THE JOUR;'I\AL O}' INFECTIOUS DISEASE. VOL. 136, SUPPLEMD1T • DECEMBER 1977 © 1977 by the University of Chicago. All rights reserved.
Alternative Approaches to Immunization of Children with Inactivated Influenza Virus Vaccines From the Influenza Research Center, the Department of Microbiology and Immunology and the Department of Pediatrics, Baylor College of Medicine, Houston, Texas; and St. Jude Children's Hospital, Memphis, Tennessee
W. Paul Glezen, Julius A. Kasel, Robert G. Webster, and Larry H. Taber
Influenza viral infections produce serious morbidity in pediatric populations [1-3]. Although the available influenza vaccines have induced protection [4], there has been a general reluctance to use them in children because of the frequency of febrile reactions [5, 6]. In an attempt to find a safe and effective method for the immunization of children, two trials were performed with inactivated vaccines against influenza A/New Jersey/8/76 (HswINI) virus. The first trial compared intranasal inoculation of
both whole and split-product commercial vaccines with im administration of each. Extensive trials of intranasal vaccination in adults have yielded variable results [7], but there has been a suggestion that children might respond more consistently [8]. The second trial com pared the reactogenicity and antigenicity of two im doses of a new purified subunit vaccine, HANAflu, with that of two small doses of whole-virus vaccine and with that of two doses of a mixture of the two preparations. In general, prior studies have shown that the licensed split-product vaccines are less reactogenic than whole-virus vaccines [9]; however, the licensed split-product vaccines contain not only the essential surface antigens, hemagglutinin and neuraminidase, but also various amounts of viral matrix protein and nucleoprotein thought not to be necessary for immunity. The HANAflu preparations virtually exclude these extraneous proteins and concentrate the hemagglutinin and neuraminidase spikes in stellate aggregates (l0]. Clinical studies of H3N2 HANAflu vaccines demonstrated that they were safe and
This work was supported by contract no. NIH AI 42528 from the Development and Applications Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health. We thank Perry Weston, principal, and the students of the High School for Health Professions (Houston, Texas); Robert Hager, director, and the children at Esperanza School (Houston); and Gloria Kliebert, director, and the children of Southhampton School (Houston) for their cooperation. Please address requests for reprints to Dr. W. P. Glezen, Department of Microbiology and Immunology, Baylor College of Medicine, 1200 Moursund Avenue, Houston, Texas 77080.
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The route of inoculation and the method of preparation of influenza virus vaccines were investigated. In the first trial a single dose of commercial whole-virus or splitproduct vaccine against influenza A/New Jersey/8/76 (HswlNl) virus was administered to children aged five to 10 years either intramuscularly (im) or intranasally. In the second trial a new purified hemagglutinin and neuraminidase vaccine (HANAflu) was tested in students aged 15-20 years (an un primed population). In the first trial 200 chick cell-agglutinating (CCA) units of whole-virus vaccine given im stimulated a significant antibody response in all children tested. One dose of 200 CCA units of split-product vaccine given im or 400 CCA units of whole-virus vaccine given intranasally stimulated at least some serum antibody in 30%-40% of the children, but 400 CCA units of split-product vaccine administered intranasally resulted in no detectable antibody. In the second trial subjects received im injections of two doses of 400 CCA units of the purified subunit vaccine (HANAflu), two doses of 40 CCA units of whole-virus vaccine, or a mixture of 360 CCA units of HANAflu and 40 CCA units of whole-virus vaccine. Less than one-half of the subjects who received the HANAflu preparation alone developed titers of :=:1 :40, whereas almost 90% of those receiving the mixture of HANAflu and whole-virus vaccine developed titers of :=:1:40.
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Table 1. Reactions to inactivated whole-virus (MerrellNational Laboratories, Cincinnati, Ohio) and splitproduct (Wyeth Laboratories, Philadelphia, Pa.) vaccines against influenza A/New Jersey/8/76 (Hsw 1N1) virus administered im or in tranasa1ly to children aged five to 10 years.
Route of administration, vaccine
Reaction index Systemic
Local
200 200
0.10 0.25
0.10 0.42
0/10 0/12
400 400
0.25 0.25
0.17 0.17
0/12 0/12
*CCA = chick cell-agglutinating.
potent [11] and encouraged us to test a similar vaccine prepared from the recombinant X-53 virus with the hemagglutinin and neuraminidase of A/New Jersey /8/76 (HswINl). The results of these two studies suggest avenues for further exploration in the search for better methods for pediatric immunizations. Materials and Methods
Subjects. Children aged five to 10 years were recruited for the intranasal study from two small private schools with summer programs. Parents were informed by letter about the study, and the investigators met with them at the schools to complete the consent forms. In the second trial (HANAflu), students aged 15-20 years were given an explanatory letter and consent form which they completed, if interested. The consent form was countersigned by a parent for all students under 18 years of age. The telephone number of the investigators was included in the letter so that students or parents could call to ask any questions about the study. Vaccines. Vaccines for the first trial were produced commercially and provided by the Bureau of Biologics (Food and Drug Administration, Bethesda, Md.). Whole-virus vaccine was manufactured by Merrell-National Laboratories (Cincinnati, Ohio), and the split-product vaccine by Wyeth Laboratories (Philadelphia, Pa.). The vaccine preparations for the HANAflu
Route of administration, vaccine Intramuscular Whole-virus Split-product Intranasal Whole-virus Split-product
Dose No. of (CCA subunits) * jects
No. of subjects with indicated Reciprocal titer geometric ;;;;.10 ;;;;.40 mean titer
200 200
7 9
7 3
6 1
65.6 9.3
400 400
12 12
5 0
1 0
7.9
*CCA = chick cell-agglutinating.
trial were prepared (by R. G. W.) at the St. Jude Children's Research Hospital in Memphis. Systemic reactions to vaccines were graded, and reaction indexes were calculated by methods described for all pediatric evaluations [12]. The local reactions were graded on a scale of 1 for subjective pain only, 2 for an area of redness or swelling 100 F{ no. of subjects
400 40
0.11 0.22
0.18 0.33
0/17 0/18
360-40
1.00
0.47
2/17t
Dose (CCA units]"
Reaction index
*CCA = chick cell-agglutinating. tOne of the two subjects with fever probably had a viral gastroenteri tis.
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In tramuscular Whole-virus Split-product Intranasal Whole-virus Split-product
Dose (CCA units) *
No. with fever >100 F{ no. of children
Table 2. Comparison of titers of HAl antibody after a single dose of inactivated whole-virus (MerrellNational Laboratories, Cincinnati, Ohio) or splitproduct (Wyeth Laboratories, Philadelphia, Pa.) vaccine against influenza A/New Jersey/8/76 (Hsw INl) virus administered im or intranasally to children five to 10 years of age.
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Table 4. Comparison of titers of HAl antibody after vaccination with a purified hemagglutinin and neuraminidase (HANAflu) vaccine against influenza A/New Jersey/8/76 (HswlNl) virus, small doses of whole-virus vaccine, or a mixture of the two vaccines in subjects aged 15- 20 years. First dose
Vaccine HANAflu Whole-virus HANAflu plus whole-virus
Dose (CCA units) *
No. of subjects
400 40 360-40
17 18 17
No. of subjects with indicated reciprocal titer
Second dose No. of subjects with indicated reciprocal titer
;;;.10
;;;.40
Reciprocal GMTt
;;;.10
;;;.40
Reciprocal GMT
2
1 2 6
6.1 10.8 18.4
13 15 17
7 6 15
17.0 19.2 51.1
11 12
by the World Health Organization (WHO) Influenza Reference Laboratory at the Center for Disease Control (Atlanta, Ga.). All sera were treated with receptor-destroying enzyme before testing. Antineuraminidase (NAI) assays were performed by the methods recommended by the WHO Influenza Reference Center. Vaccine preparations were assayed for neuraminidase activity by the method used by Laver and Kilbourne [13]. Results
Intranasal study. A total of 46 children aged five to 10 years were recruited to receive a single dose of whole-virus or split-product vaccine by either the im or the intranasal route. The im dose was 200 chick cell-agglutinating (CCA) units, Table 5. Comparison of antibody responses to neuraminidase in subjects aged 15- 20 years who received purified hemagglutinin and neuraminidase (HANAflu) vaccine against influenza A/New Jersey/ 8/76 (HswlNl) virus, a small dose of inactivated whole-virus vaccine, or a mixture of the two vaccines. No. (%) of subjects with antibody Vaccine HANAflu Whole-virus HANAflu plus whole-virus
Dose No. of (CCA units}" subjects
First dose
Second dose
400 40
17 18
1 (5.9) 2{11.1}
1 (5.9) 3 (15.7)
36(}-40
17
o
9 (52.9)
NOTE. Serum was obtained four weeks after the first dose and two weeks after the second dose. *CCA = chick cell-agglutinating.
and the intranasal dose was 400 CCA units delivered by dropping 0.25 ml into each nostril. The children were given thermometers so that their temperatures could be checked at bedtime (approximately 8 hr after inoculation), and the investigators saw each child at 24 hr to check for local reactions and repeat temperature determination. Reactions to these vaccines (table 1) were minimal; no child had a temperature of >99.8 F, and systemic complaints were minimal. All children were in school the next day and none altered their usual activities. Local reactions were limited to subjective complaints of sore arm or stuffy nose. A sample of serum was obtained from each child 21 days after inoculation, and titers of HAl antibody were determined (table 2). Six of seven children tested for antibodies after im administration of whole-virus vaccine had titers of Table 6. Neuraminidase activity of preparations of purified hemagglutinin and neuraminidase (HANAflu) and inactivated whole-virus vaccines. Vaccine type HANAflu Whole-virus HANAflu plus whole-virus HANAflu+
Antigen
Neuraminidase CCA activity units/ml" (units/ml)t
X-53 (Hsw1N1) X-53 (Hsw1N1)
800 400
X-53 (HswlNl) A/Victoria (H3N2)
72(}-80 800
101.3 8.3 89.7 96.8
*CCA = chick cell-agglutinating. tOne unit is the amount of enzyme that will release one nmole of sialic acid from the fetuin substrate in 1 min at 37 C. fUsed in the bivalent vaccine for high-risk children [13].
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NOTE. Serum was obtained four weeks after the first dose and two weeks after the second dose. *CCA = chick cell-agglutinating. r GMT = geometric mean titer.
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~l :40. No antibodies were detected in children who received the split-product vaccine intranasally; however, the responses of children who received the whole-virus vaccine intranasally were minimal but comparable to responses of children who received a single im dose of splitproduct vaccine. HANAflu study. A total of 52 students aged 15-20 years were recruited to receive two doses of HANAflu (400 CCA units), two small doses of whole-virus vaccine (40 CCA units), or a mixture of the two that contained 360 CCA units of HANAflu and 40 CCA units of whole-virus
vaccine. The reactions to these three vaccines are shown in table 3. Two students who received the combination had temperatures of >100 F within 24 hr after inoculation. One of these subjects probably had a coincidental viral gastroenteritis; he recalled having diarrheal stools a few hours before vaccination even though the onset of fever and vomiting was 8 hr after vaccination. His reaction score made up one-half of the total score for his group. As seen in table 4, two doses of HANAflu resulted in titers of ~l :40 for less than one-half of the subjects tested and were not significantly better than two doses of only 40
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Figure 1. Electron micrographs of influenza A vaccine preparations: A, purified hemagglutinin and neuraminidase (HANAflu) preparation of the X-53 recombinant virus with the hemagglutinin and neuraminidase of influenza A/New jersey/8/76 (HswlNl) virus; B, inactivated whole-virus preparation of the X-53 recombinant of influenza A/New Jersey/8/76 virus; C, mixture of X-53 HANAflu preparation and X-53 whole-virus at a ratio of 9: 1; D, HANAflu preparation of influenza A/Victoria/3/75 (H3N2) virus. The hemagglutinin and-neuraminidase spikes are much more distinct in the A/Victoria preparation than in the A/New Jersey recombinant preparation illustrated in A. (The vaccine preparations were negatively stained with 2% phosphotungstic acid and examined in a Siemens Elmiskop lA [Siemens Elmiskop, Berlin, Germany] X 27,778 by Dr. Heather Mayor, Department of Microbiology and Immunology, Baylor College of Medicine, Houston, Tex.)
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CCA units of whole-virus vaccine. The antibody response to the mixture was significantly enhanced, with 15 of 17 subjects attaining titers of~I:40.
References
Discussion
Intranasal study. In this study a single im dose of inactivated whole-virus vaccine (200 CCA units) was an adequate antigenic stimulus for children five to 10 years of age, but other studies have found a high frequency of febrile reactions to this dosage. A single intranasal dose of whole-virus vaccine (400 CCA units) gave an antibody response comparable to a single im dose of split-product vaccine (200 CCA units), which is now recommended in a two-dose schedule. Therefore, further studies might be warranted to explore the response to a second intranasal dose at least four weeks after the initial priming dose. HANAfiu study. A small amount of wholevirus vaccine mixed with the purified subunit HANAflu vaccine significantly enhanced the antibody response for both the hemagglutinin and the neuraminidase components. This possibility had been suggested by experimental vaccination of hamsters by Laver and Webster [10]. The antibody response to the A/New Jersey / 8/76 (HswINI) HANAflu preparations alone was inadequate and was confirmed by results of the bivalent trial in high-risk children [14]. In contrast, the HANAflu preparations of H3N2 viruses were potent in both adults [11] and chil-
J. D., Chanock, R. M., Parrott, R. H. Respiratory viruses and Mycoplasma pneumoniae in infants and young children during 1972. Clin. Proc. Child. Hosp. Nat!. Med. Cen. 29:228-233, 1973. Jordan, W. S., Jr., Denny, F. W., jr., Badger, G. F., Curtiss, C., Dingle, J. H., Oseasohn, R., Stevens, D. A. A study of illness in a group of Cleveland families. XVII. The occurrence of Asian influenza. Am. J. Hyg.68:190-212,1958. Hilleman, M. R., Hamparian, V. V., Ketler, A., Reilly, C. M., McClelland, L., Cornfeld, D., Stokes, J., Jr. Acute respiratory illnesses among children and adults. J.A.M.A. 180:445-453, 1962. Foy, H. M., Cooney, M. K., McMahan, R. A/Hong Kong influenza immunity three years after immunization. J.A.M.A. 226:758-761,1973. Marine, W. M., Stuart-Harris, C. Reactions and serologic responses in young children and infants after administration of inactivated monovalent influenza A vaccine. J. Pediatr. 88:26-30, 1976. Wright, P. F., Sell, S. H., 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. Waldman, R. H., Coggins, W. J. Influenza immunization: field trial on a university campus. J. Infect. Dis. 126:242-248, 1972. Fulk, R. V., Fedson, D. S., Huber, M. A., Fitzpatrick, J. R., Howar, B. F., Kasel, J. A. Antibody responses in children and elderly persons following local or parenteral administration of an inactivated influenza virus vaccine A2/Hong Kong/68 variant. J. Immunol. 102:1102-1105,1969. Phillips, C. F., Phillips, C. A., Hodgkin, W. E., Forsyth, B. R., Rubin, B. A., Geraghty, M. E. Killed subunit
I. Kim, H. W., Brandt, C. D., Arrobio,
2.
3.
4.
5.
6.
7.
8.
9.
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The NAI response followed the same pattern as did the HAl antibody response (table 5). Although the titers were relatively low, nine of 17 subjects receiving the mixture of HANAflu and whole-virus vaccine had detectable NAI antibodies two weeks after the second dose. Only one of the subjects who received two doses of pure HANAflu vaccine had detectable NAI antibodies, although the preparation had adequate neuraminidase activity (table 6). In contrast, three of 18 subjects had NAI antibodies in response to 40 CCA units of whole-virus vaccine, even though no neuraminidase activity was detectable at that concentration. Minimal levels of neuraminidase were found in the undiluted whole-virus vaccine (400 CCA unita/ml).
dren [14]. Electron microscopy of the H3N2 preparations revealed well-defined spikes in regular stellate aggregates, whereas the HswlNl preparations were fuzzy and amorphous (figure 1). It is possible that the hemagglutinin of the HswlNI virus is less stable and that the methodology for preparations of purified hemagglutinin and neuraminidase will have to be modified to achieve an adequate antigen. Continued efforts are needed to determine the optimal antigens and routes of inoculation for immunizing children. Influenza prophylaxis in this age group might not only significantly decrease morbidity but also could dampen or abort epidemics, thus decreasing risk for the entire community [15].
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influenza vaccine in children. Pediatrics 52:416-419, 1973. 10. Laver, W. G., Webster, R. G. Preparation and immunogenicity of an influenza virus hemagglutinin and neuraminidase subunit vaccine. Virology 69:511-522, 1976. II. Webster, R. G., Kasel, J. A., Couch, R. B., Laver, W. G. Influenza virus subunit vaccines. II. Immunogenicity and original antigenic sin in humans. J. Infect. Dis. 134:48-58,1976. 12. Gross, P. A. Reactogenicity and immunogenicity of bivalent influenza vaccine in one- and two-dose trials in children: a summary. J. Infect. Dis. 136 (Suppl.): S616-S625, 1977.
Glezen et al,
13. Laver, W. G., Kilbourne, E. D. Identification in a recombinant influenza virus of structural proteins derived from both parents. Virology 30:493-501, 1966. 14. Allison, J. E., Glezen, W. P., Taber, L. H., Paredes, A., Webster, R. G. Reactogenicity and immunogenicity of bivalent influenza A and monovalent influenza B virus vaccines in high-risk children. J. Infect. Dis. 136(Suppl.):S672-S676,1977. 15. Elveback, L. R., Fox, J. P., Ackerman, E., Langworthy, A., Boyd, M., Gatewood, L. An influenza simulation model for immunization studies. Am. J. Epidemiol. 103:152-165,1976.
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Alternative Approaches to Immunization of Children with Inactivated Influenza Virus Vaccines From the Influenza Research Center, the Department of Microbiology and Immunology and the Department of Pediatrics, Baylor College of Medicine, Houston, Texas; and St. Jude Children's Hospital, Memphis, Tennessee
W. Paul Glezen, Julius A. Kasel, Robert G. Webster, and Larry H. Taber
Influenza viral infections produce serious morbidity in pediatric populations [1-3]. Although the available influenza vaccines have induced protection [4], there has been a general reluctance to use them in children because of the frequency of febrile reactions [5, 6]. In an attempt to find a safe and effective method for the immunization of children, two trials were performed with inactivated vaccines against influenza A/New Jersey/8/76 (HswINI) virus. The first trial compared intranasal inoculation of
both whole and split-product commercial vaccines with im administration of each. Extensive trials of intranasal vaccination in adults have yielded variable results [7], but there has been a suggestion that children might respond more consistently [8]. The second trial com pared the reactogenicity and antigenicity of two im doses of a new purified subunit vaccine, HANAflu, with that of two small doses of whole-virus vaccine and with that of two doses of a mixture of the two preparations. In general, prior studies have shown that the licensed split-product vaccines are less reactogenic than whole-virus vaccines [9]; however, the licensed split-product vaccines contain not only the essential surface antigens, hemagglutinin and neuraminidase, but also various amounts of viral matrix protein and nucleoprotein thought not to be necessary for immunity. The HANAflu preparations virtually exclude these extraneous proteins and concentrate the hemagglutinin and neuraminidase spikes in stellate aggregates (l0]. Clinical studies of H3N2 HANAflu vaccines demonstrated that they were safe and
This work was supported by contract no. NIH AI 42528 from the Development and Applications Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health. We thank Perry Weston, principal, and the students of the High School for Health Professions (Houston, Texas); Robert Hager, director, and the children at Esperanza School (Houston); and Gloria Kliebert, director, and the children of Southhampton School (Houston) for their cooperation. Please address requests for reprints to Dr. W. P. Glezen, Department of Microbiology and Immunology, Baylor College of Medicine, 1200 Moursund Avenue, Houston, Texas 77080.
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The route of inoculation and the method of preparation of influenza virus vaccines were investigated. In the first trial a single dose of commercial whole-virus or splitproduct vaccine against influenza A/New Jersey/8/76 (HswlNl) virus was administered to children aged five to 10 years either intramuscularly (im) or intranasally. In the second trial a new purified hemagglutinin and neuraminidase vaccine (HANAflu) was tested in students aged 15-20 years (an un primed population). In the first trial 200 chick cell-agglutinating (CCA) units of whole-virus vaccine given im stimulated a significant antibody response in all children tested. One dose of 200 CCA units of split-product vaccine given im or 400 CCA units of whole-virus vaccine given intranasally stimulated at least some serum antibody in 30%-40% of the children, but 400 CCA units of split-product vaccine administered intranasally resulted in no detectable antibody. In the second trial subjects received im injections of two doses of 400 CCA units of the purified subunit vaccine (HANAflu), two doses of 40 CCA units of whole-virus vaccine, or a mixture of 360 CCA units of HANAflu and 40 CCA units of whole-virus vaccine. Less than one-half of the subjects who received the HANAflu preparation alone developed titers of :=:1 :40, whereas almost 90% of those receiving the mixture of HANAflu and whole-virus vaccine developed titers of :=:1:40.
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Table 1. Reactions to inactivated whole-virus (MerrellNational Laboratories, Cincinnati, Ohio) and splitproduct (Wyeth Laboratories, Philadelphia, Pa.) vaccines against influenza A/New Jersey/8/76 (Hsw 1N1) virus administered im or in tranasa1ly to children aged five to 10 years.
Route of administration, vaccine
Reaction index Systemic
Local
200 200
0.10 0.25
0.10 0.42
0/10 0/12
400 400
0.25 0.25
0.17 0.17
0/12 0/12
*CCA = chick cell-agglutinating.
potent [11] and encouraged us to test a similar vaccine prepared from the recombinant X-53 virus with the hemagglutinin and neuraminidase of A/New Jersey /8/76 (HswINl). The results of these two studies suggest avenues for further exploration in the search for better methods for pediatric immunizations. Materials and Methods
Subjects. Children aged five to 10 years were recruited for the intranasal study from two small private schools with summer programs. Parents were informed by letter about the study, and the investigators met with them at the schools to complete the consent forms. In the second trial (HANAflu), students aged 15-20 years were given an explanatory letter and consent form which they completed, if interested. The consent form was countersigned by a parent for all students under 18 years of age. The telephone number of the investigators was included in the letter so that students or parents could call to ask any questions about the study. Vaccines. Vaccines for the first trial were produced commercially and provided by the Bureau of Biologics (Food and Drug Administration, Bethesda, Md.). Whole-virus vaccine was manufactured by Merrell-National Laboratories (Cincinnati, Ohio), and the split-product vaccine by Wyeth Laboratories (Philadelphia, Pa.). The vaccine preparations for the HANAflu
Route of administration, vaccine Intramuscular Whole-virus Split-product Intranasal Whole-virus Split-product
Dose No. of (CCA subunits) * jects
No. of subjects with indicated Reciprocal titer geometric ;;;;.10 ;;;;.40 mean titer
200 200
7 9
7 3
6 1
65.6 9.3
400 400
12 12
5 0
1 0
7.9
*CCA = chick cell-agglutinating.
trial were prepared (by R. G. W.) at the St. Jude Children's Research Hospital in Memphis. Systemic reactions to vaccines were graded, and reaction indexes were calculated by methods described for all pediatric evaluations [12]. The local reactions were graded on a scale of 1 for subjective pain only, 2 for an area of redness or swelling 100 F{ no. of subjects
400 40
0.11 0.22
0.18 0.33
0/17 0/18
360-40
1.00
0.47
2/17t
Dose (CCA units]"
Reaction index
*CCA = chick cell-agglutinating. tOne of the two subjects with fever probably had a viral gastroenteri tis.
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In tramuscular Whole-virus Split-product Intranasal Whole-virus Split-product
Dose (CCA units) *
No. with fever >100 F{ no. of children
Table 2. Comparison of titers of HAl antibody after a single dose of inactivated whole-virus (MerrellNational Laboratories, Cincinnati, Ohio) or splitproduct (Wyeth Laboratories, Philadelphia, Pa.) vaccine against influenza A/New Jersey/8/76 (Hsw INl) virus administered im or intranasally to children five to 10 years of age.
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Table 4. Comparison of titers of HAl antibody after vaccination with a purified hemagglutinin and neuraminidase (HANAflu) vaccine against influenza A/New Jersey/8/76 (HswlNl) virus, small doses of whole-virus vaccine, or a mixture of the two vaccines in subjects aged 15- 20 years. First dose
Vaccine HANAflu Whole-virus HANAflu plus whole-virus
Dose (CCA units) *
No. of subjects
400 40 360-40
17 18 17
No. of subjects with indicated reciprocal titer
Second dose No. of subjects with indicated reciprocal titer
;;;.10
;;;.40
Reciprocal GMTt
;;;.10
;;;.40
Reciprocal GMT
2
1 2 6
6.1 10.8 18.4
13 15 17
7 6 15
17.0 19.2 51.1
11 12
by the World Health Organization (WHO) Influenza Reference Laboratory at the Center for Disease Control (Atlanta, Ga.). All sera were treated with receptor-destroying enzyme before testing. Antineuraminidase (NAI) assays were performed by the methods recommended by the WHO Influenza Reference Center. Vaccine preparations were assayed for neuraminidase activity by the method used by Laver and Kilbourne [13]. Results
Intranasal study. A total of 46 children aged five to 10 years were recruited to receive a single dose of whole-virus or split-product vaccine by either the im or the intranasal route. The im dose was 200 chick cell-agglutinating (CCA) units, Table 5. Comparison of antibody responses to neuraminidase in subjects aged 15- 20 years who received purified hemagglutinin and neuraminidase (HANAflu) vaccine against influenza A/New Jersey/ 8/76 (HswlNl) virus, a small dose of inactivated whole-virus vaccine, or a mixture of the two vaccines. No. (%) of subjects with antibody Vaccine HANAflu Whole-virus HANAflu plus whole-virus
Dose No. of (CCA units}" subjects
First dose
Second dose
400 40
17 18
1 (5.9) 2{11.1}
1 (5.9) 3 (15.7)
36(}-40
17
o
9 (52.9)
NOTE. Serum was obtained four weeks after the first dose and two weeks after the second dose. *CCA = chick cell-agglutinating.
and the intranasal dose was 400 CCA units delivered by dropping 0.25 ml into each nostril. The children were given thermometers so that their temperatures could be checked at bedtime (approximately 8 hr after inoculation), and the investigators saw each child at 24 hr to check for local reactions and repeat temperature determination. Reactions to these vaccines (table 1) were minimal; no child had a temperature of >99.8 F, and systemic complaints were minimal. All children were in school the next day and none altered their usual activities. Local reactions were limited to subjective complaints of sore arm or stuffy nose. A sample of serum was obtained from each child 21 days after inoculation, and titers of HAl antibody were determined (table 2). Six of seven children tested for antibodies after im administration of whole-virus vaccine had titers of Table 6. Neuraminidase activity of preparations of purified hemagglutinin and neuraminidase (HANAflu) and inactivated whole-virus vaccines. Vaccine type HANAflu Whole-virus HANAflu plus whole-virus HANAflu+
Antigen
Neuraminidase CCA activity units/ml" (units/ml)t
X-53 (Hsw1N1) X-53 (Hsw1N1)
800 400
X-53 (HswlNl) A/Victoria (H3N2)
72(}-80 800
101.3 8.3 89.7 96.8
*CCA = chick cell-agglutinating. tOne unit is the amount of enzyme that will release one nmole of sialic acid from the fetuin substrate in 1 min at 37 C. fUsed in the bivalent vaccine for high-risk children [13].
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NOTE. Serum was obtained four weeks after the first dose and two weeks after the second dose. *CCA = chick cell-agglutinating. r GMT = geometric mean titer.
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~l :40. No antibodies were detected in children who received the split-product vaccine intranasally; however, the responses of children who received the whole-virus vaccine intranasally were minimal but comparable to responses of children who received a single im dose of splitproduct vaccine. HANAflu study. A total of 52 students aged 15-20 years were recruited to receive two doses of HANAflu (400 CCA units), two small doses of whole-virus vaccine (40 CCA units), or a mixture of the two that contained 360 CCA units of HANAflu and 40 CCA units of whole-virus
vaccine. The reactions to these three vaccines are shown in table 3. Two students who received the combination had temperatures of >100 F within 24 hr after inoculation. One of these subjects probably had a coincidental viral gastroenteritis; he recalled having diarrheal stools a few hours before vaccination even though the onset of fever and vomiting was 8 hr after vaccination. His reaction score made up one-half of the total score for his group. As seen in table 4, two doses of HANAflu resulted in titers of ~l :40 for less than one-half of the subjects tested and were not significantly better than two doses of only 40
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Figure 1. Electron micrographs of influenza A vaccine preparations: A, purified hemagglutinin and neuraminidase (HANAflu) preparation of the X-53 recombinant virus with the hemagglutinin and neuraminidase of influenza A/New jersey/8/76 (HswlNl) virus; B, inactivated whole-virus preparation of the X-53 recombinant of influenza A/New Jersey/8/76 virus; C, mixture of X-53 HANAflu preparation and X-53 whole-virus at a ratio of 9: 1; D, HANAflu preparation of influenza A/Victoria/3/75 (H3N2) virus. The hemagglutinin and-neuraminidase spikes are much more distinct in the A/Victoria preparation than in the A/New Jersey recombinant preparation illustrated in A. (The vaccine preparations were negatively stained with 2% phosphotungstic acid and examined in a Siemens Elmiskop lA [Siemens Elmiskop, Berlin, Germany] X 27,778 by Dr. Heather Mayor, Department of Microbiology and Immunology, Baylor College of Medicine, Houston, Tex.)
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CCA units of whole-virus vaccine. The antibody response to the mixture was significantly enhanced, with 15 of 17 subjects attaining titers of~I:40.
References
Discussion
Intranasal study. In this study a single im dose of inactivated whole-virus vaccine (200 CCA units) was an adequate antigenic stimulus for children five to 10 years of age, but other studies have found a high frequency of febrile reactions to this dosage. A single intranasal dose of whole-virus vaccine (400 CCA units) gave an antibody response comparable to a single im dose of split-product vaccine (200 CCA units), which is now recommended in a two-dose schedule. Therefore, further studies might be warranted to explore the response to a second intranasal dose at least four weeks after the initial priming dose. HANAfiu study. A small amount of wholevirus vaccine mixed with the purified subunit HANAflu vaccine significantly enhanced the antibody response for both the hemagglutinin and the neuraminidase components. This possibility had been suggested by experimental vaccination of hamsters by Laver and Webster [10]. The antibody response to the A/New Jersey / 8/76 (HswINI) HANAflu preparations alone was inadequate and was confirmed by results of the bivalent trial in high-risk children [14]. In contrast, the HANAflu preparations of H3N2 viruses were potent in both adults [11] and chil-
J. D., Chanock, R. M., Parrott, R. H. Respiratory viruses and Mycoplasma pneumoniae in infants and young children during 1972. Clin. Proc. Child. Hosp. Nat!. Med. Cen. 29:228-233, 1973. Jordan, W. S., Jr., Denny, F. W., jr., Badger, G. F., Curtiss, C., Dingle, J. H., Oseasohn, R., Stevens, D. A. A study of illness in a group of Cleveland families. XVII. The occurrence of Asian influenza. Am. J. Hyg.68:190-212,1958. Hilleman, M. R., Hamparian, V. V., Ketler, A., Reilly, C. M., McClelland, L., Cornfeld, D., Stokes, J., Jr. Acute respiratory illnesses among children and adults. J.A.M.A. 180:445-453, 1962. Foy, H. M., Cooney, M. K., McMahan, R. A/Hong Kong influenza immunity three years after immunization. J.A.M.A. 226:758-761,1973. Marine, W. M., Stuart-Harris, C. Reactions and serologic responses in young children and infants after administration of inactivated monovalent influenza A vaccine. J. Pediatr. 88:26-30, 1976. Wright, P. F., Sell, S. H., 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. Waldman, R. H., Coggins, W. J. Influenza immunization: field trial on a university campus. J. Infect. Dis. 126:242-248, 1972. Fulk, R. V., Fedson, D. S., Huber, M. A., Fitzpatrick, J. R., Howar, B. F., Kasel, J. A. Antibody responses in children and elderly persons following local or parenteral administration of an inactivated influenza virus vaccine A2/Hong Kong/68 variant. J. Immunol. 102:1102-1105,1969. Phillips, C. F., Phillips, C. A., Hodgkin, W. E., Forsyth, B. R., Rubin, B. A., Geraghty, M. E. Killed subunit
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The NAI response followed the same pattern as did the HAl antibody response (table 5). Although the titers were relatively low, nine of 17 subjects receiving the mixture of HANAflu and whole-virus vaccine had detectable NAI antibodies two weeks after the second dose. Only one of the subjects who received two doses of pure HANAflu vaccine had detectable NAI antibodies, although the preparation had adequate neuraminidase activity (table 6). In contrast, three of 18 subjects had NAI antibodies in response to 40 CCA units of whole-virus vaccine, even though no neuraminidase activity was detectable at that concentration. Minimal levels of neuraminidase were found in the undiluted whole-virus vaccine (400 CCA unita/ml).
dren [14]. Electron microscopy of the H3N2 preparations revealed well-defined spikes in regular stellate aggregates, whereas the HswlNl preparations were fuzzy and amorphous (figure 1). It is possible that the hemagglutinin of the HswlNI virus is less stable and that the methodology for preparations of purified hemagglutinin and neuraminidase will have to be modified to achieve an adequate antigen. Continued efforts are needed to determine the optimal antigens and routes of inoculation for immunizing children. Influenza prophylaxis in this age group might not only significantly decrease morbidity but also could dampen or abort epidemics, thus decreasing risk for the entire community [15].
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influenza vaccine in children. Pediatrics 52:416-419, 1973. 10. Laver, W. G., Webster, R. G. Preparation and immunogenicity of an influenza virus hemagglutinin and neuraminidase subunit vaccine. Virology 69:511-522, 1976. II. Webster, R. G., Kasel, J. A., Couch, R. B., Laver, W. G. Influenza virus subunit vaccines. II. Immunogenicity and original antigenic sin in humans. J. Infect. Dis. 134:48-58,1976. 12. Gross, P. A. Reactogenicity and immunogenicity of bivalent influenza vaccine in one- and two-dose trials in children: a summary. J. Infect. Dis. 136 (Suppl.): S616-S625, 1977.
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13. Laver, W. G., Kilbourne, E. D. Identification in a recombinant influenza virus of structural proteins derived from both parents. Virology 30:493-501, 1966. 14. Allison, J. E., Glezen, W. P., Taber, L. H., Paredes, A., Webster, R. G. Reactogenicity and immunogenicity of bivalent influenza A and monovalent influenza B virus vaccines in high-risk children. J. Infect. Dis. 136(Suppl.):S672-S676,1977. 15. Elveback, L. R., Fox, J. P., Ackerman, E., Langworthy, A., Boyd, M., Gatewood, L. An influenza simulation model for immunization studies. Am. J. Epidemiol. 103:152-165,1976.
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