Potency evaluation of rabies vaccine for human use: The impact of reducing the number of animals per dilution - Part 2.

The most critical parameter for the quality control of the rabies vaccine is potency, which is evaluated by challenge test in mice while using a large animal number. Because the 3Rs concept is applied worldwide, it becomes necessary to develop alternative methods to demonstrate the production consistency of these vaccines and reduce the number of animals used for performing assays. Hence, the present study evaluated the impacts of reducing the number of mice used in the NIH test for such vaccines. A retrospective data analysis compared vaccines tested in the standard test with the results of the reduced test using only the first cages of each dilution and considering the second cages as their replicates. The relevance of the reduced assay was evaluated using Bland- Altman plot and CCC. Reliability was assessed by CV% and confidence intervals, while the impact of the reduced mouse number was evaluated by the analysis of the confidence interval of potency results and regression, linearity and parallelism parameters. The results demonstrated the feasibility of reducing to eight mice per dilution in routine assays, with complete statistical validation of the resulting potency, allowing the number of animals used for the test vaccines to be reduced by 50%.

Inactivated rabies vaccines: Standardization of an in vitro assay for residual viable virus detection.

Human rabies, a neglected viral zoonosis, is preventable through domestic animals vaccination and post-exposure prophylaxis using inactivated rabies vaccines. During vaccine production, several mandatory in vivo quality control trials, such as potency, live virus, and safety, are responsible for the use of large numbers of laboratory animals. Over the years, global organizations encouraged the development of alternative methods to reduce, replace and refine the use of animals in the pharmaceutical industry. In this study we standardized an in vitro assay for determination of residual live virus combining viral isolation techniques with direct immunofluorescence detection and viral quantification by a molecular method. Standardization of viral recovery steps and quantification by RT-qPCR were performed and the combined method was shown to be 3 fold more sensitive than the in vivo assay. It was possible to identify viral suspensions cultures, which still had residual viable rabies virus particles, evidencing the importance to implement this method in quality control schemes of rabies vaccine production. In addition, this developed assay is more practical, inexpensive and less time consuming, producing results in just 4 days, which may allow greater agility in the internal quality control of the vaccine. The in vitro method may reduce 2/3rd of laboratory animals numbers used for this purpose, since it can be applied in the intermediate quality control of inactivated rabies vaccine production.

Immunogenicity and safety of a new Vero cell rabies vaccine produced using serum-free medium.

The immunogenicity and safety of a new human rabies vaccine, produced in Vero cells by a process that does not require supplementation with human or animal derived components in production, were assessed. Thus, the objective is to produce a safer vaccine at a lower cost. A total of 296 volunteers was divided into two groups: Group 1, which received the study vaccine, and Group 2, which received the Vero cells vaccine produced by Sanofi Pasteur. Five doses were given on days 0, 3, 7, 14 and 28. Blood samples for determination of rabies virus neutralizing antibodies were collected on days 0, 14, 38 and 90. The geometric mean titers (GMT) were much higher than 0.5 IU/ml in both groups on days 14, 38 and 90, indicating seroconversion according to the World Health Organization. In Group 1, however, the GMTs were higher than in Group 2, the difference being statistically significant in the two last samples. There was no statistical difference between the groups in the ratio of individuals with titers > or =0.5 IU/ml in each sample. Pain at the injection site was the most common adverse reaction and occurred most often in Group 1 (p < 0.001). All cases had a favorable evolution. There were no severe adverse reactions. It was concluded that the new vaccine is safe and immunogenic.

Evaluation of cytokines concentration and percentage of survival of rabies virus-infected mice submitted to anti-rabies Vero-cell propagated vaccine and P. acnes.

Previously, survival of rabies infection was shown to correlate with low IL-6 serum concentration in mice subjected to post-exposure treatment with the Fuenzalida Palacios rabies vaccine in conjunction with the immunomodulator Propionibacterium acnes, previously Corynebacterium parvum. Considering the substitution of the Fuenzalida Palacios rabies vaccine by the Vero cell raised anti-rabies vaccine in almost all countries, the objective of this work was to evaluate the survival and cytokine serum concentration of rabies virus-infected mice treated with P. acnes in conjunction with or the anti-rabies-VERO vaccine. For this, Swiss mice were experimentally infected with street rabies virus and subjected to vaccine and/or P. acnes following infection. Animals were killed at different times and serum was collected to evaluate cytokines. The greatest survival was observed in animals given one or two does of P. acnes in the absence of vaccination. Animals given anti-rabies VERO vaccine alone or with three doses of P. acnes had the second highest survival rate. The group that had the highest percentage of mortality also had the highest IL-6 concentration on the 10th day, a time correlating with clinical symptoms of the animals. The results reinforce the inefficacy of anti-rabies vaccine in only one dose as a post-exposure treatment irrespective of the type of vaccine used, the immunomodulation activity of P. acnes in rabies post-exposure treatment and suggest a role for IL-6 in rabies virus pathogenesis.

Distribution of rabies virus in infected mice, vaccinated and submitted to P. acnes as immunomodulator.

The lethality and distribution of rabies virus were evaluated in swiss mice experimentally infected with street rabies virus, vaccinated and submitted to immunomodulation by P .acnes (formerly Corynebacterium parvum). The animals were sacrificed at different times,when the different tissues were collected and submitted to fluorescent antibody test (FAT) and mouse inoculation test (MIT). The group submitted to vaccination and P. acnes treatment presented a percentage of survival superior to that observed in infected mice only treated with P. acnes. Control infected animals had the lowest survival rates. The distribution of rabies virus in spleen of infected mice, vaccinated and submitted to P. acnes was superior to that verified in infected mice not treated with P.acnes. The increased survival correlated with the distribution of rabies virus in lymphoid tissues, could be interpreted as the consequence of P. acnes activity on macrophages. The results suggest the role of macrophages against rabies virus infection in mice and the importance of vaccination in the post expositive treatment of rabies.

Effect of the contents and form of rabies glycoprotein on the potency of rabies vaccination in cattle.

One of the methods used for controlling cattle rabies in Brazil consists of vaccination. Sometimes, however, rabies occurs in cattle supposedly protected. Since rabies vaccine batches are officially controlled by tests performed on laboratory animals, it is questionable whether the minimal mandatory requirements really correspond to immunogenicity in the target species. We have analyzed the association among potencies of rabies vaccines tested by the NIH test, the contents and form (free-soluble or virus-attached) of rabies glycoprotein (G) in the vaccine batches, and the virus-neutralizing antibodies (VNA) titers elicited in cattle. No correlation was found between G contents in the vaccine batches and the NIH values, whatever the presentation of G. There was no correlation either between NIH values and VNA titers elicited in cattle. There was, however, a positive correlation (r = 0.8681; p = 0.0001) between the amounts of virion-attached G present in the vaccine batches and VNA elicited in cattle. This was not observed when the same analysis was performed with total-glycoprotein or free-soluble glycoprotein. The study demonstrated that NIH values can not predict the effect of the immunogen in cattle. On the other hand, the quantification of virus-attached rabies glycoprotein has a strong correlation with VNA elicited in cattle.

Evaluation of standard reagents for radial-immunodiffusion assays. In vitro control of rabies vaccines.

The RID assay is one of the in vitro methods used for in-process control in the production of rabies vaccines for veterinary use. It has been shown to be very useful for determining antigen concentration in the final bulk product. The work presented in this paper, including the production and standardization of candidate standard reagents for use in the Radial Immunodiffusion Assay (RID) was carried out at the Pan American Institute for Food Protection and Zoonoses (INPPAZ/PAHO/WHO). The study was completed with the cooperation of the Faculty of Veterinary Sciences, National University of La Plata (NULP), Argentina, where the validation of the proposed standards and the quality control of samples from 28 different batches of rabies vaccines produced with Pasteur strain rabies virus (PV) in BHK cells were performed. The activity of the vaccines was determined by in vivo (NIH) and in vitro (RID)assays. The results of the candidate reagents for the reagent standardization tests showed stability, sensitivity and reproducibility. The Relative Potency the 1.2 between the problem vaccines and the reference vaccine was estimated by variance and regression analysis. The results of our validation study show that the INPPAZ (PAHO/WHO) is capable of producing and distributing the above-mentioned standard reagents, as well as of providing support for the incorporation of the RID technique (sensitive, rapid and inexpensive) to the laboratories that manufacture rabies vaccines in Latin America and the Caribbean.

[Thermostability of rabies vaccine, type Fuenzalida & Palacios, for human use]. / Termoestabilidade da vacina contra a raiva, tipo Fuenzalida & Palacios, uso humano.

Ten lots of Fuenzalida & Palacios type antirabies vaccine for human use, produced at the Instituto Butantan (São Paulo, Brazil) were stored at temperatures of 45, 37, 28 and 2-8 degrees C. The potency of each lot was determined in samples taken at varied time intervals using the NIH method and lots presenting antigenic values > or = 0,3 were considered satisfactory for use. After 2 hours at 45 degrees C the antigenic value of one out of 10 lots tested was found to be less than the minimum required value. At 37 degrees C all lots maintained satisfactory antigenic values until the third day of storage, whilst at 28 and 2-8 degrees C the potency was fully maintained, respectively for 10 and 360 days. At the ideal temperature of 2-8 degrees C, 100% of the tested vaccines maintained the minimum required antigenicity for a longer period (16 months) than the expiration time of 6-12 months usually recommended for this type of biological produced in Latin American and Caribbean countries. Thus, the obtained data suggested that in countries still producing Fuenzalida & Palacios type vaccine, the expiration tim could be extended to 16 months, what could prevent the unnecessary discarding of products still in useful condition.

Rabies reference vaccine for use as a regional standard for Latin America and the Caribbean countries.

A candidate rabies reference vaccine of suckling mouse brain (SMB) origin was prepared and standardized at the Pan American Zoonoses Center (PAHO/WHO) and evaluated in a collaborative study involving seven laboratories. On the basis of three different tests, its potency, immunogenicity, and stability were demonstrated to be satisfactory. The vaccine was proposed for consideration of the Latin American and Caribbean countries as a regional standard to determine the potency of SMB vaccines, the most widely used in the Region.

Rabies reference vaccine for use as a regional standard for Latin America and the Caribbean countries

A candidate rabies reference vaccine of suckling mouse brain (SMB) origin was prepared and standardized at the Pan American Zoonoses Center (PAHO/WHO) and evaluated in a collaborative study involving seven laboratories. On the basis of three different tests, its potency, immunogenicity, and stability were demonstrated to be satisfactory. The vaccine was proposed for consideration of the Latin American and Caribbean countries as a regional standard to determine the potency of SMB vaccines, the most widely used in the region. (AU)

Origen y eliminación de elementos fibrosos observados en vacuna antirrábica CRL de uso humano / Origin and elimination of fibrous-like particles observed in rabies vaccine for human use

El presente trabajo describe la aparición de una alteración organoléptica observada en algunos lotes de vacuna antirrábica a cerebro de ratón lactante producidos en el Laboratorio Central de Salud Pública del Ministerio de Salud de la Provincia de Buenos Aires, consistente en la aparición de elementos de aspecto fibroso, blanquecinos, relativamente poco perceptibles, que se mantenían en suspensión en el líquido y no eran debidos a problemas de molienda. La técnica de producción seguida era la recomendada por los autores (Fuenzalida y Palacios), con las modificaciones propuestas por el Centro Panamericano de Zoonosis, que incluía una centrifugación bajo refrigeración a 17.000 g durante 10 minutos. La alteración descripta originaba una pérdida por descarte de dosis de vacuna del 20% o mayor y una relativa disminución de la potencia de las mismas. Los resultados obtenidos permiten concluir que durante el proceso de elaboración de vacuna antirrábica CRL se debe evitar la congelación del material nervioso en suspensión, recomendándose un rango de temperatura entre 3 y 5-C para todo el proceso

Origen y eliminación de elementos fibrosos observados en vacuna antirrábica CRL de uso humano / Origin and elimination of fibrous-like particles observed in rabies vaccine for human use

El presente trabajo describe la aparición de una alteración organoléptica observada en algunos lotes de vacuna antirrábica a cerebro de ratón lactante producidos en el Laboratorio Central de Salud Pública del Ministerio de Salud de la Provincia de Buenos Aires, consistente en la aparición de elementos de aspecto fibroso, blanquecinos, relativamente poco perceptibles, que se mantenían en suspensión en el líquido y no eran debidos a problemas de molienda. La técnica de producción seguida era la recomendada por los autores (Fuenzalida y Palacios), con las modificaciones propuestas por el Centro Panamericano de Zoonosis, que incluía una centrifugación bajo refrigeración a 17.000 g durante 10 minutos. La alteración descripta originaba una pérdida por descarte de dosis de vacuna del 20% o mayor y una relativa disminución de la potencia de las mismas. Los resultados obtenidos permiten concluir que durante el proceso de elaboración de vacuna antirrábica CRL se debe evitar la congelación del material nervioso en suspensión, recomendándose un rango de temperatura entre 3 y 5-C para todo el proceso (AU)

[Rabies vaccine inactivated with ethylenimine. Duration of the immunity in dogs]. / Vacuna antirrábica inactivada con etilenimina. Duración de immunidad en perros.

The duration-of-immunity afforded by the ethylenimine inactivated rabies vaccine produced in BHK cells with the PV strain, was studied in dogs. One hundred per cent of the dogs became serologically positive 7 days after vaccination; the same percentage was still positive 3 years after vaccination with one dose of the vaccine. A good correlation was observed between the antibody profiles as determined by the titers obtained with the mouse neutralization and fluorescent field inhibition techniques. The correlation was not as good when the number of international units per ml was determined by both tests. The best correspondence between serological response and resistance to challenge was observed when the antibodies were determined by the number of international units per ml, using the neutralization test in mice. All the dogs challenged 12 and 25 months after vaccination resisted challenge; 89% (8/9) were protected 36 months after vaccination. Inactivated vaccines can be as effective to control rabies as those prepared with modified live virus; moreover, the inactivated vaccines are more stable and safer than the latter.