CA2205677A1 - Novel replication process - Google Patents

Abstract

The proposed process permits replication of human influenza virus at a low multiplicity of infection in a Vero cell line by maintaining a trypsin concentration of at least 0.05 µg/ml of the culture medium throughout the growth cycle.

Description

, . ; - CA 02205677 1997-05-16 -- -DESCRIPTION

NOVEL REpLTcAl~IoN PROCESS

~echnical Field This invention relates to a process for viral replication in m~mm~lian cells and particularly viral replication of human influenza virus in Vero cell culture~

Background Art The major influenza virus glycoprotein, hemagglutinin (HA), is synthesized in infected cells as a single polypeptide. Post-LO translational cleavage of the HA forms two subunits, HA1 and HA2,joined by a disulfide bond. Cleavage is essential to the production of infectious virus; virions cont~;n;ng uncleaved HA are non-infectious. The process can occur intracellularly or extracellularly. The HAS of human" swine, and most avian influenza L5 virus strains cannot b~e cleaved by ubiquitous intracellular proteases. Therefore, replication of these viruses in cell culture require, the addition of trypsin to the maintenance medium to - ensure HA cleavage thereby permitting activation of the progeny virus so that the infection can proceed.

For the past several decades" fertilized chicken eggs have been us,ed to produce influenza virus in large quantities. Killed influenza vaccines are purified from virus-cont~;n;ng chick embryo allantoic fluid. However, a large body of data now suggests that this is not an ideal system. Even a single passage of a human ~5 influenza virus isolate in eggs can lead to the selection of variants that differ in their antigenic specificity from the original virus. By contrast, viruses isolated and passaged exclusively in m~mm~lian cell cultures fully retain their antigenic characteristics, a feature that would prove highly advantageous in vaccine production. However, the cell lines routinely used in laboratory studies, including the favored line, Madin-Darby canine kidney (MDCK) cells, have not been certified for virus vaccine , . -~ . CA 02205677 1997-05-16 ' production.

In contrast to influenza A and B viruses grown in eggs, those isolated in ~alian host cells possess structurally homogenous hemagglutinin molecules (Has) that are identical to the predo~;n~nt Has of the original clinical isolate [Katz et al, Virology, Vol.
165 (~988), pp. 446-456; Rober1son et al, Virology, Vol. 179 (1990), pp. 35-40]. Moreover, influenza viruses grown in mammalian cells elicit neutralizing and hemagglutinin inhi~ition (HI) antibodies in human sera more readily and at higher titers than do their egg-grown counterparts. An experimentally inactivated influenza virus grown in Madin-D~rbin canine kidney (MDCK) celis introduced higher HI in neutralizing antibody titers than did egg-grown counterpart virus, and provided superior protection of ferrets against subsequent challenge with infectious virus grown in either MDCK cells or embryonated eggs tKatz et al, J. Infect.
Dis., Vol. 160 (1989), pp. 191-198; Wood et al, Virology, Vol. 171 (1989), pp. 214-221]. These observations underscore the need for a m~mm~l ian cell line that could be used to replace chicken eggs in the production of influenza virus vaccines and diagnostic reagents. ~mm~lian celi grown virus may also have advantages for easier virus purification.

Influenza viruses can be propagated in several types of primary cell cultures including chick embryo kidney, chick embryo lungs, monkey kidney, canine kidney, bovine kidney, chick kidney, guinea pig kidney, and chick embryo ibroblasts. Kowever, primary tissue cultures are unlikely to be useful as a substrate for vaccine production for several reasons, including cont~m;n~tion by various endogenous agents, the variable quality of the cells, different sensitivities to variants of the same virus, and, of course, high cost and difficulties in obtaining and preparing the tissue cultures. Diploid tissue cultures, such as WI-38, have been used to produce vaccines against poliomyelitis, adenovirus types 4 and 7, rubella, measles, and rabies viruses. Although human diploid (MRC-5) cells can support the growth of influenza viruses, such systems have stringent growth media requirements and are expensive to maintain, making them suboptimal for large-scale production of vaccines.

-. CA 02205677 1997-05-16 ' -~

Disclosure of the Invention This invention o~ a process ~~or ensuring replication of human influenza virus at a low multiplicity of infection in a mammalian cell line involves maint~in;ng a consistent m;n;~um concentration of trypsin (about 0.05 ~g/ml) in the culture medium.

Vero cells are sensitive to a spectrum of viruses, including:
entero~iruses, measles and parainfluenza viruses, herpes viruses, andenoviruses, rhabdoviruses and some arboviruses. Low-passage-number Vero cells lack tumorigeni~_ity, do not contain adventitious viruses and can support efficien1_ proli~eration of many types of viruses. This ceIl line has been used successfully for the producl_ion of vaccines against poliomyelitis and rabies. The Vero cell line is suitable for culti~ation of infectious influenza A
viruses and for primary isola,tion of currently circulating influenza A (H3N2) strains. The data in the examples on the adaptal_ion of influenza A/EnglancL/l/53 (HlNl) [HG] strain to Vero cell culture, its growth characteristics and antigenic stability, and the likelihood of obtaining high yields of viral proteins with Vero i; compared to MDCK~cells.

The first attempts to obtain high yields at low multiplicities of infection in m~mr~1;an ce]l line certified for vaccine production [Vero (WH0), a subline of African green-monkey kidney cells] were unsuccessful. Subsequent studies to identify the cause(~s) of this failure implicaled loss of trypsin from the cell maintenance medium. When infected with influenza A virus at a multiplicity of at least 0.005 TCIDso per cell, Vero (WH0) cells produced yields of virus comparable to those produced in Madin-Darby canine kidney (MDCK) cells. However, at lower multiplicities of infection, multicycle growth was blocked early in the course of infection, the progress of the cytopathic effect was stopped, and the final virus yields were low.

To test the possibility that loss of trypsin activity was responsible for the observed effect, we used a sensitive fluorogenic substrate to measure this activity in the culture fluid of Vero (WH0) cells. The results indicated a rapid decrease of __. CA 02205677 1997-05-16 ~

trypsin activity. Similar findings were made with MDCK, rhesus monkey kidney LLC-MK2, and swine]cidney cells although the rates of decrease were much slower than iII Vero (WHO) cells. The causative role of trypsin was verified in experiments in which repeated addit:ion of the enzyme to cell cultures restored multicycle virus growth and permitted high virus yields to be obtained at a low ~~
multiplicity of infection.

'rests showed that trypsin concentrations of at least 0.05 ~g/ml in the cell culture were essential for securing high virus yields and that a concentration cf about 0.l ~g/m~ was optimal when the multiplicity of infection ranged from about l x 10-5 and l x l0 TCID50 per cell; satisfactory results were obtained at about 5 x l0-~TCIDso per cell. Thus, trypsin had to be maintained from a~out 0.0 to 0.5 ~g/ml to secure adequate yields of virus.

A higher volume of maintenance medium per area of cell monol~yer also required slight improvement of multicycle virus growth at low input doses, most likely because of a lower concentration of trypsin-inactivating factor in the medium. It seems that efficient replication of virus in MDCk cells is possible becau~e of a relatively slow rate of trypsin inactivation. The level of trypsin activity necessary for efficient HA cleavage, to the extent of ensuring multicyclle virus growth, is much lower than the initial trypsin concentration in the maintenance medium, so that the infection proceeds even though the trypsin activity decreases, provided the decrease! in not so rapid as in Vero (WHO) cells.

It might prove useful, however, to supplement even MDCk cell cultures with trypsin, particularly in situations where small volumes of medium are used with large amounts of cells, such as roller cultures, etc. At the very least, trypsin activity in culture fluid should be monitored routinely.

The nature of the fac1_or(s) responsible for trypsin inactivation in cell cultures is not known. Once it is secreted into the medium and collects there, it rapidly inactivates trypsin.
The kinetics of trypsin inactivation in cell cultures appears to . ~ CA 02205677 1997-05-16 - -_ reflect the accumulation of the inhibitory factor rather than its interaction with trypsin.

Passage of the trypsin-inactivating factor through a series of gracled filters indicated a molecular mass very close to 100 kilodaltons (kDa). Alternately, the protein may consist of two fractions, one larger than 100 Kda and the other between about 50 and about 100 (Kda). Of the many inhibitors of serine proteinases that bLock the cleavage of low molecular weight substrates by trypsin only, very few have a molecular mass as high as the one ~o estimat:ed for our factor. Inter-a-trypsin inhibitor (ITI) of human plasma is represented by a native molecule of 180 Kda as well as a species of lower molecular mass that retains inhibitory activity.
Relatecl inhibitors were detected in baboon plasma. If, as suggest:ed by our molecular weight estimates, the trypsin-L5 inactivating factor in cell cu]tures belongs to the class ofproteins that inhibit pro~;n~es, our findings of a putative inhibit:or of trypsin activity may be of value in the studies of serine proteinase inhibitors. The inhibitors of this family, although numerous and extensively studied, are mostly derived from such substances as plants, bovine pancreas, human and ~";m~l plasma" tissues of invertebrate species, etc. For this reason, their structure in enzymological properties are far better known than their biosynthesis, intracellular transport and secretion me~h~ni ~r. Thus, inhibitors procluced by cultured cells may prove to be a valuable asset. In the best mode for carrying out the invention, there are described ;everal preferred embo~;~ents to illustrate the invention. However, it is to be understood that the invention is not intended to be limited to the specific embodiments contained therein.

Best Mode for Carryin~ Out the Invention The following materials and methods were used.

Example 1 Cells:

The Vero (WH0) cell line, deposit no. 1297, was obtained from ._ CA 02205677 l997-05-l6 --~ ~

the American Type Tissue Collection at the level of the 134th passage. The cells were cultivated as monolayers in Falcon Labware 250 C~3 flasks at 37~C and 5~ Co2 in a growth medium of Eagles m;ni~l essential medium (MEM) supplemented with lO% unheated fetal calf serum. For the growth of Madin-Darby canine kidney (MDCK) cells and rhesus monkey kidney (L]~C-MK2) cells, the medium used was MEM with ~ fetal calf serum heated 30 minutes at 56~C. For the cultivation of swine kidney cell line (SwK), RPMI 1640 medium was used with 5% heated fetal calf serum. For the experiments involving infection or mock-in~ection, the cells were grown either in 50 cm3 flasks or in 6-well, 24-well, and 96-well plates (Falcon Labware). Cell monolayers were washed three times with PBS and overlaid with maintenance medium. The latter had the same composition as the growth mediu1m for each cell line, the serum being omitted and 0.3% bovine serum albumin (BSA) added. Unless otherw~ise stated, the maintenance medium contained TPCK-trypsin (Worthington) at l.0 ~g/ml. Plaque assays were performed with TPCK-treated trypsin t2.5 ~g/ml).

Viruses:

~rero (WH0)-adapted influenza A/England/l/53 (HlNl) [HG~, A/FW/~/50 (HlNl), and A/Aichi/2/68-PR/8/34 (H3N2) [X-31] viruses were used. The viruses were pa~;saged 5 times in Vero (WH0) cell cultures, and the final stock virus preparations contained about 1073 to 108~ TCID50/O.2 ml and àbout 32 to about 128 HAU. In the pre1;min~ry experimen~s, the Vero ~WH0)-adapted A/Rome/49 (HlNl) strain was used (1067 TCID50/O.2 mil, about 15 to about 32 HAU). HA
and infectivity titration were performed essentially as described in "Advanced Laboratory Techniques for Influenza Diagnosis"
~ImmuI1ol. Ser. 6, pp. 51-57 (l975)~. HA titrations were done in mirot:iter plates. Infectivity was measured by an end point titra1_ion technique in MDCK cells grown in 96-well plates with CPE
evaluation at 72h postinfection.

Asses,ment of TryPsin Activity:

A highly sensi1_ive assay of trypsin activity based on application of a fluorogenic substrate, BAAMC (Na-benzoyl-L-CA 02205677 1997-05-16 . _ arginine-7-amido-4-methylcoumarin-hydro chloride; Sigma) was used.
The sub,trate was dissolved to a final concentration of about 0.2 Mm in a buffer containing 50 Mm of ~ris-Hcl, Ph about 8.0, lO Mm CaCl~ and 1% DMS0. A O.l ml sample of trypsin-cont~;n;ng cell culture fluid was added to about 0.9 ml of BAAMC solution and incubat,ed at 37OC for l hour. The samples were placed on ice and assayed in a Perkin-Elmer MPF-44B fluorescence spectrophotometer at activation and emission wavelengths of about 380 and 460 nm, respectively.

RES~,TS

Inefficient MulticYcle RePlication of Influenza in Vero (WH0) Cells:

In the attempts to passage influenza A virus in Vero (WH0) cells, dif~iculties were encountered in the production of high L5 virus yields using low multiplicities of infection (m.o.i.). When the cells were grown in 50 cm3 flasks, the m.o.i. had to be at least 0.005 TCIDsO/cell to produce m~ yields--a concentration that would be impractical for use in vaccine production. At lower input doses, the tiers were low or the viLrus failed to accumulate at all.
~0 In mosfc instances, the accumulation of virus in the cultures infecte;d at the low m.o.i. stopped after 48 hours postinfection.
The progress of the cytopathic effect also ceased. This pattern, however, occurred to a differen1_ extent in different kinds of plasticware; it was strongly expressed in 50 cm3 flasks, less strongly in 6-well plates, and even less in 24-well plates and practic:ally not at all in 96-wel] plates. The mode of infection and incubation in the experiments with all kinds of plasticware was identic:al. The only difference was the volume of maintenance medium per unit of monolayer, tha~: is, the amount of culture fluid per cell. This dependence o~ the final yields on the input dose was not observed in MDCK cells, irrespective of the plasticware used. An example of the multiplicity dependence of the final yields is presented in an expe!riment with Vero (WH0)-adapted influenza A/Rome/49 (HlNl) virus strain (Table l).

CA 02205677 1997-05-16 ~ __~

Restoration of Multicycle virus Growth bY Repeated Addition of Trypsin:

To verify the abrogation of in~luenza virus accumulation in Vero cell cultures was due to the loss of trypsin actiYity in the culture medium, several experimen1:s were performed in which trypsin concentration was restored in the course of infection by repeated~--additions of trypsin to the culture medium. This procedure led to an increase of the virus production in the cultures inf ected with low input doses, thus ensuring high final yields irrespective of the multiplicity of infection. l'he 'effect was especially evident in 50 cm3 flasks (Table 2) and 6-well plates with dense confluent monolayers (Table 3), that is, in the conditions favoring a rapid loss of trypsin activity. In ~j-well plates with non-confluent monolayers, as well as in 24-well plates, the effect was much less lS dramatic because in this case the multicycle growth o~ the virus was fairly efficient under standard conditions (Table 3).

Exam~le 2 Viruses:

';eventy-two influenza A virus strains, ohtained from the repository of St. Jude ChilcLren's Research Hospital, were invest:igated for their growth chLracteristics in Vero cells.

Vero cells wère infecte'd wit:h the A/England/l/53 (HlNl) [High GrQwth, HG] strain of influenza virus, a reassortant cont~;n;ng the gene segments coding for the two surface glycoproteins (HA and NA) from A/England/l/53 (HlNl) and the remaining six genes from A/PR/8/34 (HlNl). For the first four passages, the virus was left to adsorb for l hour at 37~C, aft:er which the monolayer was washed twice with warm phosphate buffered saline (PBS) solution to remove the unadsorbed viruses. Serum--free MEM with 0.3~ bovine serum albumin (BSA) was then added; the maintenance medium contained TPCK-treated trypsin at about l.0 ~g/ml. T~Le input dose of virus was 10-2-lO-3 PFU/cell. The material for further passage was colle,cted 72 hours postinfection (p.i.), with trypsin (final concentration, about l.0 ~g/ml) added at 48 hours p.i.. Cells were infected with serial 10-fold dilutions o~ virus, which were added to the washed cell monolayer wit~lOut previous adsorption. Virus accumulation was estimated by visual determination of the cytopathic e~fect (CPE) and HA titration of culture fluid at different times p.i. (24, 48 and 72 hours). In~ectivit~ titrations were pe!rformed in 96-well plates. Tissue culture infectious doses (TCIDsO/ml and egg infectious doses (EID)50/ml values were calculated by the formula of Karber tArch, Exp. Path. Pharmak., Vol. 162, pp.
480-48-~ (1931).

Virus-containing culture flu:ids were concentrated in an Amicon system and purified by differential sedimentation through 25-70~
sucrose gradients. Whole virus protein estimates were made by the method of Bradford (1976). To dl-termine the yield of HA protein in virus grown in Vero and MDCK cells, the virus proteins were separat:ed by gradient (4-20~) SDS-PAGE and intensity o~ Coomassie blue-sl_ained protein bands was ~uantified by densitometry.
~, Virus :Csolation From Clinical Mat:erial:

Influenza A viruses~were isolated ~rom the throat washings o~
patients with clinical signs of influenza and collected in PBS to which 0.7% BSA was added. Cell culture (both Vero and MDCK) or embryonated chicken eggs were infected directly with freshly collected (not frozen) throat washings. Chicken eggs were inocul;ated amniotically and allantoically. Clinical samples used for isolation were inoculated undiluted and at 10-1 and 10-2 dilutions and incubated for 72-96 hours. Trypsin was added at 0 and 48 hours p.i. (about 1.0 ~g/ml) and tested for virus replication with chicken and guinea pig erythrocytes. Each sample was given at least two passages in chicken eggs or cell cultures before being considered negative.

Immunoloqical Tests:

Monolayer antibodies to t:he A/Baylor/5700/82 (HlN1) and A/Baylor 11515/82 (HlN1) strains were prepared by the method of Kohler and Milstein (1976). Polyclonal antisera to influenza A/Bngland/1/53 ~irus (20 passages in Vero cells) were prepared in chickens by intravenous injection of virus-cont~; n; ng culture fluid. HA and HI reactions were performed in microtiter plates with about 0.5~ (v/v) chicke~n erythrocytes. Guinea pig erythrocytes (about 0.4% v/v) were used to analyze primary influenza A isolates from the 19cl3_l994 winter epidemic season.

Gene Amplification: - -RNA was isolated by treating virus-cont~; n; ng allantoic or culture fluids with proteinase ~; and sodium dodecyl sulfate and then ~xtracting the product with phenol-chloroform (1:1) and ethanol precipitation as previously described (Bean et al, 1980).
Viral RNA was converted to cDNA with the use of U12 (5'AGCGAAAGCAGG3') and AMV reverse transcriptase. The sequences of the oligonucleotide primers used in this study for molecular charaGterization of internal genes (PB2, PB1, PA, NS and M) are lS available on request.

Amplification proceeded through a total of 35 cycles of denaturation at 95~C (1 min), annealing at 50~C (1 min), and primer extensiion at 74~C (3 min). Amplified DNAs were analyzed by electrophoresis, visualized with ethidium bromide and then purified with either the Magic~ PCR Preps DNA purification system ~Promega, Madison, WI) or the Geneclean~ kit (BIO 101, La Jolla, CA) accorcling to the manufacturers' instructions.

Nucleotide Sequence Determination:

~ Jucleotide sequencing was performed dideoxynucleotide chain termination method with the fmol~' DNA sequencing system (Promega).
The reaction products were separated on 6% polyacrylamide-7M urea gels, 0.4 mm thick.

Morphological Observations: -For electron microscopic detection of virus particles on the30 cell surface and for comparison of cytopathological changes, Vero and ~Ck cell monolayers were infected with the Vero-adapted influenza virus strain A/England/1/53 tHG] at 10-3 PFU/cell CA 02205677 1997-05-16 --' multiplicity of infection, trypsin (about 1.0 ~g/ml) was included in the medium. Infected and control cell monolayers were fixed at 48 hours postinoculation iLn cacodylate-buffered 2.5%
glutaraldehyde, post-fixed in 1% osmium tetroxide, dehydrated in graded series of alcohols and embedded in Spurr low-viscosity embedding medium (Ladd Research Industries, Burlington, VT).
UltrathLin sections of cells were cut with a diamond knife on a Sorvall MT 6000 ultramicrotome, and the sections were e~ln;ned in a Philips EM 301 electron microscope operated at 80 kV.
Immunohistochemical assay for de*ection of apopto-tic changes in Vero and ~DCk virus-infected cells was performed with ApopTag~ In Situ Aploptosis Detection Kit-Fluorescein (ONCOR~) according to the manufac-turer's instructions.

RESUL~S

Screenin~ of Influenza A Viruses in Vero Cells "
Influenza viruses can replic:ate to high titers in a limited number of ~~m~lian cells, provided that trypsin is present for cleavage of the HA molec~le. To determine whether Vero cells are a suitable alternative system ~Eor replication of influenza A
viruses" a virus repository was ,creened and a master strain was selected that would replicate sufficiently in the m~m~lian epithe:Lial-like cell line. MDCK cells, which are widely used to isolate and culture viruses, were included in the study as a reference.

The influenza A virus str~lins that we ~m; ned had been isolated from a wide range of human and avian hosts, and represent 12 of the 14 HA (not H5 and H7) and 9 NA subtypes. Viruses were passag,ed three times in Vero and MDCk cells, and the virus yield was estimated from HA and infectivity titers. Of the 72 strains investigated, 65 (90.3~) replicated to the level that can be detected by HA titration in Vero cells after the first passage and 37 (51.4%) after the second. E~y comparison, all strains could replicate in MDCK cells during the first and second passages. Six humans and four avian influenza A viruses were selected as strains with the highest growth potential (Table 4), among which - -'. CA 02205677 1997-05-16 -~ -!.
A/England/1/s3 (HlN1) [HG] virus ~was chosen for further adaptation to Vero cells.

If the A/England/1/53 (HlN1) ~HG] virus is to be used as a master strain for generation of high growth reassortants, it is necessary to establish the genotype of this virus. We, therefore, partially sequenced the genes encoding the internal proteins and compared their nucleotide sequence with the prototype influenza strain, A/PR/8/34 (HlN1). As shown in Table 5, the A/England/1/53 tHG] strain selected for adaptation to growth in Vero cells is a reassortant between the original A/England/1/53 strain and A/PR/8/34. Six genes of the reas,ortant encoded internal proteins o~ A/PR/8~34~and two surface glycoproteins of A/England/1/53.

Infectivity of A/Enqland/1/53 ~HG~ after Serial Passaqinq:

To enhance the yield of virus in Vero cellsr we performed 20 serial passages of A/England/1/53 [HG] at limiting dilutions, comparing the results with those for the parental strain (Table 6).
Although the infectivity of the parent was lower in Vero cells than in either MDCK or chic~ n embryos, the progeny showed increased activity in Vero cells by the lOth passage, exceeding that in both reference systems. By the 20th passage, the infectivity of the virus was superior in Vero cells, but the HA titers remained comparable (64-128). The infectivity titer (TCIDso) was 26 times higher than that of the parental strain. By contrast, adaptation of replication in~Vero cells resulted in a slight attenuation of the virus in chicken embryos, as indicated by a reproducible decrease in EID50 titer from abc,ut 8.2 to about 7.7 logl0. The plaques formed by the Vero-adapted A/England/l/53 [HG] influenza strain were not as clear in Vero as in MDCK cells, and the efficiency~of the production was 10-fold lower. Plaque-forming capacity in Vero cells increased during serial passages of the ~irus but not in direct relation to the TCIDso titers. Thus, after 20 serial passages in Vero cells, the yield of infectious virus was high ky comparison with that in MDCk cells and embryonated chicken eggs.

' !
Viral Protein Yield of Influenza A/England/l/53 ~HG~:

Viral protein yield is an in~ortant feature of any system used to produce influenza virus vaccines. To establish the amount of virus-specific proteins that can be obtained from Vero cells, we compared the protein yields of ~/England/l/53 tHG] (20-passage) virus after replication in Vero and MDCK cells (Table 7).
Determination of the HA protein yield was done using SDS-PAGE
separated virus proteins and was quantitated by densitometry.
Tests of culture fluids indicated that approximately 6 x lO8 of io lnfected cells could produce 4.38 mg of virus protein in Vero and 4.13 mg in MDCk cells. It was also possible to obtain viral proteins ~rom disrupted virus-infected cells of either type; the protein yields were lower than in the supernatant but there was no significant difference between the cell types in the amount of virus protein.
"
Antigenic Stability of Vero-Adapted Influenza A/Enqland/l/53 rHGl Virus:

Because repeated passage of influenza viruses in m~m~l ian cells could lead to changes in antigenicity, it was thought that it was important to access the iLnfluence of Vero cell culture on this property. In HI tests with polyclonal chicken, rabbit and goat antisera with monoclonal antibodies to cross-reacting influenza A (~lNl) viruses, there were no appreciable differences in HA reactivity ~etween the parental strain of A/England/l/53 ~HG]
and it:s serially passaged varianlts (Table 8). This finding, which extends to antibodies specific to HlNl strains other than A/Eng:Land/53 [HG3, indicates that serial passage o~ the virus in Vero cells did not modify its H~ antigenic properties.

Primary Isolation of Influenza 2. Viruses:

Currently, MDCK cells provide the most sensiti~e host cell syste~ for the primary isolation of influenza viruses. Vero cells have been successfully used to isolate parainfluenza and mumps viruses, but they were judged unsuitable for the isolation of influenza viruses. To reassess t:his issue, we tested nine clinical specimens collected during the 19sl3-1994 epidemic season in three culture systems (Vero and MDCK cell.s and embryonated chicken eggs).
Six inf:luenza A (H3N2) strains were isolated in Vero cells; seven in MDCK cells and only two in embryonated chicken eggs (Table 9).
Two sam~les failed to yield virus in any host system. During the first passage in Vero cells, C]?E observed 48-72 hours after inoculation was the only evidence of virus reproduction. HA
activity was detectable on the second passage, and, by the third passage, the positive samples produced both CPEs and HA titers that ~o ranged ~rom 2-32. In all three culture systems, it was necessary to use guinea pig erythrocytes to determine HA titers, chicken erythrocytes failed to be agglutinated as was first described by Burnet and Bull. To examine whether replication of influenza A
(H2N2) viruses in Vero cell lines could select antigenic variants, L5 we analyzed viruses that had been passaged three times in this system. The reactivity patterns oi the HA with polyclonal antisera to reference A (H3N2~ influenza strains and monoclonal anti-HA
antibodies did not indicate differences between the strains isolated in Vero cells (results not shown). These results indicate that Vero cells would provide a u.seful and nearly as sensitive a culture system as MDCK cells for primary isolation of influenza A
(H3N2) viruses.
.
Ultrastructural Features of Virus--Infected Vero Cells:

To determine (i) if influenza, virus infected Vero cells, (ii) if viru.s is released from the api.cal surface of Vero cells as in other epithelial cells, and (iii~ if Vero cells undergo apoptosis as reported for other epithelial c.ells, we studied ultrastructural features of this system as compared to MDCK cells, following infecti.on with the A/England/1/53 tHG] influenza virus (20 passages). At the m.o.i. used, both types of cells showed nuclear and cytoplasmic inclusions typic:al of influenza virus-infected cells, as well as numerous buddi.ng virions. A~ in MDCk cells, virions were released from the apical surface of Vero cells, a feature typical of epithelial cel]s infected with influenza virus.
The buclding virions in MDCK and Vero cells appeared filamentous.
A fraction of infected cells in both systems showed cytopat:hological changes indicative of apoptosis. The nuclear . 14 CA 02205677 1997-05-16 --~

changes included fragmenitation and condensation of chromatin, margination of chromatin to the nuclear envelope, and blebbing of the nuclear envelope. The cytoplasmic changes consisted of condensation, extensive vacuolation, and blebbing and vesiculation of the plasma membrane to form "apoptotic bodies."

l'o confirm the apoptotic character of these electron microscopic alterations, the DN~ fragmentation in Vero and MDCK
cells was assayed. The histochemical assay consisted of addition of digoxigenin-labeled nucleotides to the 3'-OH ends of broken DNA
with use of ter~;nal deoxynucleotidyl transferase and detection of the added nucleotides by reactivity with fluorescein-labeled antidigoxigenin antibodies. The infected Vero and MDCK cells showed 20~ and 30~ positive cells, respectively, by this assay, wherea~s the uninfected cells were negative. The reported percentage o~ apoptotic cells in both Vero and MDCk cells may be underestimates because some of the apoptotic cells,,appeared to detach from the substratum durin,g the extensive washing required by these procedures. The label in certain cells is clearly seen o~er spherical masses ~with the nucleus, which may represent condensation of chromatin. These results suggest that a fraction of infected Vero and MDCK cells undergo endonucleolytic cleavage of DN~--a ty]pical feature of apo~ptosis.

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TABL~
;o~l ~3ri~ nz~ A/Ron~ g (H~) Y~
cPll~ ~nd in Vero (W~O) c~ ~ ditrcr~t pla~tic Y~c CC1IG P~ GW~rC IU~Ul mU]ti?1i( itY (TC~ per llask or pcr well) 10,000 1,000 100 10 48- 72 ~8 72 4~ 7;~ 4~ 12 50 cm~ fl~s 3î'' 1')8 64 128 32 12~ 16 128 Ycro (WHO) 50 cm' rlas}$ 1~ 3Z 2 ~ 1 1 - ..
~lero (~-ro~ 96-well p1alcs ND 16 I~lD 16 ND 32 l~D 3 .. . ~
;ti ' Reciprc~ of ~ ~ilor i~ cu1blre Iluid ND - noc don~
;~

~5 ~ . CA 02205677 1997-05-16 TAI~r,.R, ~
~f~ o~ rc~tul~lo~ ry~ln c~ce~tra~on on X~31 ~rus g~Ow~ cro ~IO) ccll~ ~ 5~ cm~ rla~ks .
Inpu~ ~ 1.5 ~ 10~ 1.5 ~ 10' l.S x 1~1 (TCII;~s~ 4~' 72 48 7~ 48 72 ~rypsin ~ddcd ~t O h p.i. 8'- 8 1 1 0 0 Trypsin ad~l~d at 0, 12, 24, 64 128 32 1~ 4 128.
ar,d 48 '.1 p.i, 'llour.~ I~ost-i~cction '~ccipr~;~ls of HA ~ rs TP~ - ~p~ addcd to finol con~c~ration ~f 1.~ ml o~ m~p~ ~p~ s~dclît~Ds DX ~ Y t~l of in~l~c~a ~n2s L~ Y~o ~Y~Oj oell~

. . .
EXD. ~0. ~'hu~ Pl~csStlc~ o~ ~ ~ add~ I~ut dose (T~II) olw~]l) 48- 72 48 72 ~1~ 12 ~ 72 /S~ ~ll. ~r~ O ~ p. i. 6~ 2 64 2 ~ I 1 ~'/SO ~ e 0,~24, 48 hp.i. 64 ~4 ~ 16 32 8 32 D
X~31 ~wcll1)CD5e 0 11 p.i. ~ 6 16 - X- ~1 ~wcllI~;e 0, ~4, 48 ~ p.i. 3~ , 64 32 64 16 ~4 ~ 32 ~
~, ~ ~ FWI~() ~vellNan~eD~ C~ h p.i. 16 16 16 3~ l 6 64 '7 - 8 1-0 ~wellNtln~o~ 0, ~4, 48 h eL 16 16 16 32 16 64 ~ 32 - 3 FWI~0 24-wellNo~ocnt 0 h p.i 32 32 32 32 ~6 G4 4 . 32 ~n FW/5() ~A-~Non co~lucDt ~, 24, 4~ h p.i. 32 32 ~2 3~ ~6 3~2 8 32 X-31 2~wdlN~o~flu~ O h p.i. 6~ 64 32 ~4 16 64 4 32 ~-31 ~ 0~ 8 h p.i, 64 ti4 3~ ~4 16 ~4 8 64 :;

CA 02205677 1997-05-16 ~ i Tabl~ 4. ~ighest Yields of Influenza A Viruses in Vero Cells Virus SUI)LY~ Virus I-IA Ti~cr' Inrcc~iYi~y ~i~er (logln TClDs"/ltll) I~uman viruscs A/Bcllamy/42 32 7.9 I-IlNl ~/Romc/49 64 7.7 ~I~M/1150 64 7.6 A/l~n~land/1153 64 7.5 IHG]

1~2N2 AlJapan/305157 32 ,7.7 A/Netl1er~ands/65/63 .~ 64 7.6 AYian ~ lSCSA/Chic~;/USSlVKam/l2/71 32 ND

H4N8 A/Mal Ducl~/NY1194/82 64 ND
HlON7 A/Cl~ic~/Germ/49 128 ND
~ A/Duck/Mani~oba/53 128 ND

'~iters were dctennined for the ~irst passage and measured at 72 hours postinoculation..
ND = not done, HG = High Growlh. ; ~

1~

CA 02205677 1997-05-16 - ' ~a~l~ 5. Genotyping of A/England/1/53 (HlNl) Influenza VirUs Virus G~ncs .l~ld llUC lcolidcs analy7cd P~32 PB 1 I'A NP M NS
~)69-1156 36U-53~ 20-~27 1064 1246 506-881 30-~05 1480-1580 13~-14~7 478-606 A/Eng/1/53 E 1~ E 1~ E
(original) A/Eng/1153 P P P P P p [HG~ ~

P ='gencs ~rom A/PR/8!34, E = gC:llCS ~rom oLiginal ~/Engl~nd/1/53, HG = Migll Growtll.
The origin o~ H.4 and NA was determined by I~I and NI tests.

-' CA 02205677 1997-05-16 .~--:
~ ,, . ~

Table 6Growth Charac~eristics of Inf~luenza A/England/1/53 (HlN1~ [HG3 -~
Virus After 10 and 2 0 Passages in Vero Cells ~irus 1-~ Titcr Infcc~iYi~y Tit~r (logl~) Plaque-Forming Units~
(logl"PFU/
TCID~/ml MD~K Vero 13IDs~ l MI)CK Vero A/England/1/53 64 7.70 6.9'; 8.20 8.70 7.18 ~IG~ parent (lP) A/Fn~l~n~l/1/53 64 7.20 7.9') 7.70 8.57 7.65 ~HG] (lOP) , A/Englalld/1/53 6~-128 7.57 8.37 ?.70 8;65 7.70 ~HG] (20P) HC~ = Higll GrowLll; P = passages in Vero cells ~Measurcd at 72 hours postinocu~ation ~1 , .

~- . CA 02205677 1997-05-16 ,~

.

Ta~le 7'. Viral Protein Yield of In~luenza A/:E:ngland/1/53 (HlNl~ ~HG] in Vero a.nd MDCK Cells ,;i;

I-Ios~ Sys~e~latcrial Proteill ~ield' (~ng) InYestigatcd Wllole ~lrus IIA

Culture fluid 4.38 0.98 era Cells 1.25 0,36 Culture fluid 4.13 1.18 MDC:E~ Cells 1.40 0-4Z

The concen~ra.ted Yirus was d~rlvcd from 1 liLer of culLure fluid oF sonicated il~ec(ed cells 6.1 x 108 ~ero and ~.8 x 10~ MDCK cells werc initIally in~ected.
'Quantit:ation o~ I-IA protein was dctermine(l by densiLometry a~ter polyacrylamidc gel -eiec~rophoresis .

T~3L~ 8- ~ntigenic ~nalysis o~ ell2a ~ g~and/11~3 ~G]
Virus Passaged in ~ero Cells it llS ~IT Titers~
Polycl~al an~isera to: ~n~ monoclotlal antil~odies to: ; :
- AfBay10rt11515/~2 ~ aylorlj7001~ ¦
~/Eng/53 [HG;~lWS~t/33 ~ 11147 ~/USS~/90177 chic~ serunl rabbit serum goat serum rabbit serum ~-32 ~B-33 216 ~4/1 31/I ' A/En~153 160 lOO 400 1600 400 3200 400 1600 200 ~t, [HG~
A/~ ?/53 !60 100 20û .It)(lU 400 1600 400 800 200 ,_ (2P) w ..
A/Eng/53 160 10û . ~Oû 16ûO 200 3200 400 800 20û ~
(7P) . A/En(T/53 320 lûO 200 1600 ~oo 3200~ 400 160û 200~i~
(lOP) A/Eng/53 320 100 40û 1600 200 - 3~00 400 1600 200-(20P) HG - High Growth; P - passage in Vero cells 'Reciprocal of the highest dilution of antibody ir;nibitiDg 4 H~ uDits of Yirus ' . ' CA 02205677 1997-05-16 ~
.

Tal~1~ g . Primary Isolation of Influenza A ~H3N2 ) Viruses in Vero Ce] ls During 1993-1994 Epidemic Season Strain ~I~ T~tcr at 48 ~nd 72 ]Hours ~lrus Isola~ion Pos~inrcction in V~ro Cclls~
Is~ ~nd 3rd~
r)assage passage passage 48ll 7~I1 4811 72ll 4~3h 7211 Yero Ml)CKl ~ggs~
AIMem/ll93 < < 2 2 ~3 I6 + + +
A/Mem/ l/94 < < < 4 ~ 4 + + +
A/Mem/'7!94 < < ~ 4 C 8 +
AlM~ml7/94 < < 8 ~ 16 - 8 32 (:~linical Sample ~1 < < < < ~ < - -01112/~4 A/Meml1 1/g4 < < < 2 2 ,:16 +
Clinical Sample ~2 < < < < <: <
01/12/C~4 A/~lem/12/94 < < < < ': < - +
A/Mem/16194 < < 8 16 8, 32 ~ ~

< --HA Liter less tllan 1:2, + - vil~s isolatcd and typed as influenza A (H3N2), - = virus not isolated ~I~A titraCion ~vas pcr~ol~ned wiill 0.~% (V/V) guinc:a pig erythrocytcs Yirus was de~cclcd af~cr ~hc ~lrst passa~e in MDCK: cells ~Yirus was detectcd on the sccond amniotic passage .

~- - CA 02205677 1997-05-16 Various modifications of the process of the in~ention may be made without departing ~rom thle spirit thereof and it is to be understood that the invention iLs intended to be limited only as defined in the appended claims.

. 25

Claims (5)

1. A process of replication, of human influenza virus in Vero cell culture comprising infecting the Vero cells with the influenza virus in the presence of a minimum concentration of trypsin of about 0.05 µg/ml in the culture medium throughout the influenza virus growth cycle.

2. The process of Claim 1 wherein the Vero cells are infected with influenza virus at a multiplicity of infection between about 1 x 10-5 and about 1 x 10-6 TCID50 per cell.

3. The process of Claim 1 wherein the multiplicity of infection is between about 1 x 10-5 and about 1 x 10-6 TCID
50 per cell.

4. The process of Claim 1 wherein the trypsin is regularly added during the replication of the Vero cells to the culture medium to maintain the trypsin concentration greater than 0.05 µg/ml.

5. The process of Claim 1 wherein the concentration of trypsin is maintained between about 0.05 and about 0.5 µg/ml throughout the growth cycle.