Growth of hepatitus A virus in vero cells

Hepatitus A virus is grown in Vero cells after passage in in vitro cell culture. Virus replication and continued passage in Vero cells requires extended incubation times, up to about four weeks, for early passages and incubation temperatures no higher than about 32.degree. C. Continued passage results in a significant decrease in incubation time and an increase in virus yield. Cultivation of hepatitus A virus in Vero cells meets the development requirements for an inactivated human vaccine.

BACKGROUND OF THE INVENTION 
In vitro growth of hepatitis A virus was first accomplished with virus 
inocula prepared by prior in vivo passage of hepatitis A virus in certain 
sub-human primates, Provost et al., U.S. Pat. No. 4,164,566. Subsequently, 
a procedure was described for the growth of hepatitis A virus in vitro by 
direct inoculation of cell cultures with human clinical specimens 
containing the hepatitis A virus, Provost et al., European patent 
application No. 025745. Hepatitis A virus isolates may be initiated to in 
vitro growth in cell culture by either of the described techniques. The in 
vitro cultured hepatitis A virus can then be adapted to growth in several 
types of cell cultures by serial passages. Such adaptive passaging results 
in increased hepatitis A virus yields which are absolutely necessary for 
the development of inactivated viral vaccines. In vitro growth of 
hepatitis A virus has allowed the adaptation of the virus to growth in 
cell lines which are acceptable for the production of vaccines, e.g., 
human fetal diploid lung cells and MRC-5 cells. The MRC-5 cells are 
suitable for the production of live hepatitis A virus vaccine but are less 
suitable for inactivated vaccine production. The Vero cell line is more 
advantageous for the growth of virus in the quantities necessary for 
inactivated vaccine production. Vero cells are transformed but 
non-tumorigenic cells derived from cercopithicus monkey kidney, Yasumura 
et al., Nippon Rinsko 21:1201-1215 (1963). Vero cells are available in 
larger quantities than are MRC-5 cells and are more readily adaptable to 
large scale cell culture techniques than are MRC-5 cells. 
Attempts to grow hepatitis A virus in Vero cell cultures under conventional 
growth conditions, e.g., cultivation at about 37.degree. C., have resulted 
in little or no production of virus particles, Locarnini et al., J. 
Virology 37:216-225 (1981). The ability to propagate hepatitis A virus in 
Vero cells would greatly enhance the development of an inactivated vaccine 
since Vero cells are available in sufficient quantity for mass culture, 
are considered an acceptable cell substrate for inactivated vaccine 
production and are technologically advantageous when compared with other 
acceptable cell lines. 
OBJECTS OF THE INVENTION 
It is, accordingly, an object of the present invention to provide a 
commercially feasible method for the large-scale propagation of hepatitis 
A virus suitable for inactivated vaccine development. Another object is to 
provide conditions for the successful cultivation of hepatitis A virus in 
Vero cell cultures. These and other objects of the present invention will 
be apparent from the following description. 
SUMMARY OF THE INVENTION 
Hepatitis A virus is grown in Vero cells after passage in in vitro cell 
culture. Virus replication and continued passage in Vero cells requires 
extended incubation times, up to about four weeks, for early passages and 
incubation temperatures no higher than about 32.degree. C. Continued 
passage results in a significant decrease in incubation time and an 
increase in virus yield. Cultivation of hepatitis A virus in Vero cells 
meets the development requirements for human vaccine.

DETAILED DESCRIPTION 
The present invention relates to a commercially feasible method for the 
propagation of hepatitis A virus in continuously cultured Vero cells. The 
Vero cell line was derived from transformed cercopithecus monkey kidney 
tissue and has become an acceptable cell line for human vaccine 
production. 
It has now been shown that hepatitis A virus can be grown in Vero cell 
cultures. Propagation in Vero cells requires a suitable viral inoculum to 
initiate infection of the Vero cells. The suitable inoculum may include 
the product of successful infection and growth of the hepatitis A virus in 
cell cultures, e.g., low passage, normal fetal rhesus monkey kidney-6 
cells or transformed rhesus monkey kidney cells, LLC-MK2, at either about 
32.degree. C. or about 35.degree. C., or it may be virus-containing 
clinical specimens, e.g. blood, liver, stool, derived from infected humans 
or sub-human primates. The Vero cells are used as confluent cell 
monolayers and may be prepared from a wide range of cell passage levels, 
e.g., from about 100 to about 200 passages. Any suitable nutrient medium 
may be used, for example, Eagle's minimal essential medium containing 
Earle's salts supplemented with about 0.2% to about 0.5% fetal calf serum. 
Hepatitis A virus inoculum is prepared by freeze-thawing and sonication of 
cell monolayers to form an infective homogenate. 
Typically about 1 part of the homogenized virus infected culture is used as 
inoculum for from about 100 to about 1000 parts by volume of new cell 
culture. The inoculated cultures are incubated in an atmosphere of about 
5% CO.sub.2 in air and at a temperature no higher than about 32.degree. 
C., typically at from about 29.degree. C. to about 33.degree. C., with 
about 32.degree. C. being preferred. Hepatitis A virus infected Vero cell 
monolayers receive fresh nutrient media with fetal calf serum 
approximately every fifth day following inoculation. All monolayers are 
periodically fixed and examined for the presence of hepatitis A viral 
antigen by direct immunofluorescence. Infected cell cultures, cell 
monolayers plus nutrient medium, are homogenized by freezing-thawing and 
sonication and examined for antigen by radioimmuno assay. Early passages 
of the virus require extended incubation times, about 4 weeks, for the 
development of weakly positive results as determined by both assays. 
Hepatitis A virus yields from Vero cells grown in 850 cm.sup.2 roller 
bottles containing about 200 ml of nutrient medium are estimated to be 
about 20 to about 50 micrograms. When suitably purified and inactivated 
the antigenic material can be used as a vaccine. In marmoset studies, it 
has been shown that quantities of formalin-inactivated hepatitis A viral 
antigen as low as 3.times.1 nanogram can immunize against hepatitis A 
virus infection. Consequently, about 1000 such doses can be prepared from 
a single roller bottle of Vero cell culture. 
Other cell cultures, e.g. low passage fetal rhesus monkey kidney cells, 
primary African green monkey kidney cells, transformed rhesus monkey 
kidney cells (LLC-MK2), and human diploid lung fibroblast cells (MRC-5, 
Wl-38) can all support growth of hepatitis A virus. Yields of hepatitis A 
virus antigen grown on these cell cultures approximate the yields 
described for the virus maintained on Vero cells. The Vero cell culture 
system is unique in that it requires incubation at temperatures no higher 
than about 32.degree. C. None of the other potential hepatitis A virus 
substrates have this temperature restriction on viral growth. Also the 
Vero cell culture system is unique among transformed cells in that is has 
been accepted as a human vaccine substrate. Thus, the Vero cell line is 
preferable to other transformed cells such as LLC-MK2. Transformed cells 
such as the Vero cell line have an infinite lifetime and will always be 
adequate for large-scale vaccine manufacture. This is the main advantage 
of the Vero cells over conventional cells such as MRC-5. 
EXAMPLE 1 
Adaptation of Hepatitis A Virus to Growth in Vero Cells 
Hepatitis A virus was isolated and grown in marmosets prior to adaptation 
to cell culture. Initially, fetal rhesus monkey kidney-6 (FRhK-6) cell 
culture monolayers were inoculated with hepatitis A virus obtained from an 
extract of infectious marmoset liver. The homogenized liver was clarified 
and inoculated, generally 1 part liver extract to from about 20 to 1000 
parts by volume of fresh culture fluid to obtain a viral concentration of 
2.5.times.10.sup.5 infectious dose units per ml. The infected cell 
cultures were grown at 35.degree. C. in 25 cm.sup.2 culture flasks 
containing 5 ml of a nutrient medium consisting of Eagle's minimal 
essential media supplemented with 0.5% fetal calf serum. The nutrient 
medium was replaced at 5 day intervals. Viral growth was shown by various 
assays for the presence of viral antigens and by the ability to serially 
passage the viral agent in FRhK-6 cells. Hepatitis A virus for antigen 
assay and as an inoculum for subsequent cell cultures was released from 
the cultured cells by twice freezing and thawing the contents of the 
culture flask and by sonication. This homogenized product was clarified by 
centrifugation and the supernatant liquid was used to determine viral 
antigen concentration and as the hepatitis A virus inoculum. A virus 
homogenate prepared from the eighth serial passage in FRhK-6 cells was 
used to inoculate LLC-MK2 cells, transformed rhesus monkey kidney, 1 part 
homogenized cell culture by volume to 20 to 1000 parts Eagle's minimal 
essential medium by volume to obtain a viral concentration of 
2.5.times.10.sup.5 infectious dose units per ml. The infected LLC-MK2 cell 
cultures were maintained under the same conditions as those used for 
infected FRhK-6 cells except that the fetal calf serum concentration was 
lowered to 0.2%. Successful growth of hepatitis A virus in LLC-MK2 cell 
cultures was also demonstrated by various assays for hepatitis A anigen 
and by the ability to successfully serially transmit the viral infection 
in LLC-MK2 cells. 
Vero cell cultures were inoculated with hepatitis A virus from the fifth 
serial passage in LLC-MK2 cells. The infected Vero cell cultures received 
1 part homogenized cell culture by volume to about 20 to 1000 parts by 
volume of fresh Eagle's minimal essential medium supplemented with 0.5% 
fetal calf serum to obtain a viral concentration of 2.5.times.10.sup.5 
infectious dose units per ml. The initial cultures were incubated at 
35.degree. C. in an atmosphere of 5% CO.sub.2 in air. Viral growth was 
severely limited at this temperature and the infection could not be 
transmitted to other Vero cell cultures at 35.degree. C. When similarly 
infected Vero cell cultures were incubated at 32.degree. C. viral antigens 
were detected and infective virus could be passaged. Incubation periods 
were reduced over the course of about 10 passages from 4 weeks to 2 weeks 
while viral antigen yields were increased from a trace to about 100 
nanograms of hepatitis A virus antigen per millileter of homogenized cell 
culture. 
EXAMPLE 2 
Large-Scale Growth of Hepatitis A Virus in Vero Cells 
Vero cells were grown to confluency in roller bottles, 850 cm.sup.2, under 
the conditions described in Example 1. The culture fluid was removed and 
the Vero cell monolayers were inoculated with 15 ml of a 1 to 75 dilution 
of the homogenized virus product, containing about 20 nanograms of viral 
antigen, from the eighteenth passage of hepatitis A virus in Vero cells as 
described in Example 1. The inoculum in fresh nutrient medium was absorbed 
to the Vero cell culture for about 4 hours at 32.degree. C. and then 185 
ml of nutrient medium was added and the roller bottle cultures were 
incubated at 32.degree. C. The nutrient medium, 200 ml, was replaced at 5 
day intervals. The cultures were terminated at day 21 by the 
homogenization process as in Example 1. In an alternate experiment 
hepatitis A virus was obtained by discarding the culture fluids and 
collecting the cell monolayer by scraping and subsequent homogenization. 
Typical estimates of hepatitis A viral antigen yields per roller bottle 
culture were about 50 micrograms, representing a 2500-fold increase in 
viral product over inoculated viral antigen.