ISRAEL JOURNAL OF
VETERINARY MEDICINE
Vol. 55(4) 2000
A WORKING CELL BANK OF BHK-21 CELLS
FOR RABIES VACCINE PRODUCTION IN CUBA
G. P. G—mez, E. A. Hernandez, O. F. Perera, A. Z. Fuentes and N. Rodr’guez.
Finlay Institute. Center for Research and Production of Sera and Vaccines. Ave. 27 no.19805,
La Coronela, La Lisa. A.p. 16017. Cod. 11600. Havana City, Cuba.
Abstract
The use of BHK-21 cells for producing foot and mouth disease and rabies
vaccines was proposed because of the significant advantages that this cell system
could provide. The objective of this work was the characterization of a BHK-21
working cell bank (WCB) to produce rabies vaccine in Cuba for veterinary use. The
cell bank was examined for freedom from adventitious agents. In vivo assays included
the inoculation of various systems such as guinea pigs, adult mice, suckling mice,
rabbits and embryonated chicken eggs. Animals were observed for 28 days post
inoculation. Sera were then collected and examined for different viruses. In vitro
safety testing was also performed in three test systems. Standardized culture methods
were employed to search for bacterial, fungal and mycoplasmal contaminants. The
cell bank was also examined for retroviruses by electron microscopy. Finally a nonspecific safety test was employed to verify that the cell bank components were not
allergic in the species tested. The cell bank was free of bacteria, fungi, mycoplasma,
haemadsorbent haemagglutinating and cytopathic viruses. It was showed to be free of
mouse viruses. Electron microscopic evaluation did not reveal particles resembling
type A or type C retroviruses. We conclude that the cell bank is safe for use for
producing veterinary vaccines.
BHK-21 cells; Working Cell Bank; haemadsorbtion; haemagglutination;
electron microscopy; adventitious agents, safety.
Key words:
Introduction
One of the main aims of the World Health Organization (WHO) and the International
Conference on Harmonization (ICH) is to reach international consensus on the basic
acceptance criteria for biological products. These include general and international
requirements for the production of different classes of biological substances such as
vaccines, interferon, hormones, blood and its derivatives.
The use of BHK-21 cell for producing foot and mouth disease vaccine and rabies vaccine was
proposed because of the significant advantages that a cell system could provide (1). The
origin and history of the cells are well known (2). Current vaccine production is based on a
well-defined seed lot system consisting of master, working and extended cell banks (3).
The objective of this work was the characterization of a BHK-21 working cell bank (WCB) to
produce rabies vaccine in Cuba. This is the first Cuban example of employment of a
continuos cell line for vaccine production.
Materials and Methods
Cells
WCB of BHK-21 cells to be tested. BHK-21 C13 (Baby Hamster Kidney) (kidney, Syrian or
golden hamster, Mesocricetus auratus). ATCC CCL-10.
Vero (Kidney, African green monkey, Cercopithecus aethiops). ATCC CCL-81.
MRC-5 (Lung, diploid, human) ATCC CCL-171.
Morphology
Optical and electron microscopy were carried out in an Olympus CK-2 BIP2 and a JEOL JEM
2000 EX transmission electron microscope respectively (4, 5).
Viability
Cell count by dye exclusion (6).
Sterility
The test was carried out in compliance with the United States Pharmacopeal Convention XXII
(7).
Adventitious contaminants
Vero, MRC-5 and BHK-21 cells were employed and daily observation was carried out for 14
days. The growth medium was 199, Eagle Diploid and GMEM (ICN Flow) respectively and
supplemented with 10% of fetal serum bovine (Gibco) The three systems were inoculated
with the lysates of 107 cells from the WCB (4,8-10).
Haemadsorption and haemagglutination tests were carried out at the end of the observation
period. A measles virus was used as a positive control (4,10,11).
Both techniques were performed using Hartley-Duncan guinea pigs, Lohman chicken and
African green monkey erythrocytes, specifically for the haemadsorption test, incubation
periods of 30 min at 3-40C and repeated after 30 min at 34-370 C according to Lee (12), at room
temperature after 30 additional min. (8,9,13).
Transmission electron microscopy was used to detect type A and type C retrovirus-like
particles.
Embryonated chicken eggs.
All embryonated Lohman chicken eggs were inoculated via yolk sac or allantoic cavity
and incubated at 370C, for 7 days. The eggs were candled daily. (10,11,14).
The fluids were collected at the end of the incubation period and tested for
haemagglutination with guinea pig, chicken and monkey erythrocytes at different
temperatures (40, 220 and 370C). The positive control was Influenza virus.
Animals
Suckling and adult mice: OF1 cells (106) were inoculated by intracerebral and intramuscular
routes.
Guinea pig (Hartley Duncan) and rabbits (F1) were inoculated by intramuscular route with
106 cells.
All animals, except suckling mice, were observed daily for 28 days and their body weighs
were checked each week.
The sera of adult mice were tested by immunofluorescence (IF) to detect LCM
(Lymphocytic choriomeningitis), MAD (mouse adenovirus), PVM (mouse pneumonia virus),
REO and Sendai virus and by ELISA for M. Pulmonis using a kit produced by the Center for
the Production of Laboratory Animals (CENPALAB). All techniques were carried out in
compliance with the Guide for the Care and Use of Laboratory Animals (15).
Results and Discussion
In Vitro assays
Traditional methods of evaluating the safety of the BHK-21 cell line have been employed in
this work.
As expected, the morphology of BHK cells was shown to be fibroblast-like. This is a
pseudodiploid line. The growth characteristics of thawed cells in an inoculum of 4 x 10 5 viable
cells per 25 cm2 flask containing 10 ml of growth medium produced 1,4 x 10 6 viable cells in 4
days, according to the ATCC, 1994 (2). The viability of the WCB was 94% by dye exclusion.
The growth of contaminating microorganisms such as bacteria, fungi and mycoplasmas was
not observed.
The results of inoculating Vero, BHK-21 and MRC-5 were negative. Cytopathic effect
(CPE) on inoculated and non-inoculated cells was not observed. The tests to detect
haemagglutination and haemadsorption were negative; these tests are according to Steur and
Ostrove (14). Thus the WCB was free from haemadsorbent, haemagglutinating and
cytopathic virus.
Retroviruses are the major example of an endogenous virus. Electron microscopy
evaluation did not reveal particles resembling type A or type C retroviruses. Such particles
have been previously observed in CHO-K1 cells (1,16). They are also not detectable by
electron microscopy in culture fluids, which were negatively stained after 15,000-fold
concentration, however, we recommend a test for viral reverse transcriptase.
In vivo assays
All embryonated chicken eggs used by both routes of inoculation remained alive during the
observation period. On the other hand, the presence of haemagglutinins in allantoic fluids and
yolk sac was not detected using erythrocytes from the species mentioned above at different
temperatures. There are convincing data to support that the WCB is free from myxoviruses
and arboviruses. These results are in accordance with the recommendation of Lee (12). As
expected, there was haemagglutinin in the positive control.
There has been no evidence of adverse effects in any animal after inoculation with cell culture
lysates and culture fluids in the three species tested and all the animals were still alive at the
end of the observation period.
Non-specific innocuousness.
The weights of the inoculated animals were higher than those of the control animals. The
animals of each species remained healthy and we could say that the WCB is free from
adventitious agents. In suckling mice no evidence of possible contamination of the test
material with Coxsakie viruses was found. In adult mice no evidence of possible
contamination of the test material with Lymphocytic choriomeninigitis virus was detected by
immunofluorecence (IF), and Reo 3, Sendai, mouse adenoviruses and pneumonia virus of
mice were not detected. In guinea pigs no evidence of clinical and pathological signs
demonstrating the presence of Mycobacterium tuberculosis was found and neither was
Lymphocytic choriomeninigitis virus detected by IF.
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