Shinobu Abe

Evaluation of Immunogenicity and Protective Properties of Inactivated Poliovirus Vaccines: A New Surrogate Method for Predicting Vaccine Efficacy

An assay for the evaluation of protective properties of inactivated poliovirus vaccines (IPVs) in transgenic (Tg) mice susceptible to poliovirus has been developed and optimized for type 2 IPV. This method was used to compare the immunogenicity and protective properties of experimental IPV produced from the attenuated Sabin strain (sIPV) with those of conventional IPV (cIPV) produced from the wild-type (wt) poliovirus MEF-1 strain. Modified enzyme-linked immunosorbent assays (ELISAs) were used to measure immune response in serum and saliva samples from test mice. Tg mice were vaccinated and were challenged either with wt poliovirus or virulent poliovirus derived from the vaccine strain. Compared with cIPV, sIPV induced lower levels of antibodies and did not completely protect mice against challenge with wt virus but did protect mice against challenge with the virulent vaccine-derived strain. This may be due to an 18% nucleotide difference between the MEF-1 and Sabin 2 strains, resulting in 72 amino acid substitutions and leading to antigenic dissimilarity. Immunological properties of both strains, revealed by cross-neutralization tests and ELISAs, confirmed that MEF-1 possesses broader immunogenicity than does Sabin 2. This animal model may be used for the assessment of new IPVs and of combination vaccines containing an IPV component.

Transgenic mice as an alternative to monkeys for neurovirulence testing of live oral poliovirus vaccine: validation by a WHO collaborative study

OBJECTIVE Extensive WHO collaborative studies were performed to evaluate the suitability of transgenic mice susceptible to poliovirus (TgPVR mice, strain 21, bred and provided by the Central Institute for Experimental Animals, Japan) as an alternative to monkeys in the neurovirulence test (NVT) of oral poliovirus vaccine (OPV). METHODS Nine laboratories participated in the collaborative study on testing neurovirulence of 94 preparations of OPV and vaccine derivatives of all three serotypes in TgPVR21 mice. FINDINGS Statistical analysis of the data demonstrated that the TgPVR21 mouse NVT was of comparable sensitivity and reproducibility to the conventional WHO NVT in simians. A statistical model for acceptance/rejection of OPV lots in the mouse test was developed, validated, and shown to be suitable for all three vaccine types. The assessment of the transgenic mouse NVT is based on clinical evaluation of paralysed mice. Unlike the monkey NVT, histological examination of central nervous system tissue of each mouse offered no advantage over careful and detailed clinical observation. CONCLUSIONS Based on data from the collaborative studies the WHO Expert Committee for Biological Standardization approved the mouse NVT as an alternative to the monkey test for all three OPV types and defined a standard implementation process for laboratories that wish to use the test. This represents the first successful introduction of transgenic animals into control of biologicals.

Role of the Alpha/Beta Interferon Response in the Acquisition of Susceptibility to Poliovirus by Kidney Cells in Culture

ABSTRACT Replication of poliovirus (PV) is restricted to a few sites, including the brain and spinal cord. However, this neurotropism is not conserved in cultured cells. Monkey kidney cells become susceptible to PV infection after cultivation in vitro, and cell lines of monolayer cultures from almost any tissue of primates are susceptible to PV infection. These observations suggest that cellular changes during cultivation are required for acquisition of susceptibility. The molecular basis for the cellular changes during this process is not known. We investigated the relationship between PV susceptibility and interferon (IFN) response in primary cultured kidney and liver cells derived from transgenic mice expressing human PV receptor and in several primate cell lines. Both kidneys and liver in vivo showed rapid IFN response within 6 h postinfection. However, monkey and mouse kidney cells in culture and primate cell lines, which were susceptible to PV, did not show such rapid response or showed no response at all. On the other hand, primary cultured liver cells, which were partially resistant to infection, showed rapid IFN induction. The loss of IFN inducibility in kidney cells was associated with a decrease in expression of IFN-stimulated genes involved in IFN response. Mouse kidney cells pretreated with a small dose of IFN, in turn, restored IFN inducibility and resistance to PV. These results strongly suggest that the cells in culture acquire PV susceptibility during the process of cultivation by losing rapid IFN response that has been normally maintained in extraneural tissues in vivo.

An Infectious cDNA clone of the poliovirus sabin strain could be used as a stable repository and inoculum for the oral polio live vaccine

Viruses were recovered from HeLa S3 cells and African green monkey kidney (AGMK) cells transfected with an infectious cDNA clone of poliovirus vaccine Sabin 1 strain. The viruses recovered from the different DNA-transfected cells were tested for the biological characteristics of temperature sensitivity (rct marker), plaque size, and bicarbonate concentration dependency (d marker). The results revealed that the above properties were similar to those obtained from tests on the Sabin 1 vaccine reference strain. The recovered viruses and the vaccine reference virus were passaged in AGMK cells at an elevated temperature of 37.5 degrees, and the passaged isolates were tested for the rct marker. The virus recovered from AGMK cells had the most stable rct phenotype while the virus from HeLa S3 cells had a similar stability to that of the reference virus, suggesting that the virus from AGMK cells would be more suitable as a vaccine strain than the other two viruses. Furthermore, an infectious cDNA clone of high specific infectivity, constructed by introducing SV40 large T antigen into the plasmid, was used for production of high titers of virus after transfection. The results of in vitro biological tests on the recovered virus suggested that virus produced in the transfected AGMK cells also had the high quality that is desirable in vaccine stocks. Monkey neurovirulence tests performed with these recovered viruses revealed that the recovered viruses were weakly neurovirulent, similar to the vaccine reference virus. The infectious cDNA clone of the poliovirus vaccine strain could therefore be used to generate a possible inoculum of the oral polio live vaccine. Our findings strongly suggest that an infectious cDNA clone of poliovirus RNA may be used to preserve the constancy and quality of the present seed viruses of the Sabin 1 vaccine strain.

Further Development of a New Transgenic Mouse Test for the Evaluation of the Immunogenicity and Protective Properties of Inactivated Poliovirus Vaccine

Recently, we developed and optimized a new method for the evaluation of the protective properties of serotype 2 inactivated poliovirus vaccines (IPV). The method is based on the immunization and subsequent challenge of transgenic (Tg) mice susceptible to poliovirus. We describe a similar method for the assessment of the protectiveness of serotype 1 IPV and demonstrate that experimental IPV produced from attenuated Sabin strain (sIPV) of serotype 1 poliovirus induced serum neutralizing antibodies, immunoglobulin (Ig) G, IgM, and salivary IgA at titers comparable to those induced by conventional IPV (cIPV) produced from the wild-type Mahoney strain. In contrast to our previous results with serotype 2 sIPV, serotype 1 sIPV provided even better protection of Tg mice than cIPV against challenge with wild-type Mahoney strain.

Evaluation of an enzyme-linked immunosorbent assay based on binding inhibition for type-specific quantification of poliovirus neutralization-relevant antibodies.

To detect neutralization‐relevant antibodies against 3 types of poliovirus (PV) without using tissue cultures and live viruses, an enzyme‐linked immunosorbent assay (ELISA) based on monoclonal antibody‐binding inhibition was evaluated using sera from 80 vaccinated Japanese children and 60 Pakistani poliomyelitis patients. Compared with the neutralization test, the sensitivity of the inhibition ELISA was 100% (111/111) for detection of anti‐PV1 antibody, 98.3% (118/120) for anti‐PV2, and 96.5% (82/85) for anti‐PV3, and the specificity was 93.1% (27/29), 100% (20/20), and 92.7% (51/55), respectively. Thus, the inhibition ELISA showed excellent potential as a seroepidemiologic tool in both vaccinated and naturally‐infected populations.

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 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.

The Development of New Poliovirus Vaccines Based on Molecular Cloning

Poliovirus, known since 1908 to be the causative agent of poliomyelitis (Paul, 1971), is a human enterovirus of the Picornaviridae. This virus consists of a single-stranded RNA of plus-strand polarity and 60 copies each of four capsid proteins, VP1, VP2, VP3, and VP4; it occurs in three serologically distinct types, i.e., type 1, type 2, and type 3.

Poliovirus (Sabin strain) multiplication in human intestinal tract after oral polio vaccination

Fecal specimens from a baby vaccine were collected every day from 1 to 51 days after primary vaccination and from 0 to 15 days after secondary vaccination. Polioviruses were isolated with GMK-2 cell line from 10% emulsion of the feces and titrated the virus contents in the emulsion of the feces. The isolated viruses were tested the reproductive capacity at 39.0 degrees C and 39.5 degrees C by the plaque method with primary cynomologous monkey kidney cells. Viruses were isolated from the feces during 28 days for type 1, 39 days for type 2 and 36 days for type 3 after primary vaccination, however, only type 1 viruses were isolated during 7 days after secondary vaccination. The multiplication of type 3 viruses in the intestine were increased after diminished the multiplication of type 1 and type 2. In plaque formation capacity at 39.0 degrees C and 39.5 degrees C, the isolates had shown to differ clearly among the types of poliovirus. After primary vaccination, type 1 isolates were not produced the plaques at 39.0 degrees C and 39.5 degrees C. Although type 2 isolates were not formed the plaques until the 14th day at 39.5 degrees C, the plaque formation capacity of the these isolates were increased gradually i.e.; on the 20th day (10(0.88) PFU/ml), the 26th day (10(2.00) PFU/ml) and the 39th day (10(2.63) PFU/ml) at 39.5 degrees C, and all of type 2 isolates tested were showed the plaque formation capacity (10(2.88 approximately 10(3.76) PFU/ml) at 39.0 degrees C. Type 3 isolates were formed plaques at 39.0 degrees C and 39.5 degrees C from the 7th day. After the secondary vaccination, type 1 isolates (7th day) was a little changed them. Neutralizing antibody titers were shown that type 1 was 320, type 2 was 110 and type 3 was 60 after 1 year of the second administration. These titers were closely similar the geometric mean titers of 2 year old babies in Japan.

[Application of frozen stored various animal red blood cells for the virological and serological tests (author's transl)].

各種動物赤血球を用いておこなうウイルス学的, 血清学的試験に, 凍結保存赤血球を利用する目的で, 解凍赤血球の性能について試験をした結果, ヒト, ミドリザル, ヒツジ, モルモット, ニワトリおよび1日ビナ赤血球は, 非凍結赤血球と同様, 各種試験に使用できる成績を得た. すなわち, 上記各種動物凍結保存赤血球は, おのおの, 赤血球吸着, 赤血球凝集, 補体結合および免疫粘着反応に関与する特異的な性能を長期間保持していることを確認した.1. ヒトO型, ミドリザル, ヒツジおよびモルモット赤血球の血球沈渣を28w/v% glycerol, 3% mannitol, 0.65% NaCl液と等量混合し, 急速凍結, 液体窒素保存で長期に亘り保存できた.2. ニワトリおよび1日ビナ赤血球については, 血球沈渣を51.5w/v% dimethylsulfoxide (DMSO), 8% glucose, 1% fructose および0.3% EDTA-2Naからなる凍害防止液とを等量混合し, 緩速凍結後, -85℃で保存する方法が有効であった.3. 急速解凍後の脱グリセリンあるいは脱DMSOは十分量の16w/v% mannitol, 0.9% NaCl液で希釈する方法がよく, 等張液で洗浄し, 目的とする緩衝液におきかえた時点での回収率は, ヒト, サル, モルモットでは80%以上, ヒツジ, ニワトリでは65～80%, 1日ビナでは40～50%であった.4. 解凍赤血球は, 浸透圧に対する抵抗性が減弱する動物種もあったが, 少なくとも各種ウイルス学的, 血清学的反応に関与する表面構造は, 凍結, 解凍により障害を受けておらず, 非凍結の同種赤血球に匹敵する性能を示した.