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Poor immune responses to influenza vaccination in infants
Vaccine 22 (2004) 3404–3410
Poor immune responses to influenza vaccination in infants Takuji Kumagai a,∗ , Kazushige Nagai b , Toyo Okui c , Hiroyuki Tsutsumi b , Nobuo Nagata a , Shoki Yano c , Tetsuo Nakayama d , Yoshinobu Okuno e , Hitoshi Kamiya f a
Pediatric Allergy and Infectious Diseases Society of Sapporo, Kumagai Pediatric Clinic, W-6, Momijidai, Atsubetsu-Ku, Sapporo 004-0013, Japan b Department of Pediatrics, Sapporo Medical University School of Medicine, S1-W17, Chuo-Ku, Sapporo 060-0061, Japan c The Hokkaido Institute of Public Health, N19-W12, Kita-Ku, Sapporo 060-0819, Japan d Laboratory of Viral Infection I, Kitasato Institute for Life Sciences, 5-9-1, Shirokane, Minato-Ku, Tokyo 108-8462, Japan e The Osaka Institute of Public Health, 1-3-69, Nakamichi, Higashinari-Ku, Osaka 537-0025, Japan f Department of Pediatrics, Mie National Hospital, 357 Kubota-Cho, Ohsato, Tsu, Mie 514-0125, Japan Received 27 October 2003; received in revised form 25 February 2004; accepted 25 February 2004 Available online 26 March 2004
Abstract Twenty-two and 37 infants and young children received two doses of influenza HA vaccine before the 2001–2002 influenza season and before the 2002–2003 season, respectively. Two or three serial specimens were obtained, before and 1 month after the first vaccination as well as 1 month after the second vaccination. Infants showed a significantly poor HI antibody rise and lymphocyte response compared with young children aged ≥12 months. Time kinetics of the lymphoproliferative responses to influenza antigen among young children varied whereas their activities in infants were typically negative before immunization and increased after vaccination. Infants responded poorly to HA influenza vaccine compared with young children. © 2004 Elsevier Ltd. All rights reserved. Keywords: Influenza vaccine; Antibody; Lymphocyte transformation
1. Introduction Until recently, influenza vaccination has been recommended for adults over the age of 65 years, high-risk children of 6 months of age or older, and those who might transmit influenza virus to high-risk persons [1], and the immune response to influenza vaccine in adults, including the elderly and older children, is well documented [1,2]. However, children aged 6–23 months have a substantially increased risk for influenza-related hospitalization, and the desirability of universal vaccination for this age group has been suggested [3,4]. In this context, the Advisory Committee on Immunization Practices (ACIP) of the Centers for Disease Control and Prevention (CDC) has revised its policy and in a new recommendation encourages influenza vaccination for all children 6–23 months of age for the 2002–2003 winter season [5]. Besides this new recommendation in the United States, the number of young children and infants given influenza HA vaccine in Japan has increased significantly during the last several years, possibly
∗
Corresponding author. Tel.: +81-11-897-1118; fax: +81-11-897-7572. E-mail address: [email protected] (T. Kumagai).
0264-410X/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2004.02.030
due to concerns regarding encephalopathy complicating influenza. However, the characteristics of vaccine immunity in infants and young children remain incompletely understood. An overall progressively reduced antibody response to influenza vaccine was observed with decreasing age, especially at 6.5 months to 3 years of age [6,7]. The poor immune response to influenza HA vaccine (ether-split viral product) in immunologically naive persons has been left unresolved because young children have not been given influenza vaccine routinely until recently [8]. Encephalopathy complicating influenza occurs predominantly in young children and infants in Japan, raising substantial concern as to how to deliver immunity to this age group, especially the infants. To establish the best system to deliver the most efficacious immunity, a detailed laboratory-based vaccine immunogenicity study would be required. To clarify whether the present influenza HA vaccine provides effective priming and antibody response in Japanese young children and infants, we investigated the antibody and cellular immune responses to influenza antigen in sequentially obtained specimens from subjects inoculated with two doses of influenza HA vaccine. We also evaluated the relationship between the antibody response and the induction of cell-mediated immunity.
T. Kumagai et al. / Vaccine 22 (2004) 3404–3410
2. Materials and methods 2.1. Subjects Before the 2001–2002 influenza season, 22 healthy children (11 boys, 11 girls) with a mean age of 14.7 months (range, 6–31 months, 10 children were under 12 months of age), received two doses of influenza HA vaccine. Pre and post-vaccination peripheral blood specimens were obtained before and 1 month after the second vaccination. For the 2002–2003 influenza season, 37 children (21 boys, 16 girls) with a mean age of 14.8 months (range 6–23 months, 11 children were under 12 months of age) were also inoculated with two doses of influenza HA vaccine. Serial specimens were obtained, prior to, 1 month after the first vaccination and 1 month after the second vaccination. In addition, nine consecutive specimens were obtained once a week before and after two serial immunizations from one adult (38 years old, female) and three children (14, 12, 11 years old, two females and one male) in the 2001–2002 season in order to identify the best sampling points and to set up the system. Informed consent was obtained from the parents of the subjects after full explanation and consent was also obtained from all children aged 6 years or more. 2.2. Influenza vaccine and viruses used for lymphoproliferative response and antibody assays A commercially available subunit vaccine was used in this study. The vaccine of 0.5 ml contained 15 g hemagglutinin of the following virus strains: A/New Caledonia/20/99 (H1N1), A/Panama/2007/99 (H3N2), B/Johannesburg/5/99 for the 2001–2002 season, and A/New Caledonia/20/99 (H1N1), A/Panama/2007/99 (H3N2), B/Shandong/7/97 for the 2002–2003 season. In Japan, the recommended dose of influenza vaccine for infants 6 months or more, children younger than 6 years and those 6–13 years of age is 0.1 (3 g HA), 0.2 (6 g HA ), and 0.3 (9 g HA )ml per dose, respectively. Thus, 0.1 ml or 0.2 ml per dose was given to infants and young children, respectively. Viruses corresponding to vaccine strains in each season were propagated in the allantoic cavity of 10-day-old embryonated eggs. After 3–4 days incubation, the allantoic fluid was harvested and clarified by centrifugation at 1800 × g. Viruses were further concentrated through zonal centrifugation and inactivated with formaldehyde. Single radial immunodiffusion assay was performed to determine HA concentration. Stock solution contained 1500–5000 g HA per milliliter in concentration and the final concentration of formaldehyde was 0.01%. Viruses used as antigen for lymphoproliferative responses were suspended in 0.1 M phosphate buffer solution (pH 7.2) with 50% sucrose and were stored in aliquots at 4 ◦ C in order to avoid agglutination, then diluted to optimal concentrations in each experiment.
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2.3. Determination of hemagglutination-inhibition antibody specific to influenza virus The serum samples were stored at −35 ◦ C until testing. The HI test was performed by a standard microtiter assay with human erythrocytes after removal of nonspecific inhibitors with receptor-destroying enzyme and of cold agglutinins by hemadsorption at 4 ◦ C. For each antigen, all specimens were tested on the same day using identical reagents. 2.4. In vitro lymphocyte proliferation test (LPT) Whole blood microculture assay was performed to determine the lymphoproliferative response to influenza virus antigen [9]. Heparinized venous blood was diluted to 1:10 in RPMI 1640 medium supplemented with kanamycin and HEPES buffer. A 200 l volume of the diluted blood with or without 20 l of influenza antigens of various concentrations was cultured in quadruplicate in an incubator with an atmosphere of 5% CO2 at 37 ◦ C for 7 days. The cultures were labeled with tritiated thymidine for the final 24 h, washed and harvested in a multiple automated sample harvester. The radioactivity was measured and the results were expressed by ratios of tritiated thymidine incorporation in antigen-stimulated cultures to that in control cultures (stimulation index). A stimulation index of >3.0 was considered a significant response. 2.5. Statistical analysis All statistical analyses were performed using the SAS statistical software (SAS Institute Inc, Cary, N.C.). The differences between values of the HI titers as well as the SIs at the beginning of the study and at follow up were compared by means of the unpaired t-test or by Wilcoxon’s rank sum test. Welch’s t-test was utilized for unequal variances. For calculations, an HI titer of five was arbitrarily assigned to sera with a titer of <10. Spearman’s rank correlation test was used to examine the association between lymphocyte activity and antibody response. A significance level was set at P < 0.05. 3. Results 3.1. Antibody response and LPT in an adult and older children (Fig. 1) Nine consecutive specimens were obtained from one adult and three older children. The level of HI titer to all of the three subtype antigens in case 4 and that to one of the three subtype antigens in the other three subjects before the first vaccination was ≥ 1:40. Booster responses were observed after the first immunization but were less pronounced. In addition, the expected amount of booster response was not
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to determine an “optimal sampling point” after vaccination, thus we decided to obtain specimens prior to, 4 weeks after the first vaccination, and 4 weeks after the second vaccination. 3.2. Antibody response in young children and infants
Fig. 1. Acquisition of HI antibody and lymphocyte responses directed against influenza virus antigen in adult and older children inoculated with influenza vaccine. Trivalent influenza HA vaccine was administered on day 0 and 31. Abscissa represents days after vaccination and the ordinate the HI titer and the stimulation index (SI) of the lymphoproliferative response to influenza virus antigen. Open, solid, and hatched columns show HI titer to H1N1, H3N2, and B antigens, respectively. Open and shaded circles, and the triangles represent stimulation indices to H1N1, H3N2, and B antigens, respectively. Numbers correspond to age (years) of subjects and M or F represents gender. The horizontal line shows the point of 1:40 in HI titer.
obtained after the second vaccination. Once relatively high titers of antibody response were obtained even before or after the first vaccination, further antibody titer elevation was not observed after subsequent vaccination. LPT showed rather heterogeneous responses. Cases 2 and 3 showed positive responses before the first vaccination and substantial booster responses after the first vaccination, but no obvious booster response after the second vaccination. In contrast, LPT activity in cases 1 and 4 declined after the first vaccination and then gradually increased, they again decreased after the second immunization, before increasing again. From these observations, it was difficult
In the 2001–2002 season (Fig. 2A), only three out of 10 infants acquired ≥ 1:40 in HI titer to all three subtype antigens (H1N1, H3N2, B). Of the remaining seven recipients, one acquired the same antibody level to H1N1 and H3N2, three to H1N1 and B, one to only H1N1 and two to none. In contrast, 10 of 12 older recipients obtained an HI titer of ≥ 1:40. One showed antibody rise to H1N1 and B and another one to only B. Statistical analysis shows that the antibody rise to H3N2 and B antigens in infants was significantly lower than in subjects aged 12 months or older (P = 0.0380, P = 0.0020, respectively). In the 2002–2003 season (Fig. 2B), only 1 among 11 infants developed an HI titer of ≥ 1:40 to all three subtype antigens. Among the remaining 10 recipients, one acquired an HI titer of ≥ 1:40 to both H1N1 and H3N2, another one obtained it to only H1N1 and none showed an antibody rise to B. In addition, all infants needed two consecutive doses to obtain antibody of ≥ 1:40 to any subtype. In subjects aged 12 months or older, nine out of 26 obtained ≥ 1:40 in HI titer to all three subtype antigens. Among the remaining 17 recipients, nine acquired an HI titer of ≥ 1:40 to both H1N1 and H3N2, another five and one obtained it to only H1N1 and H3N2, respectively. Two showed no significant antibody rise to any subtype antigens. None of the 17 recipients showed an antibody rise to B subtype. Regarding the data for the 2002–2003 season, statistical analysis shows that the levels of antibody rise to H1N1, H3N2, and B antigens in infants were significantly lower than those in subjects aged 12 months or older (P < 0.0001, P = 0.0004, P = 0.0009, respectively). Concerning the time kinetics of antibody titer, 8 of 23 children who showed an antibody elevation to H1N1 obtained an HI titer of ≥ 1:40 after just one dose of vaccination whereas another 13 needed two doses of vaccination to reach that level. Two in this group had a high antibody titer before vaccination. Also, 6 of 19 children who showed an antibody rise to H3N2 acquired the same level of antibody after one dose of vaccination whereas 11 needed two doses. Two showed a high antibody level to H3N2 before vaccination. 3.3. Lymphoproliferative response in young children and infants Regarding the specific lymphocyte responses in the 2001–2002 season (Fig. 3A), positive responses to all three subtype antigens after vaccination were observed in nine out of 12 young children, of the remainder, one showed a positive response to H1N1, H3N2, one to H1N1, B and one to only B antigen. In 10 infants, nine developed responses to
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Fig. 2. Kinetics of HI antibody titer directed against influenza virus antigen in young children (top) and infants (bottom) inoculated with influenza vaccine in the 2001–2002 (A) and the 2002–2003 influenza season (B). Trivalent influenza HA vaccine was administered on the day of the first sampling and 4 weeks after the first vaccination. Abscissa represents weeks after vaccination and ordinate the HI titer. Number of open circles shows scattergram in each sampling point. The horizontal line defines the point of 1:40 in HI titer.
all three subtype antigens and the remaining one showed a positive response to H1N1, B. In the 2002–2003 (Fig. 3B), all young children and eight out of 11 infants reacted positively to all three subtype antigens after vaccination. In the remaining three infants, one showed a positive response to H1N1 and B, one to only H1N1 and one did not respond to any antigens. Statistical analyses showed that the rise in LPT activity to all three subtype antigens after vaccination in infants and young children was not significantly different
for the 2001/2002 season whereas LPT in young children showed significantly higher activity compared with infants in the 2002/2003 season (P = 0.0065, 0.0450, 0.0474 for H1N1, H3N2, and B antigens, respectively. The time kinetics of LPT activity looked rather simple by evaluation using two point sampling in the 2001–2002 season, however, three point sampling in 2002–2003 revealed that the kinetics of LPT during serial vaccination were rather complicated especially in young children. The time kinetics
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Fig. 3. Kinetics of lymphoproliferative response expressed as stimulation indices directed against influenza virus antigen in young children (top) and infants (bottom) inoculated with influenza vaccine in the 2001–2002 (A) and the 2002–2003 influenza season (B). Trivalent influenza HA vaccine was administered on the day of the first sampling and 4 weeks after the first vaccination. Abscissa represents weeks after vaccination and ordinate stimulation index.
of LPT among young children varied, some children showed the expected immune response such as low or negative before vaccination with an increase after the first vaccination, and a further increase after the second vaccination. However other children showed an unexpected kinetic pattern, similar to that observed in a group of older individuals, in that they already had a positive response before immu-
nization which declined after the first immunization and increased again after the second vaccination. Others also showed a decreased response after the second vaccination. On the other hand, LPT activity in infants was typically negative or low before immunization and increased after vaccination with a further increase after the second vaccination, resembling a primary immune response.
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3.4. Relationship between antibody response and specific lymphocyte activity In the 2001–2002 season, the correlation coefficients between HI titer and lymphocyte activity for three subtype antigens at the second sampling point were 0.1709, 0.0284, −0.0300 for H1N1, H3N2, B, respectively, and in the 2002–2003 season, the largest correlation coefficients for three subtype antigens were obtained at the second sampling point, those are 0.4820, 0.5980, 0.1638 for H1N1, H3N2, B, respectively. It seems that there is only a weak relationship between HI titer and S.I. in H1N1 and H3N2 on the basis of the correlation coefficients around 0.5.
4. Discussion Our main purpose was to determine whether the present HA vaccination provides young children and especially infants with protective antibody as well as specific lymphocyte activity. We also evaluated whether the antibody response was associated with the development of specific lymphocyte activity. We observed significantly poorer immune responses to vaccination in infants compared to young children. Furthermore, only a few infants achieved an antibody response of ≥ 1:40 to any subtype even after two consecutive doses, whereas in young children about 30% acquired antibody at the same level after one dose. Influenza HA vaccine is known to be poorly immunogenic in young children [6,7,10], causing one vaccine manufacturer in the United States to withdraw from manufacturing HA split vaccine [8]. In our study, a substantial number of infants failed to mount an antibody response even after two serial dose vaccination. Respecting host factors related to immunological mechanisms in infants, Clerici et al. [11] found functional defects in T helper cells of infants involving antigen presentation and immunological memory. Regarding the dosage required to induce a primary antibody response in unprimed subjects, it is known that two to three times as much as the regular dosage is needed such that 50 g per dose or more of HA is required [12,13]. Furthermore, in the Hong Kong influenza epidemics of 1968/1969, a 70% reduction in the attack rate was observed by inoculation of 10 times the regular vaccine dose whereas the regular dosage caused no reduction [14]. Thus, the poor antibody response to vaccination observed in our study as well as by other authors might reflect both insufficient dosage resulting in poor immunogenicity, and deficits in the infant immune system. In this regard, the relationship between T helper cell activity and the antibody response in individuals following vaccination is interesting. However, we found only a weak correlation between the lymphocyte response and the amount of antibody to H1N1 and H3N2 antigens elicited by vaccination. We could not find a definitive factor accounting for this weak interrelationship, possibly the antigenic epitopes recognized by the humoral immune system and those
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of cell-mediated immunity are different. More sophisticated monitoring of T helper immunity such as CD45 isotype evaluation for morphological aspects and determination of IL4, IL-5, and interferon gamma production for functional characteristics may elucidate its relationship with antibody production [15]. Detailed kinetics studies, such as nine consecutive sampling in individuals, have not been previously conducted. As one would expect, once relatively high titers of antibody were obtained before or after the first vaccination, a further elevation of antibody titer was not induced by the following vaccination. Some subjects showed an unexpected and superficially incoherent response pattern in which LPT activity declined after the first vaccination and then gradually increased, with a similar response after the second vaccination. Similar kinetic patterns were also observed in some young children. Concerning this point, one might question the stability of our system, however, robustness of the assay system is easily confirmed by the fact that each value of LPT fits stable lines or curves consecutively in each subject. The mechanism of this kinetic response pattern is unknown, possibly compartmentalization or redistribution of lymphocytes after sensitization might be involved [16]. A potential weakness of the present study is its small sample size. However, a high level of statistical significance for some of the differences in antibody responses was observed even with this small cohort, suggesting that a type I error (i.e., finding differences where none exist) is unlikely and that the results are robust enough to demonstrate that infants immunized with influenza vaccine acquired a poor immune response compared to young children. Our data raise the issue of whether a two dose regimen of influenza vaccination is appropriate for the primed population such as older children and adults. Also, based on the poor immunogenicity of the present HA vaccine in the younger age group, especially infants, even higher doses should be considered if we continue to employ HA split vaccine. Apart from the issue regarding the present vaccine preparation, alternative influenza vaccines are available, one is a whole virion vaccine which is approved in the United States, while another is a newly developed live attenuated influenza vaccine (LAIV) [8,17]. Nasal administration of highly purified whole virion vaccine, instead of the parenteral route, has been proposed [18]. More recently, ACIP published supplemental recommendations on the use of LAIV, however, its usage is currently limited to children aged 5 years or older and adults less than 50 years of age [19]. These newly developed vaccine preparations encourage us to reappraise current HA influenza vaccination policies.
Acknowledgements The authors thank Fujio Matsuo, B.S. (The Chemo-SeroTherapeutic Research Institute, Kumamoto, Japan) for his help in statistical analyses. We are indebted to Professor
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Peter M. Olley (Emeritus Professor of Pediatrics, University of Alberta at Edmonton) for his invaluable help in revising the manuscript. References [1] Prevention and control of influenza: recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Morb Mortal Wkly Rep 48 (1999) 1–28. [2] Edwards KM, Dupont WD, Westrich MK, Plummer Jr WD, Palmer PS, Wright PF. A randomized controlled trial of cold-adapted and inactivated vaccines for the prevention of influenza A disease. J Infect Dis 1994;169:68–76. [3] Neuzil KM, Mellen BG, Wright PF, Mitchel Jr EF, Griffin MR. The effect of influenza on hospitalizations, outpatient visits, and courses of antibiotics in children [see comments]. N Engl J Med 2000;342:225–31. [4] Izurieta HS, Thompson WW, Kramarz P, Shay DK, Davis RL, DeStefano F, et al. Influenza and the rates of hospitalization for respiratory disease among infants and young children. N Engl J Med 2000;342:232–9. [5] Bridges CB, Fukuda K, Uyeki TM, Cox NJ, Singleton JA. Prevention and control of influenza. Recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm Rep 2002;51:1–31. [6] Hilleman MR. Serologic responses to split and whole swine influenza virus vaccines in light of the next influenza pandemic. J Infect Dis 1977;136(Suppl):S683–5. [7] Parkman PD, Hopps HE, Rastogi SC, Meyer Jr HM. Summary of clinical trials of influenza virus vaccines in adults. J Infect Dis 1977;136(Suppl):S722–30. [8] Hilleman MR. Realities and enigmas of human viral influenza: pathogenesis, epidemiology and control. Vaccine 2002;20:3068–87. [9] Kumagai T, Chiba Y, Wataya Y, Hanazono H, Chiba S, Nakao T. Development and characteristics of the cellular immune response to infection with varicella-zoster virus. J Infect Dis 1980;141:7–13.
[10] Wood JM. Developing vaccines against pandemic influenza. Philos Trans R Soc London B, Biol Sci 2001;356:1953–60. [11] Clerici M, DePalma L, Roilides E, Baker R, Shearer GM. Analysis of T helper and antigen-presenting cell functions in cord blood and peripheral blood leukocytes from healthy children of different ages. J Clin Invest 1993;91:2829–36. [12] Mostow SR, Schoenbaum SC, Dowdle WR, Coleman MT, Kaye HS, Hierholzer JC. Studies on inactivated influenza vaccines. I.I., Effect of increasing dosage on antibody response and adverse reactions in man. Am J Epidemiol 1970;92:248–56. [13] Palache AM, Beyer WE, Luchters G, Volker R, Sprenger MJ, Masurel N. Influenza vaccines: the effect of vaccine dose on antibody response in primed populations during the ongoing interpandemic period. A review of the literature. Vaccine 1993;11:892–908. [14] Schoenbaum SC, Mostow SR, Dowdle WR, Coleman MT, Kaye HS. Studies with inactivated influenza vaccines purified by zonal centrifugation. 2. Efficacy. Bull World Health Organ 1969;41: 531–5. [15] Kufer P, Zettl F, Borschert K, Lutterbuse R, Kischel R, Riethmuller G. Minimal costimulatory requirements for T cell priming and TH1 differentiation: activation of naive human T lymphocytes by tumor cells armed with bifunctional antibody constructs. Cancer Immun 2001;1:10. [16] Gallichan WS, Rosenthal KL. Long-lived cytotoxic T lymphocyte memory in mucosal tissues after mucosal but not systemic immunization. J Exp Med 1996;184:1879–90. [17] Prevention and control of influenza. Recommendations of the Advisory Committee on Immunization Practices (ACIP). Centers for Disease Control and Prevention. MMWR Recomm Rep 52 (2003) 1–34. [18] Takada A, Matsushita S, Ninomiya A, Kawaoka Y, Kida H. Intranasal immunization with formalin-inactivated virus vaccine induces a broad spectrum of heterosubtypic immunity against influenza A virus infection in mice. Vaccine 2003;21:3212–8. [19] Using live, attenuated influenza vaccine for prevention and control of influenza: supplemented recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Morb Mortal Wkly Rep 52 (2003) 1–8.
Poor immune responses to influenza vaccination in infants Takuji Kumagai a,∗ , Kazushige Nagai b , Toyo Okui c , Hiroyuki Tsutsumi b , Nobuo Nagata a , Shoki Yano c , Tetsuo Nakayama d , Yoshinobu Okuno e , Hitoshi Kamiya f a
Pediatric Allergy and Infectious Diseases Society of Sapporo, Kumagai Pediatric Clinic, W-6, Momijidai, Atsubetsu-Ku, Sapporo 004-0013, Japan b Department of Pediatrics, Sapporo Medical University School of Medicine, S1-W17, Chuo-Ku, Sapporo 060-0061, Japan c The Hokkaido Institute of Public Health, N19-W12, Kita-Ku, Sapporo 060-0819, Japan d Laboratory of Viral Infection I, Kitasato Institute for Life Sciences, 5-9-1, Shirokane, Minato-Ku, Tokyo 108-8462, Japan e The Osaka Institute of Public Health, 1-3-69, Nakamichi, Higashinari-Ku, Osaka 537-0025, Japan f Department of Pediatrics, Mie National Hospital, 357 Kubota-Cho, Ohsato, Tsu, Mie 514-0125, Japan Received 27 October 2003; received in revised form 25 February 2004; accepted 25 February 2004 Available online 26 March 2004
Abstract Twenty-two and 37 infants and young children received two doses of influenza HA vaccine before the 2001–2002 influenza season and before the 2002–2003 season, respectively. Two or three serial specimens were obtained, before and 1 month after the first vaccination as well as 1 month after the second vaccination. Infants showed a significantly poor HI antibody rise and lymphocyte response compared with young children aged ≥12 months. Time kinetics of the lymphoproliferative responses to influenza antigen among young children varied whereas their activities in infants were typically negative before immunization and increased after vaccination. Infants responded poorly to HA influenza vaccine compared with young children. © 2004 Elsevier Ltd. All rights reserved. Keywords: Influenza vaccine; Antibody; Lymphocyte transformation
1. Introduction Until recently, influenza vaccination has been recommended for adults over the age of 65 years, high-risk children of 6 months of age or older, and those who might transmit influenza virus to high-risk persons [1], and the immune response to influenza vaccine in adults, including the elderly and older children, is well documented [1,2]. However, children aged 6–23 months have a substantially increased risk for influenza-related hospitalization, and the desirability of universal vaccination for this age group has been suggested [3,4]. In this context, the Advisory Committee on Immunization Practices (ACIP) of the Centers for Disease Control and Prevention (CDC) has revised its policy and in a new recommendation encourages influenza vaccination for all children 6–23 months of age for the 2002–2003 winter season [5]. Besides this new recommendation in the United States, the number of young children and infants given influenza HA vaccine in Japan has increased significantly during the last several years, possibly
∗
Corresponding author. Tel.: +81-11-897-1118; fax: +81-11-897-7572. E-mail address: [email protected] (T. Kumagai).
0264-410X/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2004.02.030
due to concerns regarding encephalopathy complicating influenza. However, the characteristics of vaccine immunity in infants and young children remain incompletely understood. An overall progressively reduced antibody response to influenza vaccine was observed with decreasing age, especially at 6.5 months to 3 years of age [6,7]. The poor immune response to influenza HA vaccine (ether-split viral product) in immunologically naive persons has been left unresolved because young children have not been given influenza vaccine routinely until recently [8]. Encephalopathy complicating influenza occurs predominantly in young children and infants in Japan, raising substantial concern as to how to deliver immunity to this age group, especially the infants. To establish the best system to deliver the most efficacious immunity, a detailed laboratory-based vaccine immunogenicity study would be required. To clarify whether the present influenza HA vaccine provides effective priming and antibody response in Japanese young children and infants, we investigated the antibody and cellular immune responses to influenza antigen in sequentially obtained specimens from subjects inoculated with two doses of influenza HA vaccine. We also evaluated the relationship between the antibody response and the induction of cell-mediated immunity.
T. Kumagai et al. / Vaccine 22 (2004) 3404–3410
2. Materials and methods 2.1. Subjects Before the 2001–2002 influenza season, 22 healthy children (11 boys, 11 girls) with a mean age of 14.7 months (range, 6–31 months, 10 children were under 12 months of age), received two doses of influenza HA vaccine. Pre and post-vaccination peripheral blood specimens were obtained before and 1 month after the second vaccination. For the 2002–2003 influenza season, 37 children (21 boys, 16 girls) with a mean age of 14.8 months (range 6–23 months, 11 children were under 12 months of age) were also inoculated with two doses of influenza HA vaccine. Serial specimens were obtained, prior to, 1 month after the first vaccination and 1 month after the second vaccination. In addition, nine consecutive specimens were obtained once a week before and after two serial immunizations from one adult (38 years old, female) and three children (14, 12, 11 years old, two females and one male) in the 2001–2002 season in order to identify the best sampling points and to set up the system. Informed consent was obtained from the parents of the subjects after full explanation and consent was also obtained from all children aged 6 years or more. 2.2. Influenza vaccine and viruses used for lymphoproliferative response and antibody assays A commercially available subunit vaccine was used in this study. The vaccine of 0.5 ml contained 15 g hemagglutinin of the following virus strains: A/New Caledonia/20/99 (H1N1), A/Panama/2007/99 (H3N2), B/Johannesburg/5/99 for the 2001–2002 season, and A/New Caledonia/20/99 (H1N1), A/Panama/2007/99 (H3N2), B/Shandong/7/97 for the 2002–2003 season. In Japan, the recommended dose of influenza vaccine for infants 6 months or more, children younger than 6 years and those 6–13 years of age is 0.1 (3 g HA), 0.2 (6 g HA ), and 0.3 (9 g HA )ml per dose, respectively. Thus, 0.1 ml or 0.2 ml per dose was given to infants and young children, respectively. Viruses corresponding to vaccine strains in each season were propagated in the allantoic cavity of 10-day-old embryonated eggs. After 3–4 days incubation, the allantoic fluid was harvested and clarified by centrifugation at 1800 × g. Viruses were further concentrated through zonal centrifugation and inactivated with formaldehyde. Single radial immunodiffusion assay was performed to determine HA concentration. Stock solution contained 1500–5000 g HA per milliliter in concentration and the final concentration of formaldehyde was 0.01%. Viruses used as antigen for lymphoproliferative responses were suspended in 0.1 M phosphate buffer solution (pH 7.2) with 50% sucrose and were stored in aliquots at 4 ◦ C in order to avoid agglutination, then diluted to optimal concentrations in each experiment.
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2.3. Determination of hemagglutination-inhibition antibody specific to influenza virus The serum samples were stored at −35 ◦ C until testing. The HI test was performed by a standard microtiter assay with human erythrocytes after removal of nonspecific inhibitors with receptor-destroying enzyme and of cold agglutinins by hemadsorption at 4 ◦ C. For each antigen, all specimens were tested on the same day using identical reagents. 2.4. In vitro lymphocyte proliferation test (LPT) Whole blood microculture assay was performed to determine the lymphoproliferative response to influenza virus antigen [9]. Heparinized venous blood was diluted to 1:10 in RPMI 1640 medium supplemented with kanamycin and HEPES buffer. A 200 l volume of the diluted blood with or without 20 l of influenza antigens of various concentrations was cultured in quadruplicate in an incubator with an atmosphere of 5% CO2 at 37 ◦ C for 7 days. The cultures were labeled with tritiated thymidine for the final 24 h, washed and harvested in a multiple automated sample harvester. The radioactivity was measured and the results were expressed by ratios of tritiated thymidine incorporation in antigen-stimulated cultures to that in control cultures (stimulation index). A stimulation index of >3.0 was considered a significant response. 2.5. Statistical analysis All statistical analyses were performed using the SAS statistical software (SAS Institute Inc, Cary, N.C.). The differences between values of the HI titers as well as the SIs at the beginning of the study and at follow up were compared by means of the unpaired t-test or by Wilcoxon’s rank sum test. Welch’s t-test was utilized for unequal variances. For calculations, an HI titer of five was arbitrarily assigned to sera with a titer of <10. Spearman’s rank correlation test was used to examine the association between lymphocyte activity and antibody response. A significance level was set at P < 0.05. 3. Results 3.1. Antibody response and LPT in an adult and older children (Fig. 1) Nine consecutive specimens were obtained from one adult and three older children. The level of HI titer to all of the three subtype antigens in case 4 and that to one of the three subtype antigens in the other three subjects before the first vaccination was ≥ 1:40. Booster responses were observed after the first immunization but were less pronounced. In addition, the expected amount of booster response was not
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to determine an “optimal sampling point” after vaccination, thus we decided to obtain specimens prior to, 4 weeks after the first vaccination, and 4 weeks after the second vaccination. 3.2. Antibody response in young children and infants
Fig. 1. Acquisition of HI antibody and lymphocyte responses directed against influenza virus antigen in adult and older children inoculated with influenza vaccine. Trivalent influenza HA vaccine was administered on day 0 and 31. Abscissa represents days after vaccination and the ordinate the HI titer and the stimulation index (SI) of the lymphoproliferative response to influenza virus antigen. Open, solid, and hatched columns show HI titer to H1N1, H3N2, and B antigens, respectively. Open and shaded circles, and the triangles represent stimulation indices to H1N1, H3N2, and B antigens, respectively. Numbers correspond to age (years) of subjects and M or F represents gender. The horizontal line shows the point of 1:40 in HI titer.
obtained after the second vaccination. Once relatively high titers of antibody response were obtained even before or after the first vaccination, further antibody titer elevation was not observed after subsequent vaccination. LPT showed rather heterogeneous responses. Cases 2 and 3 showed positive responses before the first vaccination and substantial booster responses after the first vaccination, but no obvious booster response after the second vaccination. In contrast, LPT activity in cases 1 and 4 declined after the first vaccination and then gradually increased, they again decreased after the second immunization, before increasing again. From these observations, it was difficult
In the 2001–2002 season (Fig. 2A), only three out of 10 infants acquired ≥ 1:40 in HI titer to all three subtype antigens (H1N1, H3N2, B). Of the remaining seven recipients, one acquired the same antibody level to H1N1 and H3N2, three to H1N1 and B, one to only H1N1 and two to none. In contrast, 10 of 12 older recipients obtained an HI titer of ≥ 1:40. One showed antibody rise to H1N1 and B and another one to only B. Statistical analysis shows that the antibody rise to H3N2 and B antigens in infants was significantly lower than in subjects aged 12 months or older (P = 0.0380, P = 0.0020, respectively). In the 2002–2003 season (Fig. 2B), only 1 among 11 infants developed an HI titer of ≥ 1:40 to all three subtype antigens. Among the remaining 10 recipients, one acquired an HI titer of ≥ 1:40 to both H1N1 and H3N2, another one obtained it to only H1N1 and none showed an antibody rise to B. In addition, all infants needed two consecutive doses to obtain antibody of ≥ 1:40 to any subtype. In subjects aged 12 months or older, nine out of 26 obtained ≥ 1:40 in HI titer to all three subtype antigens. Among the remaining 17 recipients, nine acquired an HI titer of ≥ 1:40 to both H1N1 and H3N2, another five and one obtained it to only H1N1 and H3N2, respectively. Two showed no significant antibody rise to any subtype antigens. None of the 17 recipients showed an antibody rise to B subtype. Regarding the data for the 2002–2003 season, statistical analysis shows that the levels of antibody rise to H1N1, H3N2, and B antigens in infants were significantly lower than those in subjects aged 12 months or older (P < 0.0001, P = 0.0004, P = 0.0009, respectively). Concerning the time kinetics of antibody titer, 8 of 23 children who showed an antibody elevation to H1N1 obtained an HI titer of ≥ 1:40 after just one dose of vaccination whereas another 13 needed two doses of vaccination to reach that level. Two in this group had a high antibody titer before vaccination. Also, 6 of 19 children who showed an antibody rise to H3N2 acquired the same level of antibody after one dose of vaccination whereas 11 needed two doses. Two showed a high antibody level to H3N2 before vaccination. 3.3. Lymphoproliferative response in young children and infants Regarding the specific lymphocyte responses in the 2001–2002 season (Fig. 3A), positive responses to all three subtype antigens after vaccination were observed in nine out of 12 young children, of the remainder, one showed a positive response to H1N1, H3N2, one to H1N1, B and one to only B antigen. In 10 infants, nine developed responses to
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Fig. 2. Kinetics of HI antibody titer directed against influenza virus antigen in young children (top) and infants (bottom) inoculated with influenza vaccine in the 2001–2002 (A) and the 2002–2003 influenza season (B). Trivalent influenza HA vaccine was administered on the day of the first sampling and 4 weeks after the first vaccination. Abscissa represents weeks after vaccination and ordinate the HI titer. Number of open circles shows scattergram in each sampling point. The horizontal line defines the point of 1:40 in HI titer.
all three subtype antigens and the remaining one showed a positive response to H1N1, B. In the 2002–2003 (Fig. 3B), all young children and eight out of 11 infants reacted positively to all three subtype antigens after vaccination. In the remaining three infants, one showed a positive response to H1N1 and B, one to only H1N1 and one did not respond to any antigens. Statistical analyses showed that the rise in LPT activity to all three subtype antigens after vaccination in infants and young children was not significantly different
for the 2001/2002 season whereas LPT in young children showed significantly higher activity compared with infants in the 2002/2003 season (P = 0.0065, 0.0450, 0.0474 for H1N1, H3N2, and B antigens, respectively. The time kinetics of LPT activity looked rather simple by evaluation using two point sampling in the 2001–2002 season, however, three point sampling in 2002–2003 revealed that the kinetics of LPT during serial vaccination were rather complicated especially in young children. The time kinetics
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Fig. 3. Kinetics of lymphoproliferative response expressed as stimulation indices directed against influenza virus antigen in young children (top) and infants (bottom) inoculated with influenza vaccine in the 2001–2002 (A) and the 2002–2003 influenza season (B). Trivalent influenza HA vaccine was administered on the day of the first sampling and 4 weeks after the first vaccination. Abscissa represents weeks after vaccination and ordinate stimulation index.
of LPT among young children varied, some children showed the expected immune response such as low or negative before vaccination with an increase after the first vaccination, and a further increase after the second vaccination. However other children showed an unexpected kinetic pattern, similar to that observed in a group of older individuals, in that they already had a positive response before immu-
nization which declined after the first immunization and increased again after the second vaccination. Others also showed a decreased response after the second vaccination. On the other hand, LPT activity in infants was typically negative or low before immunization and increased after vaccination with a further increase after the second vaccination, resembling a primary immune response.
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3.4. Relationship between antibody response and specific lymphocyte activity In the 2001–2002 season, the correlation coefficients between HI titer and lymphocyte activity for three subtype antigens at the second sampling point were 0.1709, 0.0284, −0.0300 for H1N1, H3N2, B, respectively, and in the 2002–2003 season, the largest correlation coefficients for three subtype antigens were obtained at the second sampling point, those are 0.4820, 0.5980, 0.1638 for H1N1, H3N2, B, respectively. It seems that there is only a weak relationship between HI titer and S.I. in H1N1 and H3N2 on the basis of the correlation coefficients around 0.5.
4. Discussion Our main purpose was to determine whether the present HA vaccination provides young children and especially infants with protective antibody as well as specific lymphocyte activity. We also evaluated whether the antibody response was associated with the development of specific lymphocyte activity. We observed significantly poorer immune responses to vaccination in infants compared to young children. Furthermore, only a few infants achieved an antibody response of ≥ 1:40 to any subtype even after two consecutive doses, whereas in young children about 30% acquired antibody at the same level after one dose. Influenza HA vaccine is known to be poorly immunogenic in young children [6,7,10], causing one vaccine manufacturer in the United States to withdraw from manufacturing HA split vaccine [8]. In our study, a substantial number of infants failed to mount an antibody response even after two serial dose vaccination. Respecting host factors related to immunological mechanisms in infants, Clerici et al. [11] found functional defects in T helper cells of infants involving antigen presentation and immunological memory. Regarding the dosage required to induce a primary antibody response in unprimed subjects, it is known that two to three times as much as the regular dosage is needed such that 50 g per dose or more of HA is required [12,13]. Furthermore, in the Hong Kong influenza epidemics of 1968/1969, a 70% reduction in the attack rate was observed by inoculation of 10 times the regular vaccine dose whereas the regular dosage caused no reduction [14]. Thus, the poor antibody response to vaccination observed in our study as well as by other authors might reflect both insufficient dosage resulting in poor immunogenicity, and deficits in the infant immune system. In this regard, the relationship between T helper cell activity and the antibody response in individuals following vaccination is interesting. However, we found only a weak correlation between the lymphocyte response and the amount of antibody to H1N1 and H3N2 antigens elicited by vaccination. We could not find a definitive factor accounting for this weak interrelationship, possibly the antigenic epitopes recognized by the humoral immune system and those
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of cell-mediated immunity are different. More sophisticated monitoring of T helper immunity such as CD45 isotype evaluation for morphological aspects and determination of IL4, IL-5, and interferon gamma production for functional characteristics may elucidate its relationship with antibody production [15]. Detailed kinetics studies, such as nine consecutive sampling in individuals, have not been previously conducted. As one would expect, once relatively high titers of antibody were obtained before or after the first vaccination, a further elevation of antibody titer was not induced by the following vaccination. Some subjects showed an unexpected and superficially incoherent response pattern in which LPT activity declined after the first vaccination and then gradually increased, with a similar response after the second vaccination. Similar kinetic patterns were also observed in some young children. Concerning this point, one might question the stability of our system, however, robustness of the assay system is easily confirmed by the fact that each value of LPT fits stable lines or curves consecutively in each subject. The mechanism of this kinetic response pattern is unknown, possibly compartmentalization or redistribution of lymphocytes after sensitization might be involved [16]. A potential weakness of the present study is its small sample size. However, a high level of statistical significance for some of the differences in antibody responses was observed even with this small cohort, suggesting that a type I error (i.e., finding differences where none exist) is unlikely and that the results are robust enough to demonstrate that infants immunized with influenza vaccine acquired a poor immune response compared to young children. Our data raise the issue of whether a two dose regimen of influenza vaccination is appropriate for the primed population such as older children and adults. Also, based on the poor immunogenicity of the present HA vaccine in the younger age group, especially infants, even higher doses should be considered if we continue to employ HA split vaccine. Apart from the issue regarding the present vaccine preparation, alternative influenza vaccines are available, one is a whole virion vaccine which is approved in the United States, while another is a newly developed live attenuated influenza vaccine (LAIV) [8,17]. Nasal administration of highly purified whole virion vaccine, instead of the parenteral route, has been proposed [18]. More recently, ACIP published supplemental recommendations on the use of LAIV, however, its usage is currently limited to children aged 5 years or older and adults less than 50 years of age [19]. These newly developed vaccine preparations encourage us to reappraise current HA influenza vaccination policies.
Acknowledgements The authors thank Fujio Matsuo, B.S. (The Chemo-SeroTherapeutic Research Institute, Kumamoto, Japan) for his help in statistical analyses. We are indebted to Professor
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