is easy to perform and yields accurate and reproducible results in our laboratory. In addition, the relevance of the results of this test to human immunogenicity has been well demonstrated by the fact that vaccines, such as those produced by MSD, that have a high ratio of hemagglutinin to CCA units are also those that are most immunogenic in individuals with little prior antigenic experience with influenza, either through natural infection
Downloaded from http://jid.oxfordjournals.org/ at Florida Atlantic University on August 29, 2015
.s-/ / i/ I &/ / ~ e. J?!; I ~g cf/ 150 million doses of vaccine should be useful in estimating the capability to provide vaccines when future pandemics occur.
References 1. Miller, G. L., Stanley, W. M. Quantitative aspects of the red blood cell agglutination test for influenza virus.J. Exp. Med. 79:185-195,1944. 2. Tauraso, N. M., O'Brien, T. C., Seligmann, E. B. Problems of influenza vaccine standardization. Bull. W.H.O. 41:479-506, 1969. 3. Laurell, C.-B. Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Ann. Biochem. 15:45-52, 1966. 4. Mayner, R. E., Blackburn, R. J., Barry, D. W. Quantitation of influenza vaccine hemagglutinin by immunoelectrophoresis. In International Association of Biological Standardization. Symposium on influenza immunization. Karger, Basel, 1977 (in press). 5. Brand, C. M., Skehel, J. J. Crystalline antigen from influenza virus envelope. Nature [New Biol.] 238: 145-147, 1972. 6. Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. Protein measurement with the Folin phenol reagent. J. BioI. Chern. 193:265-275, 1951. 7. Hochstein, H. D., Elin, R. J., Cooper, J. F., Seligmann, E. B., Wolff, S. M. Further developments of limulus
Downloaded from http://jid.oxfordjournals.org/ at Florida Atlantic University on August 29, 2015
or immunization [23]. The quantity of hemagglutinin, however, is not the sole determining factor in the induction of HAl antibody in such individuals since split-virus vaccines containing similar amounts of hemagglutinin as a whole-virus product induce lower levels of antibody in immunological "virgins" [23]. Since the ratios of hemagglutinin to immunogenicity for each individual manufacturer's vaccine are fairly constant for each age group with differing antigenic experiences, it is now theoretically possible to predict, with a fair degree of certainty, the immunogenicity of particular influenza vaccines for a population of defined age and prior influenza experience. Manufacturers of influenza vaccine as well as the BOB have now begun using the immunoelectrophoresis test as a laboratory measure of potency of influenza A/New Jersey and other antigens. Should the test continue to yield accurate, reproducible, reliable, and relevant results, then consideration will be given to the use of immunoelectrophoresis as the official test of influenza vaccine potency. In responding to the threat of the A/Hong Kong (H3N2) influenza epidemic of 1968, manufacturers produced only about 10 million doses per month [24]; similarly, --50 million doses were prepared in the five-month period before the 1957 epidemic [25]. Past experience had indicated that three to four months elapse between the first receipt of a strain and the production of a significant number of doses [24]. Manufacturers were given approved influenza A/New Jersey / 8/76 virus seed stocks on February 20, 1976. The exact amount of lead time now required by the manufacturers to produce sufficient basic stocks of allantoic fluid vaccine concentrates this year cannot be accurately calculated because of the delays engendered by the requirement for the passage of indemnification legislation [26]; however, an estimate of one to two months can be made. This estimate is supported by the fact that >5,000 doses were available as early as April 1976 for the clinical trials conducted under the auspices of the U.S. Public Health Service [27, 28]. The ability of the manufacturers to produce, test, and distribute 10 million doses of vaccine per week for at least several months is now well established. There appear to be several reasons
Barry et al.
S414
8.
9.
10.
12.
13. 14.
15.
16. 17.
18.
19. Inactivated Influenza Vaccine, General Safety Test. Federal Register 39:40014-40016, 1974. 20. Barry, D. W., Mayner, R. E., Staton, E., Dunlap, R. C., Rastogi, S. C., Hannah, J. E., Blackburn, R. J., Nortman, D. F., Graze, P. R. Comparative trial of influenza vaccines I. Immunogenicity of whole virus and split product virus in man. Am. J. Epidemi61. 104:34-46, 1976. 21. Dunlap, R. C., Brown, E. R., Barry, D. W. Determination of the viral particle content of influenza vaccines by electron microscopy. J. BioI. Stand. 3:281289,1975. 22. Schild, G. C., Wood, J. M., Newman, R. W. A singleradial-immunodiffusion technique for the assay of influenza haem agglutinin antigen. Bull. W.H.O. 52: 223-231, 1975. 23. Ennis, F; A., Mayner, R. E., Barry, D. W., Manischewitz, J. E., Dunlap, R. C., Verbonitz, M. W., Bozeman, F. M., Schild, G. C. Correlation of laboratory studies with clinical responses to A/New Jersey influenza vaccines. J. Infect. Dis. 136(Suppl.):S397-S406, 1977. 24. Murray" R. Production and testing in the U.S.A. of influenza virus vaccine made from the Hong Kong variant in 1968-69. Bull. W.H.O. 41:495-496, 1969. 25. Murray, R. Some problems in the standardization and control of influenza vaccine in 1957. Am. Rev. Respir. Dis. 83:160-167,1961. 26. Public Law 94-380. 94th Congress, S. 3735, August 12, 1976. U.S. Government Printing Office, Washington, D.C. 27. Seal, J. R., Sencer, D. J., Meyer, H. M., Jr. A status report in national immunization against influenza. ]. Infect. Dis. 133:715-720, 1976. 28. Parkman, P. D., Galasso, G. J., Top, F. H., Noble, G. R. Summary of clinical trials of influenza vaccines. J. Infect. Dis. 134:100-107,1976. 29. Kilbourne, E. D. Future influenza vaccines and the use of genetic recombinants. Bull. W.H.O. 41:643645,1969.
Downloaded from http://jid.oxfordjournals.org/ at Florida Atlantic University on August 29, 2015
11.
amebocyte lysate test. Bull. Parenter. Drug Assoc. 27:139-148,1973. Nash, T. The colorimetric estimation of formaldehyde by the means of the Hantzsch reaction. Biochem.J.55:416-421,1953. Association of Official Analytical Chemists. Official methods of analysis of the AOAC. Association of Official Analytical Chemists, Washington, D.C., 1975, p.451. Rains, T. C., Menis, O. V. Determination of submicrogram amounts of mercury in standard reference materials by flameless atomic absorption spectometry. J. Assoc. Off. Anal. Chem. 55:1339,1972. Code of Federal Regulations, Title 21, Sections 610.11, 610.1Ia, and 610.12. U.S. Government Printing Office, Washington, D.C. Barry, D. W., Mayner, R. E., Hochstein, R. D., Dunlap, R. C., Rastogi, S. C., Hannah, J. E., Blackburn, R. J., Sullivan, J. L., Gerety, R. J. Comparative trial of influenza vaccines. II. Adverse reactions in children and adults. Am. J. Epidemiol. 104:47-59,1976. Peck, F. B. Purified influenza virus vaccine. J .A.M.A. 206:2277-2282, 1968. Reimer, C. B., Baker, R. S., Van Frank, R. M., Newlin, T. E., Cline, G. R, Anderson, N. G. Purification of large quantities of influenza virus by density gradient centrifugation. J. Virol. 1:1207-1216, 1967. O'Brien, T. C., Maloney, C. J., Tauraso, N. M. Quantitation of residual host protein in chicken embryoderived vaccines by radial immunodiffusion. Appl. Microbiol. 21:780-782,1971. Salk, J. E. Reactions to concentrated influenza virus vaccines. J. Immunol. 58:369-395,1948. Wright, P. F., Sell, S. H. W., Thompson, J., Karzon, D. T. Clinical reactions and serologic response following inactivated monovalent influenza type B vaccine in young children and infants. J. Pediatr. 88:31-35, 1976. Inactivated Influenza Vaccine, General Safety Test. Federal Register 39:27916-27918, 1974.
SUPPLE:\IE~T
• DECE:\IBER 1977
Evaluation and Control of Vaccines for the National Influenza Immunization Program David W. Barry, Ronald E. Mayner, Jules M. Meisler, and Edward B. Seligmann, Jr.
From the Division of Virology, the Division of Compliance, and the Division of Control Activities, Bureau of Biologics, Food and Drug Administration, Bethesda, Maryland
The safety, purity, and potency of monovalent influenza A/New Jersey and bivalent A/New Jersey and A/Victoria virus vaccines are controlled by licensing procedures for manufacturers of influenza vaccine and by lot-by-lot testing of these vaccines by the manufacturers and the Bureau of Biologics (BOB) of the Food and Drug Administration. The composition and immunogenicity of experimental influenza A/New Jersey and A/Victoria vaccines were the subjects of extensive clinical trials during the spring, summer, and fall of 1976. The experimental vaccines may now be compared with licensed influenza vaccines released for use in the National Influenza Immunization Program (NIIP). The large number of licensed lots produced for the 1976-1977 influenza season and the magnitude of testing by the manufacturers and the BOB have permitted accurate determinations of the various components of influenza vaccines and We thank Ms. Eva Brown for performance of the chick cell-agglutination tests, Dr. H. D. Hochstein for endotoxin analysis, and Dr. Joan C. May for the thimerosal and formaldehyde assays. Please address requests for reprints to Dr. David W. Barry, Division of Virology, Bureau of Biologics, Food and Drug Administration, Bethesda, Maryland 20014.
have allowed us to examine the validity of our current procedures for the testing and evaluation of these vaccines. In addition, we have been able to gain experience with a newer method of potency testing, which may be a more accurate and reliable in vitro correlate of influenza vaccine immunogenicity. The NIIP of 1976 has also provided us with precise information concerning the maximal production capability of manufacturers of influenza vaccines in the United Sates as well as the capacity of the government to evaluate and distribute these vaccines. Since the availability and administration of sufficient quantities of an effective vaccine are of prime importance in the prevention of deaths during influenza epidemics, a careful review of the time course of vaccine production, testing, and distribution this year may provide data that will guide us in preparing for future pandemics. Materials and Methods
Vaccines. Influenza vaccine manufacturers participating in the NIIP were Merck Sharp and Dohme (MSD; West Point, Pa.), Merrell-National Laboratories (MN; Cincinnati, Ohio),
S407
Downloaded from http://jid.oxfordjournals.org/ at Florida Atlantic University on August 29, 2015
The National Influenza Immunization Program of 1976 offered an ideal opportunity to test the capability of the system in the United States for production and distribution of maximal amounts of inactivated influenza virus vaccine of carefully regulated quality. The four licensed manufacturers were able to produce and distribute> 10 million doses of vaccine per week over a 14-week period. Assays showed that the quality of these vaccines was comparable to or exceeded that of vaccines produced in recent years under less stressful circumstances. Because of the extensive clinical trials conducted as part of the program, it was possible to make an unprecedented evaluation of the significance of various laboratory tests of vaccines in relation to their pertinence in prediction of immunogenicity and reactivity for humans. This experience demonstrated the superiority of immunodiffusion methods as compared with the standard chick cell-agglutination method for assay of vaccine potency. Qualitative differences in immunogenicity between whole-virus and disruptedvirus vaccines were recognized that are not measured by in vitro potency tests. The results also indicated that influenza viral components are responsible for most febrile reactions to the vaccine.
5408
min (Sigma Chemical Co., St. Louis, Mo.) as a standard. Because thimerosal increases the amount of protein detected by this method at a rate of 0.74 JLg of protein / JLg of thimerosal (R. E. Mayner, unpublished observations), this amount was calculated and subtracted from the absolute protein values. Limulus amebocyte lysate assays. Assays for endotoxin were performed according to methods previously described [7] with use of a single lot of lysate no. 2 (BOB), Reference Endotoxin lot no. IB, (BOB), and Reference Influenza Vaccine for Endotoxin Assay lot no. E-I (BOB). This vaccine contained 1.043 JLg of endotoxinjtl.li-ml dose with use of the present lysate lot no. 2 (mean of 186 tests). Four replicate tests were performed on each vaccine and on each of the references. The concentration of endotoxin in the test vaccine relative to that in the reference vaccine was obtained by dividing the geometric mean of the test vaccine by that of the reference vaccine. In addi tion, the concentration of endotoxin in the test vaccine was expressed in JLg per 0.5-ml dose of vaccine by means of parallel titration of the reference endotoxin. Formaldehyde assays. Quantitation of free formaldehyde was performed according to the colorimetric method of Nash with use of Hantzsch reagent [8]. Thimerosal. The thimerosal content of vaccines was determined as mercury by the cold vapor atomic adsorption method [9, 10] with use of a Coleman MAS-50 mercury analyzer (PerkinElmer Co., Norwalk, Conn.). Vaccine samples were cold-digested in a mixture of 5% potassium permanganate and concentrated sulfuric acid prior to analysis. Other tests. Tests for sterility and general safety in guinea pigs and mice were performed as prescribed in the Code of Federal Regulations
[11]. Production time figures. Manufacturers were contacted to determine the week during which an individual lot had been shipped from its factory, warehouse, or distribution center to a location designated by the Center for Disease Control (CDC; Atlanta, Ga.). Vaccination rates were obtained from the CDC weekly assessment report on influenza immunization. The mean time required for production, testing, and distribution
Downloaded from http://jid.oxfordjournals.org/ at Florida Atlantic University on August 29, 2015
Parke, Davis and Company (PD; Detroit, Mich.), and Wyeth Laboratories (Philadelphia, Pa.). MSD and MN produce zonally purified, wholevirus vaccines, and PD manufactures a zonally purified, polysorbate 80- and ether-disrupted virus vaccine. Wyeth disrupts the virus with polysorbate 80 and tri-N-butylphosphate after chromatographic purification. Sam ples of bulk lots were shipped or delivered to the BOB as part of normal testing and evaluation-for-approval procedures. Only tests that had been completed by December 16, 1976 are included in the present report. Chick cell-agglutination (CCA) potency testing. CCA determinations were performed as previously described [1] and were modified [2] to include a standard reference (Reference Influenza Vaccine for CCA Assay, lot no. 1975; BOB, Bethesda, Md.). Ratios of test vaccine to reference vaccine thus obtained were multiplied by the assigned value of reference vaccine (2,400 CCA units/rnl), and the results were expressed as CCA units per adult dose (0.5 ml). Laurell rocket immunoelectrophoresis test. Quantitative immunoelectrophoresis was performed according to the method of Laurell [3] as modified for hemagglutinin quantitation [4]. Briefly, 1% agarose slabs containing hyperimmune goat antisera to bromelain-purified influenza A/New Jersey /76 hemagglutinin [5] were cast in plexiglass templates. Vaccines were treated with Triton X-IOO® (final concentration, 0.4%; Fisher Scientific Co., Silver Spring, Md.) for 30 min at room temperature (about 24 C), and 10-JLI aliquots were dispensed into wells (diameter, 3 mm) punched into immunoelectrophoresis slabs. Duplicate samples were electrophoresed for 8 hr at 350 V, 40 rnA at 0.1 microfarad with use of an Ortec" power supply (Ortec Inc., Oak Ridge, Tenn.). Plates were fixed and stained for 15 min in 0.1% amido black in 2.5e;to acetic acid and 35% methanol. Gels were destained in tap water, and rocket-shaped zones of antigen-antibody complexes were measured. Estimates of sample hemagglutinin content were calculated by least-squares regression based on four dilutions of a standardized reference vaccine. Total protein. Protein. determinations were performed according to the method of Lowry et al. [6] with use of crystallized bovine serum albu-
Barry et al.
Vaccine Evaluation for National Immunization
5409
was quantitated by comparison of the cumulative number of doses in each stage at weekly intervals. Results
Table 1. Tests performed on bulk lots of influenza virus vaccine by the manufacturer and by the Bureau of Biologics (BOB), Food and Drug Administration. Performed by Manufacturer Test Sterility (7) * Guinea pig safety (7) Mouse safety (7) Identity Virus inactivation (6) Potency (CCA)+ Formaldehyde Endotoxin (LAL) § Proteinll Thimerosal Barium# Polysorbate 80# Tri-N-butylphosphate # Immunoelectrophoresisll
Bulk lot Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No
Final BOB container (bulk lot) Yes Yes Yes Yes No No No No No No No No No No
Yest Yest Yest Yest Yes Yest Yest Yest Yest Yes Yes No No Yes
*Numbers in parentheses indicate minimal number of days required to perform the test. Tests without numbers required one day. tTest applied to all lots tested by the BOB. tCCA = chick cell agglutination. § LAL = limulus amebocyte lysate test. IINot required by regulation. #Not applicable to all manufacturers.
Downloaded from http://jid.oxfordjournals.org/ at Florida Atlantic University on August 29, 2015
Tests performed on each lot of influenza vaccine by the manufacturer and the BOB are indicated in table 1. The manufacturer performed all tests on each bulk lot and on samples of final container material where applicable. All lots were tested by the BOB for sterility, guinea pig and mouse safety, identity, potency, and content of formaldehyde, endotoxin, and protein. Random analyses of thimerosal and virus inactivation were also performed by the BOB. Analyses of barium, polysorbate 80, and tri-N-butylphosphate were performed by the manufacturer only when these materials had been used in vaccine production. Some of the tests, such as for sterility, virus inactivation, and guinea pig and mouse safety, require six to seven days for completion, whereas it was possible to complete the other tests in one day. All lots submitted to the BOB passed the tests for sterility,
guinea pig and mouse safety, identity, and inactivation. The quantity of various components in the influenza vaccines are shown in table 2. Protein determinations showed a wide diversity of values ranging from a low mean value of 36.9 fLg per dose for the MN monovalent influenza A/New Jersey virus vaccine to a high of 193.1 /-Lg per dose for the Wyeth monovalent vaccine. A similar range was noted for the bivalent influenza AI New J ersey-AjVictoria virus vaccines. Mean formaldehyde concentrations ranged between 2.1 and 15.6 fLg per dose and were consistent with the individual manufacturer's license protocol for the production of influenza vaccine. Thimerosal values were all within the required range of 50 ± 10 /-LgjO.5-ml dose. Endotoxin levels were generally lower than those in vaccines studied previously [12] with mean levels of 0.007-0.082 fLg per dose. Results of the potency testing by CCA techniques showed that all monovalent vaccines had a mean somewhat greater than the minimal limit of 200 CCA units/0.5-ml adult dose. The range was rather narrow with a low of 208.8 CCA units for MSD and a high of 231.6 CCA units for Wyeth vaccines. Likewise, all of the bivalent vaccines contained somewhat more than the minimal requirement of 400 CCA units. Since CCA testing is the most common source of differences in results between the manufacturers and the BOB and the most common cause of rejection of a lot, and since it has been relied upon as the measure of vaccine potency, a more detailed analysis of CCA testing is presented in table 3. Results of tests performed by the BOB differed significantly from those of tests performed by MSD and MN on their monovalent vaccines and those by MN on their bivalent vaccine. No significant differences between PD and the BOB were found in the testing of monovalent or bivalent vaccines; however, wide differences were noted between the results of the BO B and those of Wyeth. In table 4 data are presented indicating the amount of influenza A/New Jersey hemagglutinin in the monovalent and bivalent vaccines produced by the various manufacturers. As was true for the vaccines used in the clinical trials, MSD vaccines contained significantly more and Wyeth significantly less hemagglutinin than vaccines
5410
Barry et al.
Table 2. Quantitation of components of commercial influenza vaccines.
Vaccine, manufacturer *
Total.protein (I-Lg)
Thimerosal (I-Lg)
Chick cellagglutinating units
Endotoxin (I-Lg)
67.1 36.9 43.7 193.1
±9.7 (42) ± 7.7(73) ± 11.2 (3) ± 37.6·(8)
3.2 2.1 10.5 15.6
(42) (71) (2) (8)
49 51 45 51
(10) (12) (1) (5)
0.023 0.040 0.082 0.007
(40) (72) (3) (8)
208.8 219.6 226.8 231.6
± 10.8 (47) ± 18.0 (78) ± 1.2 (5) ± 34.8 (10)
157.4 91.6 51.0 242.3
± 19.0 (10) ± 14.9 (10) ± 4.6 (28) ± 22.7 (16)
3.9 5.2 11.2 15.1
(9) (10) (26) (16)
51 51 49 46
(2) (1) (7) (5)
0.042 0.020 0.058 0.009
(9) (10) (28) (16)
430.8 426.0 436.8 448.8
± 26.4 ± 21.6 ± 32.4 ± 61.2
(10) (10) (29) (16)
NOTE. Data are given as means or means ± so per 0.5-ml adult dose of vaccine. Numbers in parentheses indicate the number of lots tested by the Bureau of Biologics. *MSD = Merck Sharp and Dohrne, MN = Merrell-National Laboratories, and PD = Parke, Davis and Company.
produced by the other manufacturers. Values for each individual manufacturer showed little variation as indicated by the relatively small SE values. The number of vaccine lots submitted to the BOB and the number voluntarily withdrawn by the manufacturer from consideration for release as of December 16, 1976 are shown in table 5. These voluntary withdrawals were based on the finding by the BOB of lots that were found to contain below the minimal requirement of 200 CCA units for the monovalent or 400 CCA units for the bivalent vaccine. No lots were withdrawn for failure to meet requirements other than number of CCA units. The 11.6% withdrawal figure is consistent with rates of withdrawal Table 3.
occurring during previous years. These withdrawn lots represent the equivalent of --17 million doses; however, most of the lots withdrawn were subsequently fortified with virus concentrates and then resubmitted to the BOB as new lots. Therefore, the withdrawal of lots because of low potency did not appear to diminish substantially the total equivalent number of doses (150,034, 580) submitted to the BOB by December 16, 1976. As mentioned previously, tests for safety, sterility, and inactivation require six to seven days to complete and were performed by both the manufacturer and the BOB on the bulk lots. The manufacturer also tested final container mater-
Results of chick cell-agglutination (CCA) tests on influenza virus vaccines. Bureau of Biologics
Manufacturer No. of Vaccine, manufacturer * samples Monovalent MSD MN PD Wyeth Bivalent MSD MN PD Wyeth
Mean ±sot
Ranget
No. of samples
Mean ± sot
Ranget
p*
49 78 5 10
213.6 236.4 222.0 264.0
± 9.6 ± 18.0 ± 12.0 ±44.4
201.6-240.0 200.4-280.8 202.8-229.2 217.2-328.8
47 78 5 10
208.8 219.6 226.8 231.6
± 10.8 ± 18.0 ± 1.2 ± 34.8
199.2-228.0 199.2-279.6 205.2-273.6 199.2-291.6
;> 40 I 30
" ffl ! II "l / vff
20/ 10
9110
9/24
10/8 10/22 11/5 11119 12/3 12/17 12/31
WEEK ENDING:
Figure 1. Rates of production, testing, shipment, and distribution of influenza vaccines during the National Influenza Immunization Program. BoB = Bureau of Biologics.
probability and quantity of bacterial contamination during influenza vaccine production. The finding of low levels of endotoxin in present influenza vaccines gives evidence of the success of their efforts. Although the Reference Influenza Vaccine for Endotoxin Assay contains 1.043 /Lg of endotoxin per 1,200 CCA units of virus and therefore might be expected to have considerably higher values of endotoxin than present vaccines which contain only 200-400 CCA units of virus, calculations of the nanograms of endotoxin per CCA unit will indicate that this is not the sole explanation for the near absence of endotoxin in present vaccines. The reference endo-
toxin vaccine has 0.87 ng of endotoxinjCCA unit, whereas the present vaccines contain 0.030.36 ngjCCA unit and therefore only about onethird to less than one-thirtieth of the proportionate amount of endotoxin as vaccines used in the recent past . With the introduction of a standard reference into the CCA test, this measure of potency became more reliable [2], but difficulties in obtaining equivalent results between laboratories have nevertheless occasionally occurred. The present data confirm this finding and indicate that significant differences sometimes occurred between the CCA results of the BOB and those obtained by MSD and MN. However, the actual difference in means between the BOB's results and those of MN in the testing of their monovalent vaccine, for example, is only 7% of the total CCA content of the vaccine, well within the coefficient of error of the test [20]. On the other hand, the large SD value obtained both by the BOB and Wyeth during the testing of representative numbers of both monovalent and bivalent vaccines indicated difficulties on the part of both parties in the precise and consistent determination of potency of this manufacturer's vaccine. In spite of the generally good correlation between CCA results of the BOB and the manufacturers, the test nevertheless remains an indirect one, dependent in part on the affinity of a particular strain of influenza virus for chicken red blood cells. The number of viral particles in preparations containing the same number of CCA units has consequently been found to vary widely [21]. A direct measure of the viral hemagglutinin content of inactivated influenza vaccine, as suggested by Schild et al. [22], would enable us to circumvent these difficulties. We have found that the adaptation of the "rocket" electrophoresis test of Laurell [4] to the quantitation of influenza hemagglutinin is easy to perform and yields accurate and reproducible results in our laboratory. In addition, the relevance of the results of this test to human immunogenicity has been well demonstrated by the fact that vaccines, such as those produced by MSD, that have a high ratio of hemagglutinin to CCA units are also those that are most immunogenic in individuals with little prior antigenic experience with influenza, either through natural infection
Downloaded from http://jid.oxfordjournals.org/ at Florida Atlantic University on August 29, 2015
.s-/ / i/ I &/ / ~ e. J?!; I ~g cf/ 150 million doses of vaccine should be useful in estimating the capability to provide vaccines when future pandemics occur.
References 1. Miller, G. L., Stanley, W. M. Quantitative aspects of the red blood cell agglutination test for influenza virus.J. Exp. Med. 79:185-195,1944. 2. Tauraso, N. M., O'Brien, T. C., Seligmann, E. B. Problems of influenza vaccine standardization. Bull. W.H.O. 41:479-506, 1969. 3. Laurell, C.-B. Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Ann. Biochem. 15:45-52, 1966. 4. Mayner, R. E., Blackburn, R. J., Barry, D. W. Quantitation of influenza vaccine hemagglutinin by immunoelectrophoresis. In International Association of Biological Standardization. Symposium on influenza immunization. Karger, Basel, 1977 (in press). 5. Brand, C. M., Skehel, J. J. Crystalline antigen from influenza virus envelope. Nature [New Biol.] 238: 145-147, 1972. 6. Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. Protein measurement with the Folin phenol reagent. J. BioI. Chern. 193:265-275, 1951. 7. Hochstein, H. D., Elin, R. J., Cooper, J. F., Seligmann, E. B., Wolff, S. M. Further developments of limulus
Downloaded from http://jid.oxfordjournals.org/ at Florida Atlantic University on August 29, 2015
or immunization [23]. The quantity of hemagglutinin, however, is not the sole determining factor in the induction of HAl antibody in such individuals since split-virus vaccines containing similar amounts of hemagglutinin as a whole-virus product induce lower levels of antibody in immunological "virgins" [23]. Since the ratios of hemagglutinin to immunogenicity for each individual manufacturer's vaccine are fairly constant for each age group with differing antigenic experiences, it is now theoretically possible to predict, with a fair degree of certainty, the immunogenicity of particular influenza vaccines for a population of defined age and prior influenza experience. Manufacturers of influenza vaccine as well as the BOB have now begun using the immunoelectrophoresis test as a laboratory measure of potency of influenza A/New Jersey and other antigens. Should the test continue to yield accurate, reproducible, reliable, and relevant results, then consideration will be given to the use of immunoelectrophoresis as the official test of influenza vaccine potency. In responding to the threat of the A/Hong Kong (H3N2) influenza epidemic of 1968, manufacturers produced only about 10 million doses per month [24]; similarly, --50 million doses were prepared in the five-month period before the 1957 epidemic [25]. Past experience had indicated that three to four months elapse between the first receipt of a strain and the production of a significant number of doses [24]. Manufacturers were given approved influenza A/New Jersey / 8/76 virus seed stocks on February 20, 1976. The exact amount of lead time now required by the manufacturers to produce sufficient basic stocks of allantoic fluid vaccine concentrates this year cannot be accurately calculated because of the delays engendered by the requirement for the passage of indemnification legislation [26]; however, an estimate of one to two months can be made. This estimate is supported by the fact that >5,000 doses were available as early as April 1976 for the clinical trials conducted under the auspices of the U.S. Public Health Service [27, 28]. The ability of the manufacturers to produce, test, and distribute 10 million doses of vaccine per week for at least several months is now well established. There appear to be several reasons
Barry et al.
S414
8.
9.
10.
12.
13. 14.
15.
16. 17.
18.
19. Inactivated Influenza Vaccine, General Safety Test. Federal Register 39:40014-40016, 1974. 20. Barry, D. W., Mayner, R. E., Staton, E., Dunlap, R. C., Rastogi, S. C., Hannah, J. E., Blackburn, R. J., Nortman, D. F., Graze, P. R. Comparative trial of influenza vaccines I. Immunogenicity of whole virus and split product virus in man. Am. J. Epidemi61. 104:34-46, 1976. 21. Dunlap, R. C., Brown, E. R., Barry, D. W. Determination of the viral particle content of influenza vaccines by electron microscopy. J. BioI. Stand. 3:281289,1975. 22. Schild, G. C., Wood, J. M., Newman, R. W. A singleradial-immunodiffusion technique for the assay of influenza haem agglutinin antigen. Bull. W.H.O. 52: 223-231, 1975. 23. Ennis, F; A., Mayner, R. E., Barry, D. W., Manischewitz, J. E., Dunlap, R. C., Verbonitz, M. W., Bozeman, F. M., Schild, G. C. Correlation of laboratory studies with clinical responses to A/New Jersey influenza vaccines. J. Infect. Dis. 136(Suppl.):S397-S406, 1977. 24. Murray" R. Production and testing in the U.S.A. of influenza virus vaccine made from the Hong Kong variant in 1968-69. Bull. W.H.O. 41:495-496, 1969. 25. Murray, R. Some problems in the standardization and control of influenza vaccine in 1957. Am. Rev. Respir. Dis. 83:160-167,1961. 26. Public Law 94-380. 94th Congress, S. 3735, August 12, 1976. U.S. Government Printing Office, Washington, D.C. 27. Seal, J. R., Sencer, D. J., Meyer, H. M., Jr. A status report in national immunization against influenza. ]. Infect. Dis. 133:715-720, 1976. 28. Parkman, P. D., Galasso, G. J., Top, F. H., Noble, G. R. Summary of clinical trials of influenza vaccines. J. Infect. Dis. 134:100-107,1976. 29. Kilbourne, E. D. Future influenza vaccines and the use of genetic recombinants. Bull. W.H.O. 41:643645,1969.
Downloaded from http://jid.oxfordjournals.org/ at Florida Atlantic University on August 29, 2015
11.
amebocyte lysate test. Bull. Parenter. Drug Assoc. 27:139-148,1973. Nash, T. The colorimetric estimation of formaldehyde by the means of the Hantzsch reaction. Biochem.J.55:416-421,1953. Association of Official Analytical Chemists. Official methods of analysis of the AOAC. Association of Official Analytical Chemists, Washington, D.C., 1975, p.451. Rains, T. C., Menis, O. V. Determination of submicrogram amounts of mercury in standard reference materials by flameless atomic absorption spectometry. J. Assoc. Off. Anal. Chem. 55:1339,1972. Code of Federal Regulations, Title 21, Sections 610.11, 610.1Ia, and 610.12. U.S. Government Printing Office, Washington, D.C. Barry, D. W., Mayner, R. E., Hochstein, R. D., Dunlap, R. C., Rastogi, S. C., Hannah, J. E., Blackburn, R. J., Sullivan, J. L., Gerety, R. J. Comparative trial of influenza vaccines. II. Adverse reactions in children and adults. Am. J. Epidemiol. 104:47-59,1976. Peck, F. B. Purified influenza virus vaccine. J .A.M.A. 206:2277-2282, 1968. Reimer, C. B., Baker, R. S., Van Frank, R. M., Newlin, T. E., Cline, G. R, Anderson, N. G. Purification of large quantities of influenza virus by density gradient centrifugation. J. Virol. 1:1207-1216, 1967. O'Brien, T. C., Maloney, C. J., Tauraso, N. M. Quantitation of residual host protein in chicken embryoderived vaccines by radial immunodiffusion. Appl. Microbiol. 21:780-782,1971. Salk, J. E. Reactions to concentrated influenza virus vaccines. J. Immunol. 58:369-395,1948. Wright, P. F., Sell, S. H. W., Thompson, J., Karzon, D. T. Clinical reactions and serologic response following inactivated monovalent influenza type B vaccine in young children and infants. J. Pediatr. 88:31-35, 1976. Inactivated Influenza Vaccine, General Safety Test. Federal Register 39:27916-27918, 1974.
