Vaccine 32 (2014) 5163–5169

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A national reference for inactivated polio vaccine derived from Sabin strains in Japan Haruko Shirato a,1 , Yuichi Someya a,∗,1 , Masaki Ochiai b , Yoshinobu Horiuchi c , Motohide Takahashi c , Naokazu Takeda a , Kengo Wakabayashi d , Yasumitsu Ouchi d , Yoshihiro Ota d , Yoshio Tano d , Shinobu Abe d , Shudo Yamazaki d , Takaji Wakita a , the sIPV Evaluation Group of NIID-Virology II2 a

Department of Virology II, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashi-Murayama, Tokyo 208-0011, Japan Division of Quality Assurance, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashi-Murayama, Tokyo 208-0011, Japan c Department of Bacterial Pathogenesis and Infection Control, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashi-Murayama, Tokyo 208-0011, Japan d Japan Poliomyelitis Research Institute, 5-34-4 Kumegawa, Higashi-Murayama, Tokyo 189-0003, Japan b

a r t i c l e

i n f o

Article history: Received 16 April 2014 Received in revised form 1 July 2014 Accepted 17 July 2014 Available online 30 July 2014 Keywords: Inactivated polio vaccine Poliomyelitis Sabin strain National reference vaccine

a b s t r a c t As one aspect of its campaign to eradicate poliomyelitis, the World Health Organization (WHO) has encouraged development of the inactivated polio vaccine (IPV) derived from the Sabin strains (sIPV) as an option for an affordable polio vaccine, especially in low-income countries. The Japan Poliomyelitis Research Institute (JPRI) inactivated three serotypes of the Sabin strains and made sIPV preparations, including serotypes 1, 2 and 3 D-antigens in the ratio of 3:100:100. The National Institute of Infectious Diseases, Japan, assessed the immunogenic stability of these sIPV preparations in a rat potency test, according to an evaluation method recommended by the WHO. The immunogenicity of the three serotypes was maintained for at least 4 years when properly stored under −70 ◦ C. Based on these data, the sIPV preparations made by JPRI have been approved as national reference vaccines by the Japanese national control authority and used for the quality control of the tetracomponent sIPV-containing diphtheria–tetanus–acellular pertussis combination vaccines that were licensed for a routine polio immunization in Japan. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Poliomyelitis is caused by polioviruses of the family Picornaviridae. This neurological disorder manifests clinically as acute flaccid

Abbreviations: CCID50 , 50% cell culture infective dose; cIPV, conventional inactivated polio vaccine; DTaP, diphtheria–tetanus–acellular pertussis combination vaccine; DU, D-antigen unit; IPV, inactivated polio vaccine; JPRI, Japan Poliomyelitis Research Institute; NIID, National Institute of Infectious Diseases, Japan; OPV, oral polio vaccine; sDU, D-antigen unit for Sabin vaccine; sIPV, Sabinderived inactivated polio vaccine; SO, Sabin Original; VAPP, vaccine-associated paralytic poliomyelitis; VDPV, vaccine-derived polioviruses; WHO, World Health Organization. ∗ Corresponding author. Tel.: +81 42 561 0771; fax: +81 42 561 4729. E-mail address: [email protected] (Y. Someya). 1 These authors contributed equally to this work. 2 Members of the sIPV Evaluation Group of NIID-Virology II who contributed to this work are Takashi Shimoike, Tomoichiro Oka, Kosuke Murakami, Yoshiki Fujii, YoungBin Park, Reiko Takai-Todaka, Tatsuo Miyamura, Hiroyuki Shimizu, Kazuhiko Katayama from the Department of Virology II, National Institute of Infectious Diseases. http://dx.doi.org/10.1016/j.vaccine.2014.07.065 0264-410X/© 2014 Elsevier Ltd. All rights reserved.

paralysis [1,2]. No antiviral drugs against polioviruses are commercially available, but vaccines have been very effective in preventing and controlling epidemics. Since the World Health Organization (WHO) launched the global polio eradication program in 1988, the number of patients with poliomyelitis caused by wild polioviruses have steadily decreased worldwide from approximately 350,000 cases in 1988 to 406 cases in 2013 [3]. Oral polio vaccines (OPVs) have been particularly important for curtailing the epidemic of poliomyelitis. These effective and safe tools have several key advantages. They are inexpensive to produce, easy to administer, and induce much better mucosal immunity than inactivated polio vaccines (IPVs). In polio-free areas, however, concerns have been raised about the use of OPVs as a live vaccine. For example, risk of vaccine-associated paralytic poliomyelitis (VAPP) is small but not insignificant [4], and polio outbreaks caused by vaccine-derived polioviruses (VDPVs), highly neurovirulent OPV-derived variants, can occur [5]. Thus, many countries that have eradicated wild polioviruses have adopted IPVs to minimize the risks of VAPP and polio outbreaks caused by VDPVs.

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Until quite recently, only conventional IPVs (cIPVs) were used to prevent poliomyelitis. However, cIPVs are produced from virulent wild polioviruses, representing that much attention should be paid so as not to be a potential source of new infections. Thus, WHO has encouraged the development of IPVs derived from the Sabin strains (sIPVs) for as an alternative to cIPVs, especially in lowincome countries during the final stage of global polio eradication [6]. After polio eradication, wild polioviruses should be quarantined [6–9]. The attenuated mutant Sabin strains are much less neurovirulent than wild strains and have been used as the source of OPVs. It is therefore possible that they are safer than the wild strains and more suitable for production of IPVs. In the light of the VDPV outbreaks and VAPP cases, it is not yet clear how much safer they will be in practice. In addition, careful risk assessment will be needed for an appropriate containment of Sabin strains during the process for manufacturing sIPVs in the post-eradication era. It is assured that sIPVs induce neutralizing antibodies against the wild virulent poliovirus strains [10,11], indicating that sIPVs have adequate potency to be substituted for cIPVs. Worldwide three institutes have developed their own sIPVs: the Japan Poliomyelitis Research Institute (JPRI) in Japan, Intravacc (formerly part of National Institute for Public Health and the Environment and Netherlands Vaccine Institute), and the Chinese Academy of Medical Sciences in Kunming, China. Responding to a call from WHO for new polio vaccines, Intravacc developed a robust and transferable production process for sIPV. They have been promoting standardization of the quality control for sIPVs and attempting to offer technologies for sIPV production to lowor middle-income countries [12–14]. JPRI, a manufacturer of a Sabin OPV, made sIPV preparations, including all three serotypes of polioviruses in the D-antigen unit (DU) ratio of 3:100:100 (serotypes 1, 2 and 3, respectively) [15]. The first-generation preparations included 3, 100 and 100 sDU/0.5 mL for serotypes 1, 2 and 3, respectively, so that the immunogenicity of the respective serotypes in rats is nearly equal to that of cIPVs (containing 40, 8 and 32 DU/0.5 mL for serotypes 1, 2 and 3, respectively). However, it should be noted that the DU for sIPVs is not equivalent to that for cIPVs, which has been clearly shown by Simizu et al. [15]. That is, the DUs of the WHO cIPV reference (91/574) whose indicated titers were 430, 95 and 285 DU/mL for serotypes 1, 2 and 3, respectively [16], were measured as 96.2, 150 and 549 sDU/mL by in-house ELISA using anti-Sabin antibodies prepared at JPRI with each serotype of Sabin strains used as a standard substance (see Section 2 for the method of in-house ELISA). Note that “sDU” represents the D-antigen unit for sIPVs and will be used in this manuscript in order to distinguish between cIPV and sIPV and avoid misleading. This discrepancy is possibly because different antibodies and standards are used for D-antigen ELISA of sIPV and cIPV. This is also possibly because of different antigenic structure between sIPV and cIPV. In this context, to measure the D-antigen content of sIPV products from various sources, the standardization of assaying the D-antigen content and the establishment of International Reference Reagents for sIPVs are currently under investigation by the WHO Technical Working Group [17]. Here we report our evaluation of the stability of sIPV preparations made by JPRI. We monitored the D-antigen content and immunogenicity of each serotype in rats over a long period. Based on the results, the sIPV preparations were approved as national reference vaccines by the Ministry of Health, Labour and Welfare of the Japanese government, and used for quality control of the tetracomponent vaccines consisting of sIPV and a tricomponent diphtheria–tetanus–acellular pertussis combination antigen (DTaP-sIPV). Currently, two DTaP-sIPV products from the Chemo-Sero-Therapeutic Research Institute (Kaketsuken) and the Research Foundation for Microbial Diseases of Osaka University (Biken) were licensed for the production in 2012 and used for

Table 1 The D-antigen contents in the sIPV preparations. sIPV Lot

Date of preparation

04C 05J 09A 12A

Aug. 11, 2004 Oct. 20, 2005 Jul. 15, 2009 Jul. 4, 2012

D-antigen contents (sDU/0.5 mL) Type 1

Type 2

Type 3

3.9 2.7 5.5 5.7

113 105 176 199

112 108 201 192

The D-antigen contents were determined by a sandwich ELISA method against the in-house reference standard of the Sabin polioviruses as described in Section 2 and were expressed as the Sabin D-antigen unit (sDU).

a routine polio immunization for infants and young children in Japan. 2. Materials and methods 2.1. Preparation of sIPV sIPV bulk preparations were prepared as described [15]. The following Sabin strains were used: the LSc 2ab strain for serotype 1, the P712,Ch,2ab strain for serotype 2, and the Leon 12a1 b strain for serotype 3. The respective virus master seeds were established from the WHO/Behringwerke 1976 (SO+1 = Sabin Original + 1 passage) for serotype 1, the WHO/Behringwerke 1976 (SO+1) for serotype 2, and the Leon 12a1 b/KP3 (SO) for serotype 3 [17]. Each strain was independently propagated from its working seed in Vero cells that were purchased from the American Type Culture Collection (CCL81). After purification, the virus-containing fluid was inactivated by treating with 0.025% formalin for 12 days at 37 ◦ C. Each serotype of monovalent sIPV was mixed with the others to obtain the trivalent sIPV preparations. The D-antigen contents of Lots 04C, 05J, 09A, and 12A of sIPV preparations manufactured by JPRI are shown in Table 1. sIPV bulk preparations were produced under conditions of good manufacturing practice in essentially the same way as a routine production process for cIPV [18]. 2.2. D-antigen ELISA A D-antigen ELISA was performed as described [15,19]. The wells of 96-well microtiter plates (Nunc Immunoplate Maxisorp, Thermo Fisher Scientific, Waltham, MA) were coated with D-antigen-specific mouse monoclonal antibodies against each serotype of Sabin strains (MA107-8W, MA201-159 and MA303182L for serotypes 1, 2 and 3, respectively), which were isolated by JPRI [15,19]. Antibodies were diluted by a factor of 20,000 in 0.05 M sodium carbonate buffer. The plates were incubated for 3 h at 36 ◦ C and washed three times with a wash buffer (0.01 M PBS containing 0.05% Tween 20). Samples of sIPV preparations in a dilution buffer (0.01 M PBS containing 0.05% Tween 20 and 1% BSA) were placed on wells in line B on the plate, followed by serial twofold dilutions to line G. The plates were incubated overnight at 4 ◦ C and washed three times. Fifty microliter of serotype-specific neutralizing rabbit antibodies (dilution factor for serotypes 1 and 3: 1000; and serotype 2: 2000) were added to each well on the respective plates, and the plates were incubated for 1 h at 36 ◦ C. After washing the plate, 50 ␮L of anti-rabbit IgG conjugated with horseradish peroxidase (0.21 ␮g of protein/mL, ICN/Cappel, Aurora, OH) were added to each well, and the plates were incubated for 1 h at 36 ◦ C. The o-phenylenediamine chromogen and hydrogen peroxide substrate were added, and the plates were incubated for 20 min at 36 ◦ C. The reaction was stopped by adding 50 ␮L of 2 M H2 SO4 , and the absorbance at 492 nm was measured. sDUs in samples were calculated by the parallel line method [20] against an in-house reference standard virus whose sDUs were adjusted

H. Shirato et al. / Vaccine 32 (2014) 5163–5169

Neutralizing antibody titer (log 2)

10

Type 1

Type 2

10

P = 0.074

5165

Female P = 0.009

8

8

6

6

6

4

4

4

2

2

2

0

0 1 Dilution fold (3 n )

Type 3

10

2

0

0

Male

8

1

2

Dilution fold (3 n)

0

P = 0.22

0

1 2 Dilution fold (3 n)

Fig. 1. Effect of rat gender on the neutralizing antibody titers. An immunogenic test with rats examined gender bias in rats. Along with undiluted solution of Lot 04C, one-third and one-ninth dilutions were prepared. The averages of neutralizing antibody titers, expressed as log2, were plotted against the dilution fold for the dilutions administered. Closed circles and open circles indicate neutralizing antibody titers in sera from female rats and male rats, respectively. P values were determined by Student’s t test for neutralizing titers of sera from undiluted sample-injected rats, and depicted in each graph.

with reference to the WHO sIPV preparation (91/672) [19,21,22]. For calculation, the software “Bioassay Assist,” which was made by the Department of Bacterial Pathogenesis and Infection Control in the National Institute of Infectious Diseases (NIID), was used.

Pathogenesis and Infection Control in NIID. When a serum with no neutralizing antibodies was included in dilution groups, mean and variance of the groups were estimated by the maximum-likelihood method for truncated normal distribution [25,26].

2.3. Immunogenicity tests

3. Results

All animal procedures were approved by the Committees on Biosafety and Animal Handling Regulations of NIID. Animal research was undertaken in compliance with the “Fundamental guidelines for proper conduct of animal experiment and related activities in institutions under jurisdiction” issued by the Ministry of Health, Labour and Welfare, Japan. All studies with animals also adhered to the principles stated in the guidelines. The immunogenic potency of the sIPV preparations was assessed essentially according to the method described in the WHO Recommendations [23], which originated from the report by van Steenis et al. [24]. Test vaccines were diluted in minimal essential medium so as to make a series of three- or twofold dilutions. Each dilution was tested in 10 specific pathogen-free Wistar rats that were suitably housed. If not otherwise specified, 0.5 mL of each dilution of test vaccines was injected intramuscularly into a hind limb of each rat at the age of 8 weeks. After 3 weeks, rats were bled under anesthesia. Serum isolated from each blood sample was subjected to inactivation at 56 ◦ C for 30 min and used to measure neutralizing antibody titers. Each serum sample was serially twofold diluted on 96-well microtiter plates (BD Falcon, Franklin Lakes, NJ) in duplicate, and each serotype of the Sabin strains was added as a challenge virus to a final concentration of 100 CCID50 /well, followed by neutralization for 3 h at 35.5 ◦ C and overnight incubation at 4 ◦ C. HEp-2C cells purchased from the American Type Culture Collection were added as indicator cells to a final level of 104 cells/well, and the plates were incubated at 35.5 ◦ C for 7 days. By observing the presence or absence of a cytopathic effect on the cells, a geometric mean titer for each serum was determined and expressed as log2. The in-house serotype-specific reference antisera from both JPRI and NIID were also placed in each test to verify that the test was valid. Additionally, a back titration was performed on the challenge viruses used in each test. The relative immunogenic potency of test vaccines was calculated with a 95% confidence interval by the parallel line method with an Excel sheet created by the Department of Bacterial

3.1. Refining the rat immunogenicity tests The WHO recommendations for cIPVs [23] describe an in vivo immunogenicity test as an alternative to the in vitro D-antigen ELISA potency test for IPVs. In countries where cIPVs are used for vaccination, the respective national control authorities adopt the D-antigen ELISA as a potency test for cIPVs. sIPVs had not been clinically used when we started a collaborative project to establish a national reference sIPV, and there were no WHO standard vaccines to evaluate the immunogenicity of sIPVs and a correlation between D-antigen content and immune response in an animal model. Therefore, we needed to establish a national reference vaccine to validate the D-antigen contents and the immunogenic potency in rats of sIPV-containing vaccines made by different vaccine manufacturers in Japan. First, we took a careful look at the rat immunogenicity test. The WHO recommendations [23] do not specify the strain, sex and age of rats for the in vivo potency test. We chose the Wistar strain because of their ready availability. To investigate a possible gender bias in a rat model, the neutralizing antibody titers were compared in male and female groups. In this test, we made threefold serial dilutions (undiluted, one-third dilution and one-ninth dilution) of Lot 04C of an sIPV preparation (Table 1). Ten rats per group were used for each dilution. At 21 days after intramuscular injection of sIPV solution, blood was taken from each rat, and serum samples were isolated for measuring neutralizing titers against three serotypes of Sabin strains. Sera from female rats tended to have higher neutralizing titers against all three serotypes than those from male rats (Fig. 1). Comparing neutralizing titers of sera from female and male rats using the Excel sheet described in Section 2.3, female rats responded better to the sIPV preparation by a factor of 1.6 and 2.1 for serotypes 1 and 2, respectively, than male rats. By Student’s t test, the P values for the neutralizing titers of sera from rats injected with undiluted samples were 0.074 and 0.009 for

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Type 1

Type 2

10

Type 3 10

10

Neutralizing antibody titer (log 2 )

Lot 05J 8

8

8

6

6

6

4

4

4

2

2

2

0 Lot 05J Lot 09A 0

0

0 2 3 Dilution fold (2 n) 0 1

1 2

3 4

Lot 09A

0 1

1 2

2 0 3 Dilution fold (2 n)

3 4

0 1

0

1 2

2 3

3 4

Dilution fold (2 n)

Fig. 2. Immunogenic potency of Lots 05J and 09A. Four twofold dilutions of Lot 05J (closed circles) and five twofold dilutions as for Lot 09A (closed squares) were prepared, and 0.5 mL of each dilution was injected into a female rat as described in Section 2. The average neutralizing antibody titer for each dilution group was plotted against the fold dilution. The results of the first immunogenicity test of both lots are shown. The first trial of Lot 05J was performed 2 months after production, and the first trial of Lot 09A was 1 month after preparation.

3.2. Establishing the first national reference vaccine for sIPV Lot 04C was the first sIPV preparation with all three serotypes that JPRI made for use as a national reference vaccine. However, as described above, a loss of the immunogenic potency and concomitantly the D-antigen content was obvious over time and was

Lot 05J Lot 09A 10

(sDU/0.5 mL)

D-antigen content

Type 1

1

10

20

30

40

50

60

0

10

20

30

40

50

60

10

20

30

40

50

60

100

10

1000

(sDU/0.5 mL)

Type 3

0

1000

(sDU/0.5 mL)

D-antigen content

Type 2

D-antigen content

serotypes 1 and 2, respectively. Although a factor and the P value for serotype 3 could not be calculated, the average neutralizing titer of sera from female rats injected with undiluted samples was higher than that from corresponding male rats (Fig. 1). Therefore, in all of subsequent tests, only female rats were used. Unexpectedly, the immunogenic potency of Lot 04C for serotype 3 was low (Fig. 1). This might be attributed to the storage conditions. Lot 04C had been stored at 5 ± 3 ◦ C, and we observed that the immunogenicity and the D-antigen contents decreased gradually with time (data not shown). As described below (see also Fig. 2), all other sIPV preparations (e.g., Lots 05J and 09A) were stored under −70 ◦ C, and showed acceptable stability in immunogenic potency and the D-antigen contents. In general, the body weight of the rats increased with age during the growth phase. To test the effects of body weight on immune response, 7-week-old rats were purchased, and body weight of each rat was measured. At the age of 8 weeks, body weight was measured before injection. As sIPV samples, twofold serial dilutions of Lot 05J were prepared. After 3 weeks, the body weight of each rat was measured again, and blood was taken. Sera were prepared and a neutralization test was completed (Supplementary Fig. 1, Supplementary Table 1). The final body weight correlated with the weight at the time of both purchase and injection, indicating that rats grow without any incident (Supplementary Fig. 1A–C). In fact, no rats experienced a loss of body weight. When the neutralizing antibody titers for all three serotypes were plotted against body weight of each rat, no correlation was found, indicating that body weight is not a factor affecting the neutralizing titer (Supplementary Fig. 1D). Therefore, female rats were randomly placed in cages at the time of purchase without measuring body weight in later tests. Supplementary table and figure related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.vaccine. 2014.07.065.

100

10 0

Months after preparation Fig. 3. Monitoring the D-antigen content of Lots 05J and 09A after its preparation. The D-antigen content for each serotype was measured by ELISA as described in Section 2. The D-antigen content of Lot 05J (closed circles) and Lot 09A (closed squares) immediately after preparation is shown in Table 1. Lot 05J was prepared so as to contain 3, 100 and 100 sDU/0.5 mL of the D-antigens of serotypes 1, 2 and 3, respectively. On the other hand, Lot 09A was prepared so as to contain 6, 200 and 200 sDU/0.5 mL of the D-antigens of each serotype.

H. Shirato et al. / Vaccine 32 (2014) 5163–5169

5167

Type 1 10

Relative potency

NIID JPRI

Type 3 1

0.1

100

0

20

40

60

80

10

Type 2

Relative potency

10 1 1 0.1 0.1

0

20

40

60

80

Months after preparation

0

20

40

60

80

Months after preparation

Fig. 4. Monitoring the immunogenic potency of Lot 05J. Immunogenicity tests were performed at the times indicated. The immunogenicity potency at each time was expressed as the relative potency against the result of the first trial. Error bars indicate 95% confidence intervals.

attributed to storage at 5 ± 3 ◦ C. Therefore, the next preparation, Lot 05J, was stored under −70 ◦ C and tested as to the immunogenic stability. The stabilities of the D-antigen content and immunogenicity were monitored by JPRI and NIID over the long duration. Immediately after its production, Lot 05J induced high-titer neutralizing antibodies in the immunized rats (Fig. 2). The D-antigen content of each serotype in Lot 05J was maintained even after 5 years of production under −70 ◦ C (Fig. 3). Concomitantly, the immunogenicity of this lot was maintained independently of serotypes (Fig. 4). The relative potency for each serotype was plotted against the potency at the first neutralization test that was done after 2 months of production. These data indicate that Lot 05J is stable when properly stored under −70 ◦ C. Based on these data, the Lot 05J sIPV preparation was officially approved as the first national reference vaccine by the Ministry of Health, Labour and Welfare of the Japanese government, and as a unit expressing the immunogenicity in rat potency tests, 1.0 U/mL was assigned uniformly to every serotypes of Lot 05J. It should be noted that this potency unit (U/mL) is based on neutralizing antibody titers and is completely different from the unit for the D-antigen contents (sDU/mL). 3.3. Improved immunogenicity of the national reference vaccines Although Lot 05J was approved as the first national reference vaccine, it has one problem. Several sera from rats in the oneeighth- and one-fourth-dilution groups induced no or extremely low amounts of the neutralizing antibody against a serotype 3 poliovirus, resulting in a loss of a data point or large variances when calculating the relative potency. Therefore, the potency for serotype 3 was estimated with lower precision, than those for serotypes 1 and 2 (Fig. 4). To overcome this issue, the next sIPV preparation, Lot 09A, was made with twice the amount of each D-antigen as the former Lots 04C and 05J (Table 1). Lot 09A exhibited a higher immunogenic potency than Lot 05J when the test materials of the same dilution fold were compared (Fig. 2). This is attributed to the

fact that the undiluted Lot 05J is almost equal to a half dilution of Lot 09A in terms of the D-antigen contents (see Table 1). The D-antigen content and immunogenicity of Lot 09A seemed to be stably maintained for 4 years after production (Figs. 3 and 5). Lot 09A sIPV was also approved as a national reference vaccine and specified to contain 2.6, 1.7 and 2.0 U/mL for serotypes 1, 2 and 3, respectively. These values were determined by a comparison with an immunogenic potency in rats of Lot 05J, which was based on the results obtained in a collaborative study group. Notably, the immunogenic potency roughly correlated with the D-antigen content. Lot 09A was also used as the reference vaccine for in-house tests when the manufacturers evaluated their DTaP-sIPV combination vaccines. For the continuous production of reference sIPV, Lot 12A of the sIPV preparation was also approved as a successive national reference vaccine. Immunogenic potencies of 1.7, 2.3 and 1.8 U/mL for serotypes 1, 2 and 3, respectively, were determined by comparing the immunogenic potency of Lot 09A, which was correlated with the D-antigen contents (Table 1). Lot 12A is used as a reference vaccine for the quality control of the licensed DTaP-sIPV vaccines for both in-house tests by manufacturers and the national quality control tests at NIID. In addition, NIID is responsible for monitoring the long-term stability of Lot 12A with regard to the immunogenic potency, as well as Lots 05J and 09A.

4. Discussion Based on the considerations of WHO that sIPVs could be used as alternative IPVs to cIPVs after global polio eradication, JPRI made sIPV preparations in the D-antigen ratio of 3:100:100 (serotypes 1, 2 and 3, respectively) [15] that could be eventually used as a national reference vaccine for the quality control of sIPV-containing vaccine products. As reported here, NIID and JPRI demonstrated that the sIPV preparations stored under −70 ◦ C were stable when the Dantigen content and immunogenicity were monitored over time.

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NIID JPRI

Relative potency

Type 1 10

1

0.1 0

10

20

30

40

50

0

10

20

30

40

50

10

20

30

40

50

Relative potency

Type 2 10

1

0.1

Relative potency

Type 3 10

1

respectively. D-antigens of 10, 16 and 32 sDU/dose for serotypes 1, 2 and 3, developed by Intravacc were included, respectively [8], and a phase I trial in healthy adults used their sIPV products with or without aluminum adjuvant [13]. As for the case in Japan, in the phase II and III clinical trials described in Okada et al. [27], the licensed DTaP-sIPV vaccines clinically used in Japan contain 1.5, 50 and 50 sDU/dose for serotypes 1, 2 and 3, respectively, with aluminum adjuvant. Differences in the D-antigen content in sIPV vaccines from these three institutes are thought to be due to differences in antibodies used in the D-antigen determination. In addition, the Sabin strains used in three institutes for sIPV production might contain different amino acids in neutralizing epitopes during a prolonged in-house propagation even though they originated from the same strains. Alternatively, some antigenic determinants might be altered during the formalin inactivation. In any case, the quality control of these sIPVs should be standardized and validated in a uniform manner. When global poliomyelitis is finally eradicated, the sIPV itself and sIPV-containing combination vaccines will be useful options for polio vaccines alternative to OPVs and cIPVs. sIPV is an effective vaccine with an immunogenic potency equivalent to cIPVs. International reference sIPV preparation should be established: a lot of countries will adopt sIPVs for a routine vaccination against poliomyelitis. On a final note, the study on the stability of the sIPV preparations was performed within a collaborative study group for sIPV development and evaluation, comprising Kaketsuken, Biken and Takeda Pharmaceutical Company Limited (Takeda) in addition to JPRI and NIID. Kaketsuken, Biken, and Takeda also observed that both Lots 05J and 09A of the sIPV preparations were stable in terms of the immunogenic potency in rats, supporting the conclusion described in this report.

0.1 0

Months after preparation Fig. 5. Monitoring the immunogenic potency of Lot 09A. Immunogenicity tests were performed at the times indicated. Immunogenicity potency at each time was expressed as the relative potency against the result of the first trial. Error bars indicate 95% confidence intervals.

Lot 05J of the sIPV preparation was approved as the first national reference vaccine by the Japanese government and specified to contain 1.0 U/mL of the immunogenic potency in rats to each of three serotypes of poliovirus. The subsequent preparation, Lot 09A, has twice the amount of each D-antigen to improve the immunogenic potency for serotype 3 in the reference material. Consistent with this, the immunogenic potency of Lot 09A doubled (Fig. 2). NIID and JPRI demonstrated independently that this preparation was stable in terms of the D-antigen content and immunogenicity. Lot 12A is currently used as a national reference vaccine for evaluating DTaPsIPV combination vaccines that were licensed in 2012 in Japan and introduced for routine immunization for the first time. One may ask if persons administered with sIPV have protective immunity against the virulent wild polioviruses. As described by Okada et al. [27], human antibodies induced by DTaP-sIPV injection neutralized the referential virulent polioviruses and Sabin strains. Sabin strains are much less neurovirulent than wild strains, and this feature might facilitate production of Sabin-derived vaccines even in low-income countries because the strictly controlled biosafety levels for the virulent strains are not required for Sabin strains at present. However, considering the VDPV outbreaks and VAPP cases, manipulation of Sabin strains should be properly controlled. In a phase II clinical study on domestic standard sIPV in China [28], the sIPV used in the phase III study contained D-antigen amounts of 15, 32 and 45 sDU/dose for serotypes 1, 2 and 3,

Acknowledgements This work was partially supported by a Grant-in-Aid (KAKENHI Grant 24115003) for Scientific Research from the Japan Society for the Promotion of Science, Grants-in-Aid (H24-Tokubetsu-Shitei015 and H25-Shinko-Ippan-012) for Research on Emerging and Re-emerging Infectious Diseases from the Ministry of Health, Labour and Welfare of Japan, and grants from the Ministry of Health, Labour and Welfare of Japan for the Promotion of Polio Eradication, and for Sabin IPV quality control. We are grateful to all members of a collaborative study group for sIPV development and evaluation. We would like to note the extraordinary efforts and contributions of Dr. Etsuko Utagawa and Dr. Tian-Cheng Li (Department of Virology II, NIID) for rat immunogenicity tests. We are also grateful to Ms. Miyuki Ooizumi and Ms. Kyoko Konishi (Department of Virology II, NIID) for their assistance in neutralization tests. Conflict of interest: There is no conflict of interest.

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A national reference for inactivated polio vaccine derived from Sabin strains in Japan.

As one aspect of its campaign to eradicate poliomyelitis, the World Health Organization (WHO) has encouraged development of the inactivated polio vacc...
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