Saccharomyces boulardii prevents oral-poliovirus vaccine-induced IgA nephropathy in mice.

Immunoglobulin A nephropathy (IgAN) is associated with mucosal IgA defect. Probiotics regulate specific and innate immunity. We evaluated the effect of Saccharomyces boulardii on experimental IgAN in mice. Four groups of BALB/c mice (eight for each) were formed. Group 1 was immunized by oral poliovirus vaccine (OPV) at 0, 14, and 28 days. Group 2 was also given S. boulardii in addition to OPV. Group 3 was given only S. boulardii, whereas group 4 received no treatment. At week 6, after urine and serum samples were obtained for urinalysis and serum creatinine and IgA measurements, all animals were sacrificed to get their kidneys for histopathological evaluation. Urinalysis and serum creatinine levels were normal in all groups. Serum IgA level was increased only in group 1. Whereas group 1 had mesangial proliferation, histology was normal in the other groups. Predominant IgA deposition was universal in group 1, whereas it was either not present or minimal in other groups. Three mice in group 1 also had C3 deposition, which was absent in other groups. Electron microscopy revealed mesangial proliferation, matrix expansion, focal glomerular basement membrane thickening and electron-dense deposits in group 1 only, whereas the other groups were normal. In conclusion, enteral S. boulardii prevented OPV-induced IgAN in mice.

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.

The assessment of OPV vaccines by the monkey neurovirulence test: why and how to qualify the experts in histology of central nervous system.

Prior to batch release of oral poliovirus vaccines (OPV) for marketing purpose, the World Health Organisation (WHO) and European pharmacopoeia require a monkey neurovirulence test to be performed both by the manufacturer and the relevant National Control Laboratory (NCL) to assess vaccine safety as regards neurovirulence. Due to the subjectivity of histological examination and of neural lesions scoring, the French NCL has set up a proficiency testing procedure to qualify a new expert.

Analysis of the accumulation of mutants in Sabin attenuated polio vaccine viruses passaged in Vero cells.

To confirm the safety of oral poliomyelitis vaccine (OPV) cultured in Vero cells, the genetic stability of cultured polio vaccine viruses was analysed by MAPREC (mutant analysis by PCR and restriction enzyme cleavage). The rates of mutant accumulation of the viruses passaged in Vero cells under a low multiplicity of infection (MOI) condition (approximately 10(-3.5)CCID50/cell; the same as under usual OPV production conditions) were higher than those passaged in secondary cultured monkey kidney cells. However, the rates of mutant accumulation were restrained when the viruses were cultured under a high MOI condition (approximately 10(-1.5)CCID50/cell) in Vero cells. Furthermore, neurovirulence of the passaged viruses in pollovirus susceptible transgenic mice PVR-Tg21 was shown to correlate highly with the results of MAPREC. It is expected that our results will contribute to the large scale preparation of safe and effective OPV using Vero cells.

Development of a transgenic mouse neurovirulence test for oral poliovirus vaccine: international collaborative study 1993-1999.

The neurovirulence safety of oral live poliovirus vaccine (OPV) has been tested in monkeys, because only primates are sensitive to all three types of poliovirus. The genetic engineering of transgenic mice susceptible to poliovirus led to studies on the suitability of these mice for a neurovirulence test of OPV. A WHO international collaborative study started with type-3 OPV in 1993 and was completed in 1999. The study produced a voluminous set of data proving that the TgPVR21 mice, inoculated with OPV samples into the lumbar cord, provided a test for neurovirulence of OPV as sensitive and reproducible as the monkey test. A statistical decision model for acceptance/rejection of type-3 vaccines using the transgenic mouse test has been developed. The mouse neurovirulence test showed a number of essential advantages over the monkey test. This is the first example of a successful introduction of transgenic animals into control of biologicals. In October 1999, the WHO Expert Committee on Biological Standardization approved TgPVR21 mice as alternative to the monkey model for neurovirulence testing of OPV type 3. A final step of the collaborative study with OPV types 1 and 2 is in progress, and data obtained so far are promising. Two breeding stations for production of TgPVR21 mice are being established in Asia and Europe.

Development of a neurovirulent testing system for oral poliovirus vaccine with transgenic mice.

Because only primates are susceptible to polioviruses, the neurovirulent safety and consistency of oral poliovirus vaccine (OPV) were assayed in the monkey neurovirulence test. After the development of transgenic (Tg) mice carrying the gene for human poliovirus receptor (PVR), the suitability of these mice to replace monkeys for OPV testing was evaluated. Two lines of Tg mice, TgPVR1 and TgPVR21, were tested. The TgPVR21 mice, inoculated in the spinal cord, were as sensitive as monkeys in discriminating between type-3 and type-2 OPV lots that had passed and those that had failed the monkey neurovirulence test. Results of the new molecular assay by polymerase chain reaction and restriction enzyme cleavage indicated that each OPV lot contained minuscule amounts of neurovirulent revertants in the viral genome. All type-3 OPV lots that failed the monkey neurovirulence test had higher percentages of 472-C revertants than did lots that passed this test. Analysis of multiple type-3 OPV lots also indicated a good correlation between the contents of 472-C revertants and results of the TgPVR21 mouse test. An overview of a significant set of data suggests that the TgPVR21 mouse model is suitable for the evaluation of type-3 and type-2 OPV. The necessity of the TgPVR mouse test for the neurovirulence of type-1 OPV, which is the most stable of the three Sabin strains, is under consideration.

The views and policy of the Japanese control authorities on the three Rs.

NIH Japan has tested and regulated the three Rs of animal experiments in the development and control of biological products in a stepwise manner. (1) The number of monkeys was reduced from 108 to 72 for the neurovirulence test of OPV in each type, since paralysed monkeys inoculated intraspinally revealed a linear relationship between average scores of the lumbar lesion and cumulated paralysis occurrence ratio (%). (2) Rabbits for the pyrogen test were replaced by the endotoxin test for PPF, albumin and interferon products. The endotoxin is measured by the parallel line assay method using both turbidimetric kinetic and colorimetric methods. (3) Histopathological examination was introduced to the abnormal toxicity test as a refinement. Mean body weight loss of two guinea pigs inoculated with five ml. of an albumin product in each was far below the mean weight of pooled guinea pigs used (P < or = 0.01) and appeared repeatedly. The histopathological examination showed focal necrosis in the liver. This finding was suggestive of the presence of endotoxin in the product. The product contained 0.1 EU/ml of endotoxin. The same amount of the reference endotoxin produced a similar change in guinea pigs. The mechanism of the liver cell damage by endotoxin has been investigated by an in vitro method.

Evaluation of new approaches to poliovirus vaccine neurovirulence tests.

Transgenic mice that express the human receptor for poliovirus and mutant analysis by PCR followed by restriction enzyme cleavage (MAPREC) are two new approaches to poliovirus vaccine neurovirulence testing. This review assesses the validity and current status of these tests. Both approaches require further rigorous validation and development. They are most likely to be used as corollary rather than replacement tests for the current neurovirulence tests.

Development of candidates for new type 2 and type 3 oral poliovirus vaccines.

Monkey neurovirulence tests on in vitro recombinant viruses between the virulent Mahoney and the attenuated Sabin 1 strains of type 1 poliovirus revealed that a strong neurovirulence determinant(s) resided in the 5' non-coding sequence of the genome and that the surface structure of the virion particle had a little correlation with the neurovirulence or attenuation phenotype. The results suggested that new and safer vaccine strains of type 2 and type 3 polioviruses may be constructed in vitro by replacing the sequence encoding the antigenic determinants in viral capsid proteins of the Sabin 1 genome by the corresponding sequences of the type 2 and type 3 genome, respectively, because the Sabin 1 strain is the safest vaccine among the Sabin strains. We have constructed recombinant viruses, PV1/2 (SS)BB and PV1/3(SS)BN, as vaccine candidates for type 2 and type 3 oral poliovirus vaccines respectively. These recombinant viruses were fully viable and showed antigenicities and immunogenicities identical to those of type 2 and type 3 polioviruses respectively. The monkey neurovirulence tests performed on these recombinant viruses suggest that the recombinant viruses are possible candidates for new type 2 and type 3 poliovirus vaccine strains.

Some aspects of the monkey neurovirulence test used for the assessment of oral poliovirus vaccines.

Twelve years of experience in three control laboratories with 8,000 macaca monkeys for the testing of 204 monovalent lots of the three types of oral poliovirus vaccine (OPV), have shown that the new monkey neurovirulence test (MNVT) adopted by the World Health Organization (WHO) is a reproducible and sensitive assay likely to ensure the safety of this vaccine in humans. When the test vaccine and the appropriate homotypic reference vaccine were tested in a single group of monkeys, the concurrent use of the reference vaccine considerably increased the reproducibility of the test. The usefulness of the WHO MNVT was further demonstrated in our laboratory when the test was applied to types 1 and 3 vaccine strains that were passaged serially in the intestinal tract of infants. The test was suitable for detecting increased monkey neurovirulence in these human passage strains that did not reach the level of neurovirulence of the "wild" types 1 and 3 strains tested concurrently (except human passage no. 7 of the type 3 vaccine that multiplied for a cumulative 109 days in seven infants). The statistical analysis of the data showed that the old intrathalamic (IT) assay used for many years in our laboratory was considerably less sensitive than the intraspinal WHO MNVT; a test vaccine with a two-fold increase in monkey neurovirulence showed a 41% chance of failing in the IT test (using 30 monkeys per vaccine), while this chance increased to 99% in the WHO assay (using 12 monkeys for types 1 and 2 vaccines and 20 monkeys for type 3 vaccine). Indeed, three of seven type 3 lots tested in our laboratory with both assays failed in the WHO assay but all lots passed in the IT test. Since the introduction of the WHO MNVT in Canada and the United Kingdom, the number of vaccine-associated paralytic poliomyelitis cases in the population continued to remain at a low level.

Assessment of the viral RNA sequence heterogeneity for control of OPV neurovirulence.

By using sensitive mutant analysis by PCR and restriction enzyme cleavage we have found that among several positions that differ between the wild-type and attenuated type 3 poliovirus genomes, only two positions, 472 and 2493, showed variability in vaccine lots. Of these two, only position 472 correlates with neurovirulence in monkeys, while the abundance of revertants at position 2493 indicated the type of seed virus and the passage level. Conditions of cell culture influence the rate of mutant selection, suggesting that some cellular factor(s) may be involved in selection of 472-C. Determination of 472-C content predicts which vaccine lots would fail the monkey neurovirulence test. These results imply that the PCR method can be used for optimization of manufacturing conditions.

Significance of a newly identified attenuating mutation in Sabin 3 oral poliovirus vaccine.

Sequence analysis of poliovirus vaccine RNA has resulted in the identification of a new point mutation at position 2493 in Sabin 3. cDNA-derived Sabin 3 virus whose genome includes the new mutation has phenotypic properties expected for a vaccine strain. Virus engineered to contain a single base substitution at 2493 has lost the small plaque phenotype, and low neurovirulence characteristics normally associated with Sabin poliovirus strains. The significance of these observations and a potential relationship to the frequency of vaccine-associated poliomyelitis are described.

Evaluation of the neurovirulence test of oral poliovaccines in Japan during the period 1963-1982.

The present study showed that the neurovirulence test for the lesion-inducing virus dose in the spinal cord in 50% of monkeys inoculated with oral polio-vaccine (Sabin) (LID50) reflected to a large extent safety and efficacy of the vaccine upon administration to children. The degree of attenuation of the vaccine in terms of LID50 appeared to be related to the decrease in the vaccine-associated cases as well as the seroconversion rate. An exceptional case, however, was noted in which lot No. 301 was not classified into less attenuated vaccine by the neurovirulence test. This fact suggests that the method we have employed awaits further improvement.

Experimental scoliosis in primates: a neurological cause.

Although a variety of techniques have been used with varying success to induce scoliosis in animals, primates have rarely been used. A series of monkeys is presented where scoliosis developed incidentally during the routine virulence testing of live, attenuated, oral poliomyelitis vaccines by intraspinal injection. The site and extent of histological damage in the different anatomical areas of the spinal cord were examined in 25 scoliotic monkeys and 25 matched controls. Analysis of the data demonstrated that there was significantly greater damage on the convex side of the spinal cords of the scoliotic animals, particularly in the sensory areas-the posterior horn and Clarke's column. Scoliosis was not thought to be caused by clinical poliomyelitis as the involvement of the anterior horn was not significantly greater than in the scoliotic animals than in the controls. These observations are taken to support the view that scoliosis may develop as a result of asymmetrical weakness of the paraspinal muscles due to the loss of proprioceptive innervation.