Local and systemic influenza haemagglutinin-specific antibody responses following aerosol and subcutaneous administration of inactivated split influenza vaccine Yasumasa Oh, Kazuyo Ohta, Harumi Kuno-Sakai, Ryuichi Kim and Mikio Kimura* An easily administered and safe vaccine & required to produce the herd immunity necessary to control influenza epidemics worldwide. A commercial quadrivalent inactivated split influenza vaccine was administered intranasally in aerosol form to a group of 46 volunteers; other groups were given the same vaccine subcutaneously and saline intranasally. The results show that mucosal stimulation via intranasal vaccination resulted in a marked increase in local HA-specific IgA antibodies, and that this stimulation was necessary for serum HA-specific IgA responses. Serum HA-specific IgA antibody levels can be used as indicators of local antigenic stimulation, providing a method for evaluating potency and antigenicity in humans of intranasal influenza vaccine. This vaccination route shows much promise for the fiaure. Keywords: Inactivated influenza vaccine; local IgA antibody; aerosol administration; serum HI antibody; influenza haemagglutinin
INTRODUCTION Inactivated influenza vaccines administered subcutaneously or intramuscularly, either in the form of a whole virion or in the split or subunit form, have been the only licensed influenza vaccines in the United States, Europe and Japan 1. Since influenza virus proliferates over the mucosal surface and causes disease instantaneously, vaccines which will stimulate both systemic and mucosal immunity are more promising for the prevention of influenza epidemics 2. It has been reported that the parenteral administration of inactivated influenza vaccine did not induce local specific IgA responses 3. Direct antigenic stimulation of the upper respiratory tract with a vaccine may induce local immunity. Therefore, an inactivated influenza vaccine, administered directly in the upper respiratory tract, is a good candidate vaccine which may be used safely in humans. Systemic and local immune responses to topically administered inactivated influenza vaccine have not been fully investigated. There are a few clinical trials on local Department of Pediatrics, School of Medicine, Tokai University, Isehara City, Kanagawa 259-11,Japan. *To whom correspondence should be addressed. (Received 9 October 1991; revised 18 November 1991; accepted 25 December 1991) 0264-410xi92,080506--06 1992 Butterworth-HeinemannLtd 506
Vaccine, Vol. 10, Issue 8, 1992
antibody responses of humans to topically administered influenza vaccines4'5, However, the method described for determination of local antibody responses required nasal wash solution and the results obtained were expressed only in terms of end points and not in terms of units. Lack of a sensitive method of microassay of specific antibodies present on the surface of the upper respiratory tract has long been one of the major weak points in the study of local immune responses to topically administered vaccines. We have developed a very sensitive, reliable and reproducible method of quantification of local specific antibodies to influenza haemagglutinins (HA) and investigated local specific IgA antibody responses to aerosol administration of an inactivated influenza vaccine; we compared serum haemagglutinin inhibition (HI) titres and serum specific IgA, IgM and IgG antibody levels between two groups of young adults, one of which received subcutaneous inactivated influenza vaccine and the other received aerosol administration of the same vaccine. Methods of evaluation of the potency and antigenicity in humans, both local or systemic, are also required for an influenza vaccine administered intranasally. This study may furnish basic information for estimation of the degree of local and systemic antigenic stimulation of topically administered inactivated influenza vaccine.
Aerosol administration of influenza vaccine: Y. Oh et al. VOLUNTEERS
AND METHODS
Volunteers Ninety-two young adult volunteers were divided into two groups, the aerosol administration group and the subcutaneous administration group. Written informed consent was obtained from all volunteers. A further 46 young adult volunteers served as controls and received aerosol administration of saline,
Vaccine Commercially available quadrivalent inactivated split influenza vaccine (0.5 ml), which was distributed during the winter of 1988-89 [A/Yamagata/120/86 (H1N1) 200 chicken cell agglutination units (CCA)m1-1 equivalent; A/Fukuoka/C29/85 (H3N2) 200 CCA ml- 1 equivalent; A/Sichuan/2/87 (H3N2) 200 CCA m1-1 equivalent; and B / N a g a s a k i / I / 8 7 100 CCA m1-1 equivalent] was administered by either the subcutaneous or intranasal route.
Method of administration
Aerosol administration. Forty-six volunteers received the vaccine intranasally in the form of aerosol. The vaccine was sprayed on each side of the nose at a volume of 0.25 ml each by the Jacksonian type tracheal spray. The vaccine was given four times at 1 week intervals between each administration. Subcutaneous administration. Forty-six volunteers received subcutaneous administration of the vaccine 0.5 ml each, twice at a 4-week interval. Collection of the samples
Preparation of the nasal swab solution. The samples were collected from the vaccinee group before administration and 7 weeks after the fourth aerosol administration of the inactivated split vaccine. Other samples were collected from the control group before administration and 6 weeks after the second administration of the saline. A wet cotton swab, which had been soaked in 0.01 M phosphate-buffered saline (PBS), was gently rotated in the nose for 30 s. In this way at least 20 #i of mucus can be collected. The swab was mixed with 3 ml 0.01 M PBS. The solution was centrifuged for 30 rain at 3000 rev min- 1 to remove any cell debris, and N a N 3 was added at a concentration of 0.05%. The sample was stored at - 20°C until assayed.
every day for 7 days, was added to the wells, The plate was then incubated at 4~C overnight. After two washings with PBS-Tween, alkaline phosphatase labelled antihuman IgA goat serum conjugate diluted with PBS containing 1% bovine serum albumin was added. After incubation for 2 h the plate was washed twice with PBS and three times with demineralized water. Then 200 pl of the substrate solution (100mg of p-nitrophenyl phosphate disodium dissolved in 100 ml of demineralized water with 0.53 g of NazCO3, 0.02 g of MgC12 . 6HzO and 1 ml of 1 M HCI ) was applied. After allowing to stand at 37°C for 1 h, absorbancy at 405nm (A,~os) was measured using a microplate photometer. The results were expressed in terms of units using the standard solution. Total IgA concentrations of nasal swab solutions were measured by laser nephelometry (Behringwerke, Marburg, Germany) and the final results of the specific IgA values were expressed in terms of ELISA units rag- 1 of IgA.
Determination of serum class-specific anti-influenza HA antibodies. The methods were essentially the same as that described above. Alkaline phosphatase labelled anti-human IgG, lgA and IgM goat sera were used. The values were expressed in terms of ELISA units ml- t of serum.
Serum HI titres Serum HI titres were quantified by the standard method. Haemagglutinins of the subtypes of influenza virus contained in the vaccine were used as antigens of HI tests. It should be noted that expression of the dilution ofsera in Japan is different from that in the United States ; a dilution of 1:8 in the United States corresponds to a dilution of I :32 in Japan.
Statistical analysis The 7.2 test with Yates' correction was used to compare the rises in serum HI titres or in influenza HA-specific antibodies between subcutaneous and aerosol administration groups. The Spearman-Kendall test was used to evaluate a correlation between serum-specific IgA antibody levels and HA-specific IgA antibody levels of the nasal swab solution.
RESULTS
Collection ofsera. Serum samples were collected before vaccination and 7 weeks after the fourth dose of aerosol vaccine or 6 weeks after the second dose of the subcutaneous vaccine. ELISA method
ELISA of specific lgA antibodies of nasal swab solution. Details of the ELISA method have already been published 6. In brief, a Linbro-Titertek EIA microtitration plate (Flow Laboratories Inc., McLean, VA 22102, USA ) was coated with purified influenza haemagglutinin (HA) solution and was allowed to stand at 4°C for 2 days. After washing with PBS containing 0.05% Tween 20 (PBS-Tween), 50/A of nasal swab solution or standard solution, which was the nasal swab solution collected from an adult who had received aerosol influenza vaccine
Figure 1 shows serum HI titres (A/Sichuan H3N2) before and after administration of the subcutaneous (Figure la) or aerosol (Figure lb) influenza vaccine. Greater than fourfold rises in HI titres were observed in 93% (43/46) of the subcutaneous administration group. Geometrical mean prevaccination and postvaccination titres w e r e 2 3`9 and 26.9 , respectively. Greater than fourfold elevations of serum HI titres were observed in 87% (40/46) of the aerosol vaccine administration group. Geometrical mean prevaccination and postvaccination titres were 24.5 and 2 "1 , respectively. Percentages of more than fourfold rises in serum HI titres were not significantly different between the two groups (7." = 0.4926, p = 0.4827, 7.-Yates test). It should be noted that marked elevations of HI titres were observed among those with a prevaccination HI titre of less than 1:16 of the aerosol administration group.
Vaccine, Vol. 10, Issue 8, 1992 507
Aerosol administration of influenza vaccine. Y. Oh e t al. a
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t1:16. When the split influenza vaccines were administered intranasally in the form of aerosol, the vaccines had booster effects which were shown by rises in HA-specific local IgA antibodies, in serum HA-specific IgG, IgM and IgA antibodies, and in serum HI titres. Those results showed that the vaccines induced local immunoglobulin production and rises in serum-specific antibody levels even in the subjects who already had specific serum antibodies and/or local IgA antibodies to the subtypes of the virus of the vaccine. Our results demonstrated that the route of administration of the inactivated split influenza vaccine influenced the type of immune response. Little change in serum HA-specific IgA antibody was noted after subcutaneous administration of inactivated influenza vaccine. On the other hand, rises in serum HA-specific IgA antibody could be observed after aerosol administration. Rises in local HA-specific IgA antibody were observed after aerosol administration of the inactivated influenza vaccine. The existence of a correlation between rises {n local HA-specific IgA antibodies and rises in serum HA-specific IgA antibodies may suggest that local antigenic stimulation may result in production of specific IgA~producing cells. Over the last decade, simultaneous epidemics of two or three different subtypes of influenza A virus and/or influenza B virus have been noted every year throughout the world. Therefore, most people have been primed with various subtypes of influenza virus. Aerosol administration of the inactivated split influenza vaccine can stimulate both local and systemic antibody responses even in those
Aerosol administration of influenza vaccine: Y. Oh et al.
who have been exposed to similar subtypes of the influenza virus before and have some degree of serum-specific antibody and/or local specific antibody to the subtype of the virus of the vaccine. The first influenza virus was isolated from chickens with fowl plague in 1901. In 1933, Smith, Andrews and Francis succeeded in transmission of a human influenza virus to ferrets. Half a century has passed since the first influenza vaccine was administered to humans, yet no country in the world has ever succeeded in control of influenza epidemics by the use of vaccine given subcutaneously. Epidemics of influenza are very hard to control. The only possible way to control epidemics is to obtain herd immunity. To achieve this, an increase in acceptance rate is needed and it may therefore be preferable to have a choice of several influenza vaccines. Because they can be easily administered to humans and because they induce production of local specific IgA antibodies which may be able to reduce chances of shedding of the specified influenza virus, intranasal administration of either live or inactivated influenza vaccines should have a place in the prevention of influenza epidemics in the very near future.
in 28% (12/43) of the aerosol vaccine administration group and in 0% (0/24) of the control group who received aerosol saline in place of the vaccine. Those observations suggest, first, that mucosal stimulation with inactivated influenza vaccine resulted in marked increase in local HA-specific IgA antibodies. Secondly, mucosal antigenic stimulation was necessary for serum HA-specific IgA responses. Thirdly, serum HA-specific IgA antibody levels can be used as indicators of the local antigenic stimulation. Finally, the inactivated influenza split vaccine, applied directly to the upper respiratory tract, is one of the most promising influenza vaccines for the foreseeable future. It was concluded that aerosol administration of inactivated split influenza vaccine stimulated both local and systemic IgA antibody responses.
CONCLUSION
REFERENCES
Systemic and local antibody responses to the topically administered and subcutaneously administered commercially available inactivated split influenza vaccines were investigated. Greater than fourfold rises in serum HI titres (A/Sichuan/2/87 H3N2) were observed in 87% (40/46) of the aerosol and in 93% (43/46) of the subcutaneous administration group. Greater than fourfold rises in serum HA (A/Sichuan/2/87 H3N2) specific IgG antibody were observed in 48% (22/46) and 37% (17/46), in specific IgM antibody were observed in 40% (17/43) and 20% (9/45) and in specific IgA antibody responses were observed in 58% (23/'40) and 4% (2/46) of the aerosol and subcutaneous administration group, respectively. More than fourfold rises in local HA (A/Sichuan/2/87) specific IgA antibodies were observed
Immunization Practices Advisory Committee (ACIP). Prevention and control of influenza. Moth. Mort. Week. Rep. 1991, 40 RR-6, 1-15 2 Ghendon, Y. The immune response of humans to live and inactivated influenza vaccines. Adv. Exp. Med. Biol. 1989, 257, 37-45 3 Alford, R.H. Antibody in nasal secretions after intramuscular injections of influenza virus vaccine in persons with chronic bronchopulmonary disease. J. Immunol. 1969, 103, 20-24 4 Clements, M.L. and Murphy, B.R. Development and persistence of local and systemic antibody responses in adults given live attenuated or inactivated influenza A virus vaccine. J. Clin. Microbiol. 1986, 23(1), 66-72 5 Clements, M.L., Betts, R.F., Tierney, E.L. and Murphy, B.R. Serum and nasal wash antibodies associated with resistance to experimental challenge with influenza A wild-type virus. J. C/in. Microbio/. 1986, 24(1), 157-160 6 Ohta, K., Kuno-Sakai, H., Kimura, M., Oh, Y., Kim, R., Kumihashi, H. et al. Enzyme-linked immunosorbent assay of influenza specific IgA antibody in nasal mucus. Acta Paediatr. Jpn 1991, 33, 617-622
ACKNOWLEDGEMENTS This work was partially supported by Grants in Aid for Scientific Research, the Ministry of Education, Science and Culture, no. 62570439 and no. 03454270 and by Research Grants for Health Science, the Ministry of Health and Welfare, no. 176, 1989 and no. 40, 1990.
1
Vaccine, Vol. 10, Issue 8, 1992
511
INTRODUCTION Inactivated influenza vaccines administered subcutaneously or intramuscularly, either in the form of a whole virion or in the split or subunit form, have been the only licensed influenza vaccines in the United States, Europe and Japan 1. Since influenza virus proliferates over the mucosal surface and causes disease instantaneously, vaccines which will stimulate both systemic and mucosal immunity are more promising for the prevention of influenza epidemics 2. It has been reported that the parenteral administration of inactivated influenza vaccine did not induce local specific IgA responses 3. Direct antigenic stimulation of the upper respiratory tract with a vaccine may induce local immunity. Therefore, an inactivated influenza vaccine, administered directly in the upper respiratory tract, is a good candidate vaccine which may be used safely in humans. Systemic and local immune responses to topically administered inactivated influenza vaccine have not been fully investigated. There are a few clinical trials on local Department of Pediatrics, School of Medicine, Tokai University, Isehara City, Kanagawa 259-11,Japan. *To whom correspondence should be addressed. (Received 9 October 1991; revised 18 November 1991; accepted 25 December 1991) 0264-410xi92,080506--06 1992 Butterworth-HeinemannLtd 506
Vaccine, Vol. 10, Issue 8, 1992
antibody responses of humans to topically administered influenza vaccines4'5, However, the method described for determination of local antibody responses required nasal wash solution and the results obtained were expressed only in terms of end points and not in terms of units. Lack of a sensitive method of microassay of specific antibodies present on the surface of the upper respiratory tract has long been one of the major weak points in the study of local immune responses to topically administered vaccines. We have developed a very sensitive, reliable and reproducible method of quantification of local specific antibodies to influenza haemagglutinins (HA) and investigated local specific IgA antibody responses to aerosol administration of an inactivated influenza vaccine; we compared serum haemagglutinin inhibition (HI) titres and serum specific IgA, IgM and IgG antibody levels between two groups of young adults, one of which received subcutaneous inactivated influenza vaccine and the other received aerosol administration of the same vaccine. Methods of evaluation of the potency and antigenicity in humans, both local or systemic, are also required for an influenza vaccine administered intranasally. This study may furnish basic information for estimation of the degree of local and systemic antigenic stimulation of topically administered inactivated influenza vaccine.
Aerosol administration of influenza vaccine: Y. Oh et al. VOLUNTEERS
AND METHODS
Volunteers Ninety-two young adult volunteers were divided into two groups, the aerosol administration group and the subcutaneous administration group. Written informed consent was obtained from all volunteers. A further 46 young adult volunteers served as controls and received aerosol administration of saline,
Vaccine Commercially available quadrivalent inactivated split influenza vaccine (0.5 ml), which was distributed during the winter of 1988-89 [A/Yamagata/120/86 (H1N1) 200 chicken cell agglutination units (CCA)m1-1 equivalent; A/Fukuoka/C29/85 (H3N2) 200 CCA ml- 1 equivalent; A/Sichuan/2/87 (H3N2) 200 CCA m1-1 equivalent; and B / N a g a s a k i / I / 8 7 100 CCA m1-1 equivalent] was administered by either the subcutaneous or intranasal route.
Method of administration
Aerosol administration. Forty-six volunteers received the vaccine intranasally in the form of aerosol. The vaccine was sprayed on each side of the nose at a volume of 0.25 ml each by the Jacksonian type tracheal spray. The vaccine was given four times at 1 week intervals between each administration. Subcutaneous administration. Forty-six volunteers received subcutaneous administration of the vaccine 0.5 ml each, twice at a 4-week interval. Collection of the samples
Preparation of the nasal swab solution. The samples were collected from the vaccinee group before administration and 7 weeks after the fourth aerosol administration of the inactivated split vaccine. Other samples were collected from the control group before administration and 6 weeks after the second administration of the saline. A wet cotton swab, which had been soaked in 0.01 M phosphate-buffered saline (PBS), was gently rotated in the nose for 30 s. In this way at least 20 #i of mucus can be collected. The swab was mixed with 3 ml 0.01 M PBS. The solution was centrifuged for 30 rain at 3000 rev min- 1 to remove any cell debris, and N a N 3 was added at a concentration of 0.05%. The sample was stored at - 20°C until assayed.
every day for 7 days, was added to the wells, The plate was then incubated at 4~C overnight. After two washings with PBS-Tween, alkaline phosphatase labelled antihuman IgA goat serum conjugate diluted with PBS containing 1% bovine serum albumin was added. After incubation for 2 h the plate was washed twice with PBS and three times with demineralized water. Then 200 pl of the substrate solution (100mg of p-nitrophenyl phosphate disodium dissolved in 100 ml of demineralized water with 0.53 g of NazCO3, 0.02 g of MgC12 . 6HzO and 1 ml of 1 M HCI ) was applied. After allowing to stand at 37°C for 1 h, absorbancy at 405nm (A,~os) was measured using a microplate photometer. The results were expressed in terms of units using the standard solution. Total IgA concentrations of nasal swab solutions were measured by laser nephelometry (Behringwerke, Marburg, Germany) and the final results of the specific IgA values were expressed in terms of ELISA units rag- 1 of IgA.
Determination of serum class-specific anti-influenza HA antibodies. The methods were essentially the same as that described above. Alkaline phosphatase labelled anti-human IgG, lgA and IgM goat sera were used. The values were expressed in terms of ELISA units ml- t of serum.
Serum HI titres Serum HI titres were quantified by the standard method. Haemagglutinins of the subtypes of influenza virus contained in the vaccine were used as antigens of HI tests. It should be noted that expression of the dilution ofsera in Japan is different from that in the United States ; a dilution of 1:8 in the United States corresponds to a dilution of I :32 in Japan.
Statistical analysis The 7.2 test with Yates' correction was used to compare the rises in serum HI titres or in influenza HA-specific antibodies between subcutaneous and aerosol administration groups. The Spearman-Kendall test was used to evaluate a correlation between serum-specific IgA antibody levels and HA-specific IgA antibody levels of the nasal swab solution.
RESULTS
Collection ofsera. Serum samples were collected before vaccination and 7 weeks after the fourth dose of aerosol vaccine or 6 weeks after the second dose of the subcutaneous vaccine. ELISA method
ELISA of specific lgA antibodies of nasal swab solution. Details of the ELISA method have already been published 6. In brief, a Linbro-Titertek EIA microtitration plate (Flow Laboratories Inc., McLean, VA 22102, USA ) was coated with purified influenza haemagglutinin (HA) solution and was allowed to stand at 4°C for 2 days. After washing with PBS containing 0.05% Tween 20 (PBS-Tween), 50/A of nasal swab solution or standard solution, which was the nasal swab solution collected from an adult who had received aerosol influenza vaccine
Figure 1 shows serum HI titres (A/Sichuan H3N2) before and after administration of the subcutaneous (Figure la) or aerosol (Figure lb) influenza vaccine. Greater than fourfold rises in HI titres were observed in 93% (43/46) of the subcutaneous administration group. Geometrical mean prevaccination and postvaccination titres w e r e 2 3`9 and 26.9 , respectively. Greater than fourfold elevations of serum HI titres were observed in 87% (40/46) of the aerosol vaccine administration group. Geometrical mean prevaccination and postvaccination titres were 24.5 and 2 "1 , respectively. Percentages of more than fourfold rises in serum HI titres were not significantly different between the two groups (7." = 0.4926, p = 0.4827, 7.-Yates test). It should be noted that marked elevations of HI titres were observed among those with a prevaccination HI titre of less than 1:16 of the aerosol administration group.
Vaccine, Vol. 10, Issue 8, 1992 507
Aerosol administration of influenza vaccine. Y. Oh e t al. a
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~2048
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t1:16. When the split influenza vaccines were administered intranasally in the form of aerosol, the vaccines had booster effects which were shown by rises in HA-specific local IgA antibodies, in serum HA-specific IgG, IgM and IgA antibodies, and in serum HI titres. Those results showed that the vaccines induced local immunoglobulin production and rises in serum-specific antibody levels even in the subjects who already had specific serum antibodies and/or local IgA antibodies to the subtypes of the virus of the vaccine. Our results demonstrated that the route of administration of the inactivated split influenza vaccine influenced the type of immune response. Little change in serum HA-specific IgA antibody was noted after subcutaneous administration of inactivated influenza vaccine. On the other hand, rises in serum HA-specific IgA antibody could be observed after aerosol administration. Rises in local HA-specific IgA antibody were observed after aerosol administration of the inactivated influenza vaccine. The existence of a correlation between rises {n local HA-specific IgA antibodies and rises in serum HA-specific IgA antibodies may suggest that local antigenic stimulation may result in production of specific IgA~producing cells. Over the last decade, simultaneous epidemics of two or three different subtypes of influenza A virus and/or influenza B virus have been noted every year throughout the world. Therefore, most people have been primed with various subtypes of influenza virus. Aerosol administration of the inactivated split influenza vaccine can stimulate both local and systemic antibody responses even in those
Aerosol administration of influenza vaccine: Y. Oh et al.
who have been exposed to similar subtypes of the influenza virus before and have some degree of serum-specific antibody and/or local specific antibody to the subtype of the virus of the vaccine. The first influenza virus was isolated from chickens with fowl plague in 1901. In 1933, Smith, Andrews and Francis succeeded in transmission of a human influenza virus to ferrets. Half a century has passed since the first influenza vaccine was administered to humans, yet no country in the world has ever succeeded in control of influenza epidemics by the use of vaccine given subcutaneously. Epidemics of influenza are very hard to control. The only possible way to control epidemics is to obtain herd immunity. To achieve this, an increase in acceptance rate is needed and it may therefore be preferable to have a choice of several influenza vaccines. Because they can be easily administered to humans and because they induce production of local specific IgA antibodies which may be able to reduce chances of shedding of the specified influenza virus, intranasal administration of either live or inactivated influenza vaccines should have a place in the prevention of influenza epidemics in the very near future.
in 28% (12/43) of the aerosol vaccine administration group and in 0% (0/24) of the control group who received aerosol saline in place of the vaccine. Those observations suggest, first, that mucosal stimulation with inactivated influenza vaccine resulted in marked increase in local HA-specific IgA antibodies. Secondly, mucosal antigenic stimulation was necessary for serum HA-specific IgA responses. Thirdly, serum HA-specific IgA antibody levels can be used as indicators of the local antigenic stimulation. Finally, the inactivated influenza split vaccine, applied directly to the upper respiratory tract, is one of the most promising influenza vaccines for the foreseeable future. It was concluded that aerosol administration of inactivated split influenza vaccine stimulated both local and systemic IgA antibody responses.
CONCLUSION
REFERENCES
Systemic and local antibody responses to the topically administered and subcutaneously administered commercially available inactivated split influenza vaccines were investigated. Greater than fourfold rises in serum HI titres (A/Sichuan/2/87 H3N2) were observed in 87% (40/46) of the aerosol and in 93% (43/46) of the subcutaneous administration group. Greater than fourfold rises in serum HA (A/Sichuan/2/87 H3N2) specific IgG antibody were observed in 48% (22/46) and 37% (17/46), in specific IgM antibody were observed in 40% (17/43) and 20% (9/45) and in specific IgA antibody responses were observed in 58% (23/'40) and 4% (2/46) of the aerosol and subcutaneous administration group, respectively. More than fourfold rises in local HA (A/Sichuan/2/87) specific IgA antibodies were observed
Immunization Practices Advisory Committee (ACIP). Prevention and control of influenza. Moth. Mort. Week. Rep. 1991, 40 RR-6, 1-15 2 Ghendon, Y. The immune response of humans to live and inactivated influenza vaccines. Adv. Exp. Med. Biol. 1989, 257, 37-45 3 Alford, R.H. Antibody in nasal secretions after intramuscular injections of influenza virus vaccine in persons with chronic bronchopulmonary disease. J. Immunol. 1969, 103, 20-24 4 Clements, M.L. and Murphy, B.R. Development and persistence of local and systemic antibody responses in adults given live attenuated or inactivated influenza A virus vaccine. J. Clin. Microbiol. 1986, 23(1), 66-72 5 Clements, M.L., Betts, R.F., Tierney, E.L. and Murphy, B.R. Serum and nasal wash antibodies associated with resistance to experimental challenge with influenza A wild-type virus. J. C/in. Microbio/. 1986, 24(1), 157-160 6 Ohta, K., Kuno-Sakai, H., Kimura, M., Oh, Y., Kim, R., Kumihashi, H. et al. Enzyme-linked immunosorbent assay of influenza specific IgA antibody in nasal mucus. Acta Paediatr. Jpn 1991, 33, 617-622
ACKNOWLEDGEMENTS This work was partially supported by Grants in Aid for Scientific Research, the Ministry of Education, Science and Culture, no. 62570439 and no. 03454270 and by Research Grants for Health Science, the Ministry of Health and Welfare, no. 176, 1989 and no. 40, 1990.
1
Vaccine, Vol. 10, Issue 8, 1992
511
