Higher production of rabies virus in serum-free medium cell cultures on microcarriers.

Rabies virus suspensions were obtained from VERO cells cultivated on solid microcarriers in a bioreactor after infection with the Pasteur rabies virus strain (PV). Virus production-serum free medium (VP-SFM) or Leibovitz 15 (L15) medium supplemented or not with fetal calf serum (FCS) were used to cultivate the VERO cells, before and after virus infection. The cell growth was shown to reach higher densities (1.6 x 10(6) cellsmol(-l)), when VP-SFM supplemented with 1% of FCS was used during the cell growth phase of culture, and then replaced by VP-SFM alone for the virus multiplication phase. In the cultures performed from the beginning with VP-SFM, lower densities accompanied by an altered cell morphology and detachment from the microcarriers were always observed. In rabies virus infected cultures, kinetic studies showed that higher virus yields (10(4.7) FFD(50) per 0.05 ml) were always obtained in cultures performed initially on VP-SFM supplemented with 1% FCS and after infection on VP-SFM alone. In agreement with that, rabies virus production, as measured by the average of virus titers in harvests obtained at different times after infection were shown to be 5.5 times higher in the cell cultures using initially VP-SFM+1%FCS and, following infection, VP-SFM alone. Besides the advantages of using media with a well-controlled composition, these data indicate the usefulness of serum free media also in terms of virus productivity.

Obtention of rabies antigen through BHK21 cells adhered to microcarriers.

Four rabies antigen batches were produced from virus suspensions resulting from BHK21 cells adhered to microcarriers (Cytodex 1), inoculated and cultured in a bioreactor. In parallel the methodology of production of rabies virus through cultures of BHK21 cells in monolayers in bottles was used. The results obtained showed that infecting titles were 10(6.69) DL50/mL and 10(7.28) DL50/mL for suspensions cultured in bottles and in the bioreactor, respectively. The viral suspension volumes collected were on average 11,900 per batch from the bioreactor and 800mL per bottle. Ten horses were immunized with the antigen produced in the bioreactor. The means of antirabies antibody titers found were 240 and 212 IU/mL after the initial and the first booster doses, respectively. Rabies antigen with satisfactory infecting titers can be obtained on a large scale by culturing in a bioreactor inoculated BHK21 cells adhered to microcarriers.

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.

The preparation of cultured rabies virus and the production of antiserum for human use.

In this paper we describe a methodology for the preparation of the Pasteur strain of fixed rabies virus in BHK-21 clone 13 cells and also its use for the production of antisera in horses. The methodology showed here is simple, rapid, facilitates the attainment of high protective titers, and the antisera produced are of high quality.