Preparation of human rabies vaccine in VERO cell culture using a microcarrier system.

1. The rabies virus (Pasteur PV strain) was propagated in VERO cells attached to microcarriers in a 3.7-1 bioreactor. Virus titers of about 10(6) LD50/ml were obtained regularly. 2. Ultrafiltration was efficient for concentrating the virus suspensions, and the sucrose gradient reduced the residual VERO cell DNA to acceptable levels (less than 50 pg/dose). The remaining cell DNA content was evaluated by dot-blot hybridization with a probe prepared with VERO cell DNA. 3. The final virus preparations were inactivated by B-propiolactone treatment, showed a potency higher than 2.5 IU/dose and protected mice experimentally infected intracerebrally with rabies virus (CVS-13.2). 4. This methodology for the production of a rabies vaccine for human use should be of interest to countries where high technology facilities are not available.

[Immunofluorescence performed in brain of mice, infected with the CVS strain of the rabies virus, in different stages of decomposition]. / Imunofluorescência realizada em cérebros de camundongos infectados com vírus rábico--cepa CVS, em diferentes estágios de decomposição.

The efficiency of the fluorescent antibody (FA) test in detecting rabies virus antigen in decomposed specimens was evaluated in simulated conditions of the safety test recommended for the assessment of residual virus in inactivated rabies vaccines. The CVS-infected mice were submitted to different treatments combining time and temperature in order to cause different stages of carcass decomposition and, the FA test was carried out sequentially at pre-determined time intervals. For the materials stored at 25 degrees C, greater difficulties for prompt recognition of the inclusion bodies were found after 12-18h, whilst the specimens maintained at 4 degrees C, the inclusions were easily visualized for up to 48h. Brain smears of carcasses kept at -20 degrees C were suitable for adequate identification after 720 h of storage. In carcasses that had been maintained at 25 degrees C for 10 h with additional storage at 4 or -20 degrees C, rabies antigenicity could not be detected, respectively after 10 and 24 h, due to tissue decomposition. The authors recommend that the FA test, when applied as an additional tool for the control of the safety test of inactivated rabies vaccine using mice, care must be taken in order to avoid the use of decomposed materials.

[Quality of the reagents used in counterimmunoelectrophoresis technique for the determination of antirabies antibodies]. / Evaluación de la calidad de los reactivos que se utilizan en la tecnica de contrainmunoelectroforesis para la determinación de anticuerpos antirrábicos.

This study demonstrated that the antigens and indicator sera produced by the Butantan Institute may be employed with success in the counterimmunoelectrophoresis technique for the titration of rabies antibodies in sera from immunized individuals. No statistically significant differences were demonstrated between the results obtained in the standardization tests carried out at the Butantan Institute and the reference control tests performed at the Pan American Zoonoses Center. It is proposed that the Butantan Institute be in charge of the production and distribution of these reagents at the national level.

Hyperimmune antirabies sera titration by standard mouse neutralization and counterimmunoelectrophoresis tests, comparing results of different laboratories.

To determine the rabies antibody level of twenty-four hyperimmune equine sera, Standard Mouse Neutralization (SMN) and Couterimmunoelectrophoresis (CIE) tests were carried out, both at the Instituto Butantan (IB) and Instituto Panamericano de Proteccíon de Alimentos y Zoonosis (INPPAZ). Statistical analysis has shown a correlation (r) of 0.9317 between the SMN and CIE performed at the IB, while at the INPPAZ it scored 0.974. Comparison of CIE data of both laboratories yielded a correlation of 0.845. The CIE technique has shown to be a sensitive and efficient as the SMN in titrating antirabies hyperimmune equine sera. Based on CIE results, a simple, rapid and inexpensive technique, titers of sera antibody can be rellably estimated in SMN test.

[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.

Reduced schedule of human antirabies immunization with Fuenzalida & Palacios vaccine.

The present study evaluates the humoral and cellular immune responses in 35 volunteers submitted to short antirabies vaccination schedules with the Fuenzalida & Palacios vaccine based on the administration of doses on non consecutive days. The volunteers were divided into two groups. The first group received a total number of five doses given on days 0, 4, 7, 20 and 35. The other group received four doses, the first one being a double dose given on day 0 and than three other single doses on days 7, 20 and 35. The evaluation of humoral immune response was carried out by serum neutralization (SN) and indirect immunofluorescence (IIF) tests, while the cellular immune response was evaluated by lymphoblastic transformation assay (LTA) and skin test (ST). According to our results these reduced schedules elicited early and effective humoral and cellular immune responses to rabies antigen suggesting that new reduced schedules should be extensively studied in order to give the proper bases to the proposition of changes in the current long-term schedule.

Evaluacion de la calidad de los reactivos que se utilizan en la tecnica de contrainmunoelectroforesis para la determinacion de anticuerpos antirrabicos / Evaluation of the quality of the reagents employed in the counterimmunoelectrophoresis technique for the determination of rabies antibodies

En este estudio se comprobo que el Instituto Butantan produce antigenos y sueros indicadores que se pueden utilizar con exito en la prueba de contrainmunoelectroforesis para titular anticuerpos antirrabicos en personas inmunizadas. No se pudieron demostrar diferencias estadisticamente significativas entre los resultados de las pruebas de estandarizacion realizadas en el Instituto Butantan y las pruebas de control de referencia llevadas a cabo en el Centro Panamericano de Zoonosis. Se propone que el Instituto Butantan produzca y distribuya a nivel nacional los reactivos para que los laboratorios de diagnostico apliquen la tecnica de contrainmunoelectroforesis para la determinacion de anticuerpos antirrabicos.

Imunofluorescencia realizada em cerebros de camundongos infectados com virus rabico-cepa CVS, em diferentes estagios de decomposicao / Immunofluorescence performed in brain of rabies virus infected mice - CVS cepa, at different stages of decomposition

O teste de imunofluorescencia (IF) foi avaliado na deteccao de virus rabico presente em cerebros de carcacas de camundongos infectados com virus da cepa CVS, os quais foram conseguidos atraves de uma combinacao de tratamentos, em que se variaram as temperaturas (4,25 e -20 graus Celsius) e o tempo de armazenamento. No teste de IF realizado com impressoes cerebrais de carcacas que haviam sido submetidas a temperatura de 25 graus Celsius por 12-18h, houve maior dificuldade de visualizacao imediata dos corpusculos de inclusao, enquanto que nos materiais conservados a 4 graus Celsius por ate 48h, as inclusoes foram facilmente reconhecidas. Carcacas mantidas a -20 graus Celsius mantiveram-se viaveis a identificacao pela IF mesmo apos terem sido armazenadas por 720h quando foram feitas as ultimas observacoes. Em carcacas mantidas a 25 graus Celsius por 10h, com tratamento posterior a 4 e -20 graus Celsius, o antigeno rabico nao pode ser identificado atraves da IF, em consequencia da decomposicao das carcacas que ocorrem, respectivamente, apos 10 e 24h. Recomenda-se, portanto, empregar o teste de IF, em carater de rotina, no controle de qualidade da vacina contra a Raiva, no que diz respeito a prova de virus residual (teste de verificacao da inativacao viral), de vez que ele permite esclarecer mortes assintomaticas...

Preparation of human rabies vaccine in VERO cell culture using a microcarrier system

1. The rabies virus (Pasteur PV strain) was propagated in VERO cells attached to microcarriers in a 3.7-1 bioreactor. Virus titers of about 10(6) LD50/ml were obtained regularly. 2. Ultrafiltration was efficient for concentrating the virus suspensions, and the sucrose gradient reduced the residual VERO cell DNA to acceptable levels (less than 50 pg/dose). The remaining cell DNA content was evaluated by dot-blot hybridization with a probe prepared with VERO cell DNA. 3. The final virus preparations were inactivated by B-propiolactone treatment, showed a potency higher than 2.5 IU/dose and protected mice experimentally infected intracerebrally with rabies virus (CVS-13.2). 4. This methodology for the production of a rabies vaccine for human use should be of interest to countries where high technology facilities are not available