- Email: [email protected]
Development of a mammalian cell (Vero) derived candidate influenza virus vaccine
PII: SO264-410X(97)00301-0
Vaccine. Vol. 16, No. 9/10, pp. 960-968. 1998 Cl 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0264-410X198 $19+0.00
ELSEVIER
Development of a mammalian cell (Vero) derived candidate influenza virus vaccine 0 Kistner”, P.N. Barrett”?, and F. Darner”
W. Mundt*, M. Reiter”, S. Schober-Bendixen”
Influenza vaccine production is dependent on the availability of embryonated hen eggs for virus growth. This is an extremely cumbersome system with many disadvantages with respect to selection of virus variants and presence of adventitious viruses. We have developed an alternative cell culture system which allows rapid production of large volumes of vaccine. The World Health Organisation (WHO) approved Vero cell line was used in serum-free culture to grow a multitude of influenza strains to high titre. This system could be scaled-up to allow vaccine production with a 1200 litre fermenter volume. A purijication scheme was developed which resulted in a high purity whole virus vaccine. This was demonstrated to be at least as immunogenic as a conventional egg-derived preparation in a mouse model. 0 1998 Elsevier Science Ltd. All rights reserved Keywords: Vero cell; influenza vaccine: serum free
Efficient vaccine production requires the growth of large quantities of virus produced in high yields from a readily available, safe host system. Conventional methods for producing influenza vaccine have always involved the growth of the viruses in embryonated chicken eggs. This is a cumbersome process as each egg must be sterilized, candled, inoculated with virus and incubated before harvesting small volumes of allantoic fluid from each egg and pooling before puritication. In a typical chicken embryo operation, between one and two eggs are required for one dose of influenza vaccine. The lack of reliable supplies of high quality eggs results in limitations in the amount of vaccine which may be produced and creates fears that there could be major shortfalls in vaccine supply in case of a major pandemic’, when the demand for vaccine would be much higher. In addition to these logistic and supply problems, embryonated chicken eggs have other livitations as a host system for vaccine production. For example, there are increasing concerns about the presence of adventitious viruses, particularly retroviruses in eggs2, which would compromise the production of live, attenuated influenza virus vaccines. Also it has been reported that variant viruses are selected when human influenza virus is passaged in the egg. This host system induces a selective pressure for variants which grow well in the *Biomedical Research Center, Baxter-lmmuno, Uferstr. 15, Otth/Donau, Austria. tAuthor to whom all corre-
A-2304
spondence should be addressed. (Received 15 July 1997; revised version received 24 October 1997; accepted 28 October 1997)
960
Vaccine
1998 Volume
16 Number
9/l 0
embryonated egg, but which may not be re resentative 35 of the wild type virus circulating in humans . In contrast to influenza viruses grown in eggs, virus propagated exclusively in mammalian-derived tissue culture has been reported to be representative of the natural virus”. Studies in ferrets have also demonstrated that an inactivated influenza vaccine grown in Madin-Darby canine kidney cells (MDCK) induced higher mean serum hemagglutination inhibition (HI) and neutralizing antibody titres than did egg-grown vaccine and induced superior protection against subsequent challenge with infectious virus grown in either cell type’. These reports have demonstrated the possible superiority of a mammalian cell-derived vaccine and emphasize the necessity for a mammalian cell line that could be used to replace chicken eggs in the production of influenza vaccines. However, although influenza viruses can be propagated in several cell lines, none of these have been licensed for vaccine production”. This is because of concerns about tumorogenicity and presence of adventitious viruses in such continuous cell lines. The MDCK cell line appears to be a promising candidate for influenza vaccine production. A master cell bank has been generated, which was extensively characterized and demonstrated to be free of detectable infectious agents of viral or other microbial origin. Tumorogenicity studies also demonstrated that it is highly unlikely that MDCK cells arc able to grow or induce tumors in humans, but the cell line has not as yet been approved for use in human vaccine production. Clinical studies of a MDCK-derived influenza subunit vaccine have, however, been authorized. Such studies have demonstrated that the vaccine is equal
Vero a// derived influenza vaccine: 0. Kistner et al. with respect to immunogenicity and safety to a conventional egg-derived vaccine “.“‘. The continuous monkey kidney cell line, Vera, has however been licensed for use in the manufacture of human vaccines and is used by some manufacturers for the production of polio and rabies vaccines”. Vet-o cells are susceptible to a wide range of viruses, but initial attempts to grow influenza virus on these cells were unsuccessful’-.“. A recent report has however demonstrated that the Vero cell line is a suitable system for isolation and productive replication of influenza A and B viruseslJ. We report here on the development of a Vero cell system which is capable of producing high titre influenza virus at an industrial scale and on the development of a candidate inactivated influenza whole virus vaccine utilizing this system.
lished following a number of passages in this serum free medium and this cell bank was used to generate cell cultures, which were infected with influenza virus. All cultures had been passaged in serum free medium before infection with influenza virus. Vero cells were either propagated as monolayers in Roux or roller bottles or as microcarrier-based fermenter cultures under serum free conditions. Vero cell cultures were inoculated with the different influenza viruses at a multiplicity of infection (m.0.i.) of approximately 0.01 TCID5” cell and allowed to adsorb for 1 h. Porcine trypsin’ (Sigma) was then added with an activity of 20mU ml . Incubation was carried out at 32°C until development of maximum cytopathic effect or for a maximum of 72 h before harvesting. The virus yield was determined by hemagglutination (HA) assay. Growth of influenza virus in eggs
MATERIALS AND METHODS Virus strains The following influen.za viruses were kindly provided by the Center for Disea.se Control (CDC, Atlanta, GA, USA), by the Institute of Virology (University of GielJen, Germany) or by the National Institute for Biological Standards and Control (NIBSC, Potters Bar, London, UK): A/PR/‘8/34 (HlNl) = PR8, A/Brazil/ 11178 (HINl) = Brazil, A/Singapore/6/86 (HlNl) = Singapore 6, A/Taiwan/ l/86 (H 1N 1) = Taiwan, A/Texas/ 36/91 (H 1N 1) = Texas 36, X 113 (high-growth-reassortant Texas 36) A/Singapore /l/57 (H2N2) = A2 Singapore, A/Hongkong/l/68 (H3N2) = Hongkong, A/Hongkong/5/83 (H3N2) = Hongkong 5, A/Shanghai/l6/85 (H3N2) = Shanghai 16, A/Beijing/353/89 (H3N2) = Beijing 353, A/Johannesburg/33/94 (H3N2) = Johannesburg, NIB 34 and RESVIR 8 (high-growth-reassortants Johannesburg), AJWuhan/359/95 = Wuhan, A/ Nanchang/933/95 = Narrchang, RESVIR 9 (high-growthreassortant Nanchang), B/Massachusetts/71 = B/ Massachusetts, B/Panama/45/90 = B/Panama, BIHarbin/ 7194 = B/Harbin. Standard reagents The following reagents were purchased from NIBSC (Potters Bar, London, UK): purified hemagglutinin (HA) antigen from A/Texas 36/91 (HlNl, code 951510) Johannesburg/33/94 (H3N2, code 95/516), A/Nanchang/933/95 (H3N2, code 96/648), B/Harbin/ 7194 (B, code 95/52(l); specific anti-HA antisera directed against A/Texas 36/91 (HlNl, code 92/504), code A/Johannesburg/33/94 (H3N2, 951524) A/Nanchang/933/95 (H3N2, code 961724) BIHarbin/ 7/94 (B, code 95/530) and specific antisera directed against the neuraminidase from Nl, N2 and B strains: Nl (code 96/716), N2 (code 96/696), B (code 96/740). Growth of influenza virus in Vero cells The World Health Organisation (WHO)-certified Vero cell line was used for growth of influenza virus. This cell line has been fully characterized and fulfills all the requirements for (continuous cell lines used for manufacture of biological products. The Vero cells were adapted to growth in a DMEM-based medium which was totally serum free. A cell bank was estab-
Eleven-day-old embryonated hen eggs were inoculated with 0.2 ml of a virus suspension and incubated for 3 days at 33°C. Then, the eggs were cooled overnight at 4°C before harvesting the virus-containing allantoic fluid. Virus titre was determined by HA assay. Large-scale production of influenza virus in microcarrier fermenter culture A single ampoule of Vero cells with a defined passage number was thawed from liquid nitrogen and passaged in Roux and roller bottles to produce sufficient cells to inoculate a 1.5 I fermenter using Cytodex3 (Pharmacia) microcarriers. After a cell number of approximately 5 x 10” I ’ has been obtained, the microcarriers were trypsinized and cells with microcarriers were used as an inoculum for a 12 I fermenter. This in turn was used as an inoculum for a 150 I vessel which is then used to inoculate a 1200 I fermenter. When a cell density of approximately 5 x 10’ cells litre-’ has been achieved, 1.2 1 of virus inoculum was added. This was equivalent to a m.o.i. of 0.01 TCIDs,‘celll’. Incubation was carried out at 32°C for 2-3 days using trypsin at a concentration of 20 mU ml ‘. Hemagglutination (HA)-assay Influenza viruses were diluted in two-fold steps with phosphate buffered saline (PBS) and 50 ~1 of each dilution were added to 50 bll of a 1% suspension of fowl red blood cells in a V-shaped microtitre plate. After gentle agitation, the plates were left undisturbed for 30 min at room temperature and then read. The last dilution showing complete hemagglutination was taken as the end point and was expressed as hemagglutination units (HAU) per test volume (50 /II). Hemagglutination-inhibition assay (HI) Influenza viruses were diluted to give a preparation with 4 HAU per 50 ktl. The antibody preparations were diluted in two-fold steps and 50 cll of each dilution were added to 50 ltl of the virus dilution with 4 HAU in a V-shaped microtitre plate. After gentle agitation, the plates were incubated for 30 min at room temperature. Then 50 ~tl of a 1% suspension of fowl red blood cells were added to each well and the plates were left another 30 min before reading. The reciprocal value of
Vaccine 1998 Volume 16 Number 9/10
961
Vero all derived influenza vaccine: 0. Kistner et al the highest dilution of inhibited hemagglutination HI titre. Determination content
antibody which was determined
of the specific hemagglutinin
completely to hc the
antigen
The concentration of hemagglutinin in the different influenza virus antigen preparations was determined by the single radial immunodiffusion (SRD) as described by Wood et ~1.‘~ using the NIBSC standard reagents. Briefly, influenza virus preparations and reference antigen standards were lysed with Zwittergent, diluted, added to wells in agarose plates containing reference antisera against the HA proteins of the corresponding strains and incubated for 18 h at room temperature. The size of the precipitation zones, which were stained by Coomassie blue, were measured and the HA content of the virus preparations was antigen calculated by slope ratio analysis, plotting the values of the diameters of the precipitation zones against the dilutions of reference antigen and virus preparations. Neuraminidase assay The presence and identity of neuraminidase in virus preparations was determined by neuraminidase activity (NA) and neuraminidase inhibition assay (NI) as described by Aymard-Henry et af.‘” using the NIBSC standard reagents. Briefly, two-fold dilutions of virus preparations were prepared and incubated with fetuin as substrate for 18 hours at 37°C. Following sequential addition of periodate, arsenite, thiobarbituric acid and Warenoff reagents, colour development was measured by spectrophotometric analysis at 550 nm. A standard dose (which gave an extinction of 0.6-0.9) for use in the NI assay was then selected. This standard dose was incubated with serial dilutions of NIBSC anti-neuraminidase antisera and the NA assay was performed again. The antiserum dilution which resulted in a 50% inhibition of neuraminidase activity was thus determined. Preparation of mouse sera against Vero cell proteins Non-infected Vero cells were harvested, adjusted to a concentration of 2 x 10’ cells per ml and lysed in RIPA buffer (0.01 M phosphate buffer, pH 7.2+40 mM sodium fluoride +O.l% SDS +l% TritonX-100+2mM EDTA). Mice were immunized with the lysate equivalent to 2 x lo7 Vero cells in complete Freund’s adjuvant (CFA) and boostered twice at 4 week intervals with the same lysate in incomplete Freund’s adjuvant. Two weeks after the last booster, mice were bled and the derived sera were used for Westernblot analysis. PAGE and Westernblot analysis SDS-PAGE of influenza virus preparations was carried out according to Laemmli17. Westernblot was performed by electrophoretic transfer of the proteins from the gel to polyvinyl difluoride (PVDF) membrane (Millipore) for 90 minutes. After blotting, the membranes were incubated overnight with the specific NIBSC anti-HA antisera, diluted 1:lOOO in TBS/Tween or the mouse serum against Vero cell proteins, diluted
962
Vaccine
1998 Volume
16 Number
9/10
I:100 in TBS/Twcen. After intensive washing the membranes were incubated for 1 h with horseradish peroxidase conjugated rabbit anti-sheep IgG or goat anti-mouse IgG (Chemical Accurates), diluted I:5000 in TBS/Twecn. After washing the membranes again, the blot was developed by adding diaminobenzidinc and HJ02. Ten minutes later the reaction was stopped by transferring the membranes into water.
Virus purification
for antigenicity
studies
Pooled allantoic fluids from virus infected eggs or supernatants from infected Vero cell cultures were clarified by low speed centrifugation, concentrated by ultrafiltration (300 KD cut-off) before purification on a 20-60% continuous sucrose gradient. The peak fractions were pooled, diluted and inactivated with 0.025% (w/v) formalin for 24 h at 32°C. Following diafiltration to remove formalin the HA content was determined by SRD analysis.
Determination of the effective dose 50 (ED,,,) of vaccine preparations Vero cell-derived and egg-derived influenza virus vaccine preparations were diluted with PBS and adjuvanted with aluminium hydroxide [AI(,OH),] to and an give an HA-antigen content of 15 /lg ml AI concentration of 0.2%. These preparations were serially diluted 1:4 with PBS containing 0.2% AI( The HA-antigen concentrations u;ed for immunization were 15 /lg ml , 3.7: /lg ml (1:4), 0.234 /lg ml (164) and 0.938 /Lg ml (1:16), 0.059 /[g ml ’ (1:256). Groups of ten CD1 mice (6-9 weeks old) were injected subcutaneously (s.c.) with 1 ml of each dilution. Control groups were injected with PBS containing 0.2% Al(OH)3. Four weeks after the immunization the mice were boostered with the same antigen preparation and a blood sample was drawn from each mouse. Two weeks after the booster, the mice were bled by heart puncture. One part of the individual sera was used directly for antibody measurement in ELISA, another part was diluted 1:5 in PBS and heat-inactivated for one hour at 56°C before measurement of antibody titre in an HI assay using egg-derived wild type (wt) virus as antigen.
ELISA The ELISA was performed on influenza A virus coated and influenza B virus coated tiicrotitre plates (Genzyme Virotech, Russelsheim, Germany). Each serum sample was diluted 1:lO in buffer (PBS-goat serum, 0.5% Tween 20). One hundred microlitres of each sample were transferred to the wells of the microtitre plates and incubated at 37°C for 1 h. After five washing steps with PBS/Tween, each well of the plate was tilled with 100 itI of horseradish peroxidase conjugated anti-mouse IgG (Chemical Accurates, diluted 1:15 000 in dilution buffer) and incubated for 1 h at 37°C. Again the plates were washed five times using PBS/Tween. Subsequently, 200 111 of OPD substrate (10 mg o-phenylene-diamine hydrochloride) were added per well. After incubation at room temperature
Vero all derived influenza vaccine: 0. Kistner et al. for IO minutes in the dark, the reaction was stopped by addition of 50 111of 5 hl HzS04 and the optical density of each well was measured with a microplate reader (Dynatech) at 495 nm against a reference wavelength of 630 nm. All positions with an optical density of at least two times greater than that of the sera of the control animals, but with an extinction greater than or equal to 0. IO0 were considered as positive.
Statistical analysis The effective dose nlecessary to induce seroconversion in 50% of mice, measured either by ELISA or by HI assay was determined by logit analysis. The parameters of the model were estimated by the maximum likelihood method applying Newton algorithm’“.
RESULTS Vero cell line safety The Vero cell line was established in 1962 by Yasumura and Kawakita’” from the kidney of a normal adult African Green monkey. At present, the cell line is distributed by the American Type Tissue Collection (ATCC) at passage level 124 under the designation ATCC CCL 81. The WHO Vero IO-87 cell bank has been prepared by the Institut Merieux with passage level 134 and has been extensively characterized for absence of viral contamination and lack of tumorogenicity at this passage level”‘~‘“. The master cell bank (MCB) used for the production of influenza virus as described here was additionally fully characterized for the presence of a range of specific adventitious viruses. The data presented in 7”ahfr I demonstrate that the cell bank was found to be free of retroviruses, human papilloma virus, Epstein-Barr virus, cytomegalovirus and bovine viral contaminants. In addition the MCB did not contain any infectious agent of viral or other microbial origin as determined by microbiologic tests and tests in tissue culture, baby mice, adult mice, guinea pigs, rabbits and embryonated specific pathogen free (SPF) eggs. The working cell bank (WCB) and production cell bank (PCB) were similarly tested for bacterial and mycotic sterility, mycoplasma, mycobacterium tuberculosis, adventitious viruses and were found to be free of such contaminants. In addition Vero cells from the MCB did not produce palpable tumors following inoculation of athymic nude mice and observation for a 3-week period. Table 1
Virus yield in Vero cell culture The ability of a wide range of influenza A and B virus strains to grow in Vero cells under serum-free conditions was determined. Monolayer cultures in I75 cm’ Roux bottles were infected with 0.1 ml of egg-derived virus (m.o.i. 0.01 TCID,,, cell ‘) and after adsorption ,100 ml of serum free medium containing of trypsin were added. Incubation was 20 mU ml carried out under the conditions described previously and following harvesting the HA titre in cell culture medium was determined. A direct comparison was made with the virus yield obtained following infection of embryonated eggs with the same viruses. The data presented in Table 2 demonstrate that all strains tested could grow to high titre in Vero cells, with HA titres in the range of 128-256 being obtained for all strains. These titres were in almost all cases lower than those obtained in the embryonated egg, but a larger volume was obtained from the Vero cell culture (100 ml) than from the embryonated egg (circa 7 ml). The discrepancy in titre between the two systems was most evident for the high growth (HG) reassortant strains. The HG strains X 113, NIB 34, RESVIR 8 and RESVIR 9 gave HA titres between 1024 and 2048 in the embryonated egg whereas growth in Vero cell culture resulted in an HA titre of 256 for all four strains. The lowest m.o.i. required to achieve maximum virus titre in Vero culture was also investigated and determined to be approximately 0.001 TCIDS,, cellI’. With this m.o.i. the maximum titre was achieved 2-3 days post-infection. Infection with lower m.o.i.s resulted in maximum titres being achieved 5-6 days post-infection. Production of influenza virus vaccine strains in industrial scale fermenter systems Following the demonstration that a range of influenza strains could be grown to high titre in Vero cells, studies were carried out to develop a system for industrial scale vaccine production. Virus antigen production was investigated using a 1200 litre microcarrier culture fermenter system as described above. The growth of the influenza vaccine strains in use for the 1995/1996 and 1996/1997 seasons, i.e. Texas 36 (A/HlNl), Johannesburg (A/H3N2), Nanchang (A/H3N2) and BiHarbin (B), was investigated in this system. All four strains yielded HA titres of 256 in a total volume of 1200 I cell culture medium supernatant. These titres were equal to or higher than the titres obtained in monolayer cultures, demonstrating that the Vero cell technology
Analysis of the Vero master cell bank for adventitious viruses
Test
Result
Reverse transcriptase assay Human papilloma virus Epstein-Barr virus (EBV) detection Epstein-Barr nuclear antigen (EBNA) test Electron microscopy (negative stain) Thin-section electron microscopy In vitro assay for detection oi non-murine viral contaminants In viva assay for non-murine viral contaminants Detection of cytomegalovirus (CMV) by co-cultivation assay Induction of retroviral particles by Siododeoxyuridine Extended assay for bovine virus detection
Negative for Mn2+ and Mg*+ dependent reverse transcriptase assay Negative for HPV types 6, 11, 16, 18, 31, 33 and 35 EBV DNA was not detected Negative for EBNA Viral particles were not observed Viral particles were not detected No evidence of viral contamination was found No evidence of viral contamination was found CMV not detected No retroviral induction after treatment with Siododeoxyuridine No bovine viral contaminants were detected
Vaccine
1998 Volume
16 Number
9/10
963
Vero all derived influenza vaccine: 0. Kistner et al Table 2 Comparison of HA-titres obtained for different influenza virus strains grown in Vero cell cultures and in embryonated eggs Hemagglutinating Units (HAU)
Neuraminidase
Type/Subtype
Strain
Vero
Egg
A/HlNl
PR 8 Brazil Singapore 6 Taiwan Texas 36 x113 A2 Singapore Hongkong Hongkong 5 Shanghai 16 Beijing 353 Johannesburg NIB 34 RESVIR 8 Wuhan Nanchang RESVIR 9 B/Massachusetts B/Panama B/Harbin
256 128 128 128 128 256 128 128 128 256 256 256 256 256 256 256 256 128 128 256
1024 1024 128 512 256 2048 512 1024 256 128 256 128 1024 2048 512 512 2048 512 256 512
A/H2N2 A/H3N2
B
could be scaled virus yield.
up successfully
without
reduction
in
Comparison of the antigenicity of Vero cell- and egg-derived virus following immunization of mice For a comparison of the antigenicity of Vero celland egg-derived virus, mice were immunized with inactivated influenza virus preparations derived from both systems. The 1995/1996 vaccine strains Texas 36, Johannesburg and B/Harbin received as standard WHO egg isolates were passaged at least five times in the Vero cell line, and then purified by sucrose gradient centrifugation as described and inactivated by treatment with 0.025% (w/v) formalin at 32°C for 24 h. The corresponding strains X 113 (Texas 36, HG reassortant), NIB 34 (Johannesburg HG reassortant) and B/Harbin were subjected to the same number of passages in eggs and purified and inactivated identically. All preparations were adjusted to a specific HA content of 15 /fg, adjuvanted with A1(OH)3 and inoculated S.C. into groups of ten mice. Four weeks after primary immunization, the mice were boostered with the same formulation and 2 weeks later they were sacrificed. The sera from individual mice in each group were pooled and the HI titres from each pool were determined using both Vero cell-derived and egg-derived HA antigen in the assay. The data shown in Table 3 demonstrate that almost identical HI titres were obtained for the specific mouse sera against the corresponding Vero cell-derived and egg-derived influenza antigen, with little or no cross-reaction with antigen from the non-corresponding strains. For example, serum derived from mice immunized with Vero cell-derived Texas 36 had HI titres of 2560 when measured against both Vero cell and egg-derived Texas 36 antigen and had little reactivity against the Johannesburg and B/Harbin antigens. Similar results were obtained for the Johannesburg and B/Harbin strains,
964
Vaccine 1998 Volume 16 Number 9/l 0
demonstrating that limited passage not significantly alter the antigcnicity
in Veto cells did of thcsc strains.
characterization
Further characterization of the Vcro cell-derived virus was carried out by means of a comparison of the ncuraminidase antigenic properties with that of the egg-derived virus. The neuraminidasc was charactcrizcd by means of the ncuraminidase inhibition assay using specific anti-neuraminidasc scra as described above. These standard scra obtained from NIBSC, Potters Bar, were raised against ncuraminidasc from egg grown virus. The vaccine strains for the 1996/lY97 season, i.e. Texas 36, Nanchang and BiHarbin, were characterized in this manner. The data presented in Tuhle 4 demonstrated that no significant diffcrcnces could be detected between the Vero- and egg-dcrivcd virus strains in this assay. The same anti-N1 titrcs were measured for the specific set-a against both the Veto cell- and egg-derived HlNl strain and also for the N2 serum against the H3N2 strain. The anti-B serum, however, had a somewhat higher titrc against the Vcro cell-derived virus (10000) than against the egg-derived virus (3000). Also, the specific sera had little or no reactivity against the heterologous virus strains. Development of an inactivation and purification scheme for a whole virus vaccine Following the demonstration that the Vero ccl1 technology could be scaled up to a level required for industrial production, a purification scheme was established to provide a vaccine which fulfilled all the regulatory requirements for influenza whole virus vaccines. The following scheme was established for large scale routine production: the supernatants from the microcarrier cultures were clarified using a separator (Westfalia 4SA8, 5000 g, 280 I h ‘), before treatment with benzonase (1 000 units I ‘) to destroy Vero cell DNA, followed by viral inactivation with formalin (0.025% w/v end concentration) for 24 h at 32°C. The infectivity of Texas 36 and BiHarbin was reduced below the level of detection after 1 h, and that of Nanchang after 2 h formalin treatment. The inactivated material was then concentrated by ultrafiltration (200000 Da m.w. cut-off) and further clarified by protamine sulphate precipitation before purification by continuous flow zonal centrifugation over a O-50% sucrose gradient. The virus containing peak pool was then subjected to ultrafiltration and sterile filtration. Following measurement of the HA content and appropriate dilution, the vaccine was formulated with all three virus strains. The efficacy of this purification procedure in removing contaminating Vero cell protein was demonstrated by Western blot analysis of materials drawn from successive production and purification steps of the Nanchang strain. Western blot analysis using specific Nanchang anti-HA serum (NIBSC) illustrates the purification scheme used to obtain puritied material with well defined HA1 and HA2 bands (lane 5 and 6, Figure la). Analysis using an anti-Vero cell serum demonstrated that a variety of Vero specific bands were present until after the sucrose gradient purification (lane 5 and 6, Figure Ib). No Vero cell protein bands could be detected using this methodology, subsequent to this purification step.
Vero all derived influenza vaccine: 0. Kistner et al. Table 3 Hemagglutination-inhibition
(HI) titre of mouse sera immunized with different influenza virus vaccine preparations against the corresponding Vero cell-derived and egg-derived influenza viruses HI assay antigen Texas 36
Johannesburg
Type/subtype
Immunization antigen (serum)
Source
Vero
Egg
Vero
Egg
A/HlNl
Texas 36 Xl 13 (HG Texas 36) Johannesburg NIB 34 (HG Johannesburg) B/Harbin B/Harbin
Vero cells Egg Vero cells Egg Vero ceils Egg
2560 640 10 10 10 10
2560 640 40 40 10 40
10 10 5120 2560 10 10
40 80 5120 1280 40 80
A/H3N2 B
Comparison of the immunogenicity egg-derived vaccine strains
of Vero cell- and
The immunogenicity of Vero cell- and egg-derived vaccine strains for the lYY5/1996 season, i.e. Texas 36, Johannesburg and B/Ha&in, was compared by potency by studies in mice. Potency was determined immunizing mice with various dilutions of a vaccine strain and calculating the effective dose (ED,,,) which results in seroconversion in 50% of the mice. The EDS,, values calculated on the basis of the influenza ELISA titres and the HI titrcs are summarized in Tuhfr 5. In all cases the Vero cell-derived vaccine was more immunogenic than the #equivalent egg-derived vaccine strain. The EDSo values for all strains were between two to four-fold lower for the Vero-derived than those for the equivalent egg-derived strain, when calculated on the basis of induction of binding antibodies measured by ELISA. For example the values obtained for Vero- and egg-derived Texas 36 were 71 and 204 ng, respectively, i.e. a three-fold difference in potency. The potency values obtained on the basis of the HI assay determinations also demonstrate that the Vero cell-derived material was more immunogenic than the egg-derived vaccine. However, the differences varied between a slighl: advantage for the B/Harbin strain, i.e. 140 ng compared with 162 ng and a nine-fold difference for the Texas 36 strain.
B/Harbin Vero 10 10 10 10 160 160
Egg 10 10 40 40 320 320
consisting of 15 jig of HA antigen was determined (Tahlr 6). Further characterization vaccine
of the Vero cell-derived
Prior to the initiation of efficacy studies in humans, the vaccine was subjected to the standard tests required by the European Pharmacopoeia for egg-derived vaccinesZ4, to the tests required for products derived from continuous cell lines-’ and to a 1
2345678
1
2345678
(a) Influenza vaccine yield obtained using the serum free Vero cell technology
The vaccine strains in use for the 1995/1996 and 1996/1997 seasons were all grown in a 1200 I serumfree Vero cell microcarrier culture fermenter and the antigen yield was measured. All four virus strains achieved a HA titre of 256 under these conditions. The purification procedure ‘was carried out as previously described and the amount of doses of purified vaccine Table 4 Neuraminidase
inhibition (NI) titre of Vero cell-derived and egg-derived influenza virus vaccine strains against NIBSC neuraminidase specific antiserum NIBSC anti-neuraminidase antiserum
Virus
Source
Texas 36 (A/HlNl)
Egg Vero
Nanchang (A/H3N2)
Egg Vero
B/Harbin (B)
Egg Vero
Anti-N1
Anti-N2
300 300 30 <30 30 <30
30 <30 3000 3000 30 <30
Anti-NB 30 <30 30 <30 3000 10000
@I Figure 1 Westernblot analysis of the purification steps involved in the manufacture of Vero cell-derived Nanchang using (a) specific anti-HA serum and (b) specific anti-Vero cell serum. Lane 1: virus harvest, lane 2: after formalin inactivation, lane 3: after ultrafiltration, lane 4: after protamine sulphate precipitation, lanes 5, 6: purified peak fractions from sucrose gradient, lane 7: Vero cell lysate, lane 8: egg-derived Nanchang (NIBSC)
Vaccine 1998 Volume 16 Number 9/l 0
965
Vero all derived influenza vaccine: 0. Kistner et al. Table 5
Comparison of Vero cell-derived and egg-derived formalin inactivated influenza virus vaccine preparations dose (ED,,) required for seroconversion of mice, as determined by ELISA or by hemagglutination inhibition (HI) assay
in terms of effective
EDs0 (ng) Type/subtype
Antigen
Source
ELISA
HI
A/H1 Nl
Texas 36 Xl 13 (HG Texas 36) Johannesburg NIB 34 (HG Johannesburg) B/Harbin
Vero Egg Vero Egg Vero Egg
71 204 29 50 35 153
120 1092 38 89 140 162
A/H3N2 B
number of additional tests, such as pyrogenicity testing which are normally not required for influenza vaccines. The results of these tests are detailed in Tahfr 7 and demonstrate that the vaccine fulfills all of these requirements, e.g. the DNA content was below the level required for vaccines derived from continuous cell lines (100 pg dose ‘).
DISCUSSION Influenza viruses exhibit a strict species specificity and organ tropism, which is illustrated by their inability to replicate in a large number of different cell types’“. The exact mechanisms for this restriction in specific cell lines are not fully understood, but it is known that cleavage of the precursor hemagglutinin protein by an intracellular protease into the HA1 and HA2 subunits is essential for a productive infection27,2x. When a cell contain an appropriate enzyme, does not non-infectious virus with uncleaved hemagglutinin is produced. However, it has been reported that addition Table 6 Vaccine yield obtained from a 1200 I Vero cell fermenter
Virus strain
Maximum HA titre in cell medium
A/Texas 36 (HlNl) A/Johannesburg (H3N2) A/Nanchang (H3N2) B/Harbin
256 256 256 256
Table 7
Control
of trivalent
bulk
Total HA yield in purified vaccine (9)
Vaccine dose equivalents
2.80 3.07 3.15 4.98
186 660 204 660 210000 332 000
vaccine
and
final
container
product Test
Release criteria per dose
Bacterial and mycotic sterility Haemagglutinin content (SRD-assay) Protein content Sucrose content Residual protamine sulphate Residual formaldehyde Residual benzonase Residual DNA Endotoxin content &AL-assay) Pyrogenicity Abnormal toxicity Identity-haemagglutinin antigen Identity-neuraminidase antigen
Sterile 2 15 ,rg I 250 jig SlOmg 55 jrg < 5 /rg 55mg
966
Vaccine
1998 Volume
16 Number
9/10
of trypsin or a trypsin-like enzyme to the culture medium can result in extracellular cleavage and generation of infectious virus particles”‘.3”. Previously, the only classes of cultured cells used for the production of licensed human viral vaccines were primary cells and human diploid cells. Continuous cell lines were not used because of fears concerning the potential tumorogenicity and viral contamination of such cell lines. Vero cells, a continuous African Green monkey kidney cell line, have been extensively tested in this respect and a cell bank approved by WHO has been established”. This has been demonstrated to be free of viral adventitious agents and to have no tumorogenic potential at this passage level”‘-‘3. We have utilized this WHO approved cell stock to make a master cell bank. which was passaged exclusively under serum-free conditions. This cell bank has again been extensively tested and demonstrated to be free of viral adventitious agents (Tuhfr I). Testing in nude mice also demonstrated that the cell line had no tumorogenic potential at this passage level and was thus an acceptable substrate for the manufacture of human vaccines. It had, however, been previously reported that influenza A and B viruses were restricted in their growth in Vero cells”.‘3. Recent studies’“.“‘.“‘, however, have demonstrated that repeated addition of trypsin to the culture medium of influenza virus-infected Vero cells restores the multiple growth attern of influenza A and P B strains. Govorkova et al.’ demonstrated that of 72 egg-derived influenza A strains, 00% replicated to detectable HA levels in trypsin treated Vero cells after the first passage and 51% after the second passage, but no data was provided on the virus titres obtained for these strains. We report here on the ability to grow 100% of strains tested to high titre in a serum-free Vero cell culture. All of 20 strains tested gave HA titres of 128 or 256 in Vero cell culture (Tuhlr 2). Although these titres were somewhat lower than those obtained in the egg following infection with the same volume of virus, the total amount of antigen obtained in the Vero cell culture was greater due to the larger volume of medium obtained from cell culture (100 ml) compared to 7-8 ml per egg. The lowest m.o.i. required to generate maximum virus titre in the Vero system after 2-3 days incubation was determined to be in the order of 0.001 TCID,,‘cell ‘. It was not possible to make a direct comparison with the m.o.i. required for maximum virus production in the egg, due to difficulties in estimating the cell number in the egg tissue. It is, however, likely to be lower than 0.001 TCIDS,, cell ‘. i.e. the efficiency of virus production is likely to be higher in the egg system. The Vero cell system can, however, be very successfully utilized in
Vero all derived influenza vaccine: 0. Kistner et al.
large volume fermenters with subsequent advantages for industrial production. This system could he successfully scaled up to a 1200 I fermenter volume without loss in virus antigen yield (Trrhke 6) and permits for the first time the industrial scale production of influenza vaccine in an approved continuous cell line. This is of particular signiticancc in that it allows production of large quantities of vaccine using wild-type (wt) virus, i.e. without the necessity for generation of high growth reassortants. This will allow rapid production of vaccine should a pandemic strain of influenza appear, and could greatly contribute to the control of such a pandemic. Antigenic diffcrenccs have been frequently observed between the HAS of influenza viruses that had been isolated from the same clinical sample and cultivated exclusively in MDCK cells or adapted to egg growth. In addition the viruses could be clearly distinguished on the basis of their reaction with various monoclonal antibodies3’,34. We have compared the antigenicity of virus which was originally isolated in eggs and further passaged in Vero cells with that of virus which was exclusively passaged in eggs. No differences were seen in the titres of mOuse scra immunized with either eggor Vero-derived antigen, when tested in HI assays using Vera- and egg-derived antigen (7X& 3). These tindin s are in agreement with the data of Govorkova et al. b who reporteld no differences in the HA reactivity between a parental egg-derived HlNl strain and its serially Vero cell-passaged strain. Sequence analysis of the HAI by these authors also demonstrated identity of the amino acid sequence with the parental strain after 20 passages in Vero cells. Comparison of the potency of egg- and Vero cellderived antigen demonstrates, however, that the Vero cell-derived antigen has a two to four-fold increased immunogenicity on the basis of ELISA antibody measurements (Table 5). This could be due to an improved antigenicity when HA is presented associated with Vero cell membranes as opposed to chick cell membranes. It should, however, be considered that this apparent improvement could be artifactual. Determinations of HA antigen are carried out by the SRD assay using the standard antisera which are used for measurement of HA in egg-derived vaccines. These antisera are derived by immunization with egg-derived antigen and all calibration curves are constructed with these reagents. Thus it is feasible that the size of a precipitation zone generated by interaction of Veroderived antigen with specific sera raised against egg-derived antigen may not reflect the real HA antigen content of Vero-derived material. This possibility is now being investigated by the establishment of a SRD assay with antisera raised following immunization with Vero-derived HA antigen. The demonstration that the potency of the egg-derived antigen was three or ten-fold lower on the basis of the HI titre for the A strains may also not imply a real difference. This may be due to the fact that whereas immunization was carried out with the HG reassortant, the HI titre is always determined using the egg-derived wt virus antigen. Thus we see no major difference in potency for the B strain on the basis of the HI titre determinations, as immunization and HI
assay are carried out with the egg-derived wt virus antigen. A real improvement in immunogenicity may, however, be achieved when the original isolation of virus is carried out in Vero cells, thus preventing the generation of variants which are selected en the basis of their ability to grow well in eggs, but may not be representative of the circulating wt virus population. Prior to initiation of human trials, it was essential to demonstrate that the Vero cell-derived vaccine fulfilled all requirements as determined by the European the WHO and other regulatory Pharmacopoiea, organizations’4.‘5. The vaccine was demonstrated to fulfill all such requirements (Tahke 7) and clinical trials have now been initiated with a trivalent vaccine consisting of Amexas 36 (H 1N 1), A/Nanchang (H3N2) and BiHarbin in the UK and Austria.
ACKNOWLEDGEMENTS The authors are grateful to Dr J. Wood, NIBSC, Potters Bar, UK for provision of multiple virus strains, antisera and antigen reagents, for his generous assistance in establishing the SRD assay and for many fruitful discussions. The support of Mr Robert Newman, NIBSC, Potters Bar, UK in establishing the NA assay is also gratefully acknowledged. We would like to thank Dr Nancy Cox, CDC, Atlanta, USA and Dr Christoph Scholtissek, University of GieOen, Germany for the provision of many virus strains. The authors are grateful to Frau Stefanie Fabi for her support in preparing this manuscript.
REFERENCES 1 2
3
4
5
6 7
8
9
10
Rowe, P.M. Pandemic influenza (un)preparedness described. The Lancer 1995, 346, 1699. Petricciani, J.C. Regulatory philosophy and acceptability of cells for the production of biologicals. Dev. Biol. Stand. 1991, 75, 9-15. Schild, G.C., Oxford, J.S., De Jong, J.C. and Webster, R.G. Evidence for host-cell selection of influenza virus antigenic variants. Nature (London) 1983, 303, 706-709. Robertson, J.S., Bootman, J.S. and Newman, Ft. et al. Structural changes in the haemagglutinin which accompany egg adaptation of an influenza A (Hl Nl) virus. Virology 1987, 160, 31-37. Hardy, C.T., Young, S.A., Webster, R.G., Naeve. C.W. and Owens, R.F. Egg fluids and cells of the chorioallantoic membrane of embryonated chicken eggs can select different variants of Influenza A (H3N2) virus. Virology 1995, 211, 302-306. Robertson, J.S. Clinical influenza virus and the embryonated hen’s egg. Rev. Med. Viral. 1993, 3, 97-106. Katz, J.M. and Webster, R.G. Efficacy of inactivated influenza A virus (H3N2) vaccines grown in mammalian cells or embryonated eggs. J. Infect. Dis. 1989, 160, 191-198. Cell culture as a substrate for the production of influenza vaccines: memorandum from a WHO meeting. Bull. WHO 199573, 431-435. Brands, R., Palache, A.M. and van Scharrenburg, G.J.M. Madin Darby Canine Kidney (MDCK)-cells for the production of inactivated influenza subunit vaccine. Safety characteristics and clinical results in the elderly. In: Options for the Control of influenza III. Proceedings of the Third International Conference on Options for the Control of Influenza, Cairns, Australia, 4-9 May 1996 (Eds Brown, L.E., Hampson, A.W. and Webster, R.G.). Excerpta Medica, 1996, pp. 683-693. Palache, A.M., Brands, R. and van Scharrenburg, G.J.M. lmmunogenicity and reactogenicity of influenza subunit vaccines produced either in Madin Darby Kidney (MDCK)-
Vaccine 1998 Volume 16 Number 9/l 0
967
Vero all derived influenza vaccine: 0. Kistner et al.
11
12
13 14
15
16
17
18
19
20
21
968
cells or in fertilised chicken eggs. J. Infect. Dis. 1997, 176, (suppl. l), 20-23. Montagnon, B.J., Fanget, B. and Nicholas A.J. The largescale cultivation of Vero cells in microcarrier culture for virus vaccine production. Preliminary results for killed poliovirus vaccine. Dev. Biol. Stand. 1987, 47-55. Nakamura, K. and Homma, M. Protein synthesis in Vero cells abortively infected with Influenza B virus. J. Gen. Viral. 1981, 56,199-202. Lau, SC. and Scholtissek, C. Abortive infection of Vero cells by an Influenza A virus (FPV). Virology 1995, 212, 225231. Govorkova, E.A., Murk, G., Meignier, B., De Taisne, C. and Webster, R.G. African Green Monkey Kidney (Vero) cells provide an alternative host cell system for Influenza A and B virus. J. Viral. 1996, 70, 5519-5524. Wood, J.M., Schild, G.C., Newman, R.W. and Seagroatt, V. An improved single-radial-immunodiffusion technique for the assay of influenza haemagglutinin antigen: application for potency determinations of inactivated whole virus and subunit vaccines. J. Biol. Stand. 1977, 5, 237-247. Aymard-Henry, M., Coleman, M.T., Dowdle, W.R., Laver. W.G., Schild, G.C. and Webster, R.G. Influenza virus neuraminidase and neuraminidase-inhibition test procedures. Bull. WHO 1973,48,199-202. Lammli, U.K. Cleavage of structural proteins during the assembly of the head of Bacteriophage T4. Nature 1970, 227, 680-685. Cavalli Sforza, L. Biometric-Grundztige biologisch-medizinischer Statistik 3rd edition. Gustav Fischer, Stuttgart, 1972, pp. 159-186. Yasumura, Y. and Kawakita, Y. Studies on SV40 in tissue culture-Preliminary step for cancer research “in vitro” (translated from Nihon Rinsho 1963, 21, 1201-l 215 in Japanese). In: Vero Cells-Origin, Properties and Biomedical Applications (Eds Simizu, E. and Terasima, T.). Department of Microbiology, Chiba University, Japan, 1988, pp. 1-19. Horaud, F. Absence of viral sequences in the WHO-Vero cell bank: a collaborative study. Develop. Biol. Stand. 1992, 76, 43-46. Vincent-Falquet, J.C., Peyron, L., Souvras, M., Moulin, J.C., Tektoff, J. and Patet, J. Qualification of working cell banks for the Vero cell line to produce licensed human vaccines. Dev. Biol. Stand. 1989, 70, 153-l 56.
Vaccine 1998 Volume 16 Number 9/l 0
22 23
24 25
26
27
28
29
30
31
32
33
34
Levenbook, I.S., Petricciani, J.C. and Elisberg, B.L. Tumorogenicity of Vero cells. J. Biol. Stand. 1984, 12, 391-398. Furesz, J., Fanok, A., Contreras. G. and Becker, B. Tumorogenicity testing of various cell substrates for production of biologicals. Dev. Biol. Stand. 1989, 70, 233-243. European Pharmacopeia 3rd Edition, 1997, 0159 (l-4). Influenza Vaccine (whole virion, inactivated). Points to consider in the characterization of cell lines used to produce biologicals, Office of Biologics Research and Review, Centre for Drugs and Biologics, FDA (USA) 1993. Murphy, B.R. and Webster R.G. Orthomyxoviruses. In: Fields Virology Vol. 1, 3rd edition (Eds. Field, B.N., Knipe. D.M., Howl&, P.M., Chanock, R.M., Melnick, J.L.. Monath, T.P., Roizman. B. and Straus, SE.). Liooincott-Raven. Philadelphia, 1996, pp. 1397-1446. Lazarowitz, S.G. and Choppin, P.W. Enhancement of the infectivity of Influenza A and B viruses by proteolytic cleavage of the hemagglutinin polypeptide. Virology 1975, 68, 440-454. Rott, R., Reinacher, M., Orlich, M. and Klenk, H.-D. Cleavability of hemagglutinin determines spread of avian influenza viruses in the chorioallantoic membrane of chicken embryo. Arch. Viral. 1980, 65, 123-133. Klenk. H.-D.. Rott. R.. Orlich. M. and Bliidorn. J. Activation of influenza A ‘viruses by trypsin treatment. Vir&ogy 1975, 68, 4266439. Stienecke-Grober, A., Vey, M. and Angliker, H. et al. Influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin-like endoprotease. EMBO 1992, 11, 2407-2414. Govorkova, E.A., Kaverin, N.V., Gubareva, L.V., Meignier, B. and Webster, R.G. Replication of influenza A viruses in a green monkey kidney continuous cell line (Vera). J. Infect. Dis. 1995,172,250-253. Kaverin, N.V. and Webster, R.G. Impairment of multicycle influenza virus growth in Vero (WHO) cells by loss of trypsin activity. J. Viral. 1995, 89, 2700-2703. Oxford, J.S., Corcoran, T. and Knott, R. et a/. Serological studies with influenza A(H1 Nl) viruses cultivated in eggs or in a canine kidnev cell line (MDCK). Bull. WHO 1987. 65, 181-187. ’ Katz, J.M., Naeve, C.W. and Webster, R.G. Host cell-mediated variation in H3N2 influenza viruses. Virology 1987, 156, 386-395.
Vaccine. Vol. 16, No. 9/10, pp. 960-968. 1998 Cl 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0264-410X198 $19+0.00
ELSEVIER
Development of a mammalian cell (Vero) derived candidate influenza virus vaccine 0 Kistner”, P.N. Barrett”?, and F. Darner”
W. Mundt*, M. Reiter”, S. Schober-Bendixen”
Influenza vaccine production is dependent on the availability of embryonated hen eggs for virus growth. This is an extremely cumbersome system with many disadvantages with respect to selection of virus variants and presence of adventitious viruses. We have developed an alternative cell culture system which allows rapid production of large volumes of vaccine. The World Health Organisation (WHO) approved Vero cell line was used in serum-free culture to grow a multitude of influenza strains to high titre. This system could be scaled-up to allow vaccine production with a 1200 litre fermenter volume. A purijication scheme was developed which resulted in a high purity whole virus vaccine. This was demonstrated to be at least as immunogenic as a conventional egg-derived preparation in a mouse model. 0 1998 Elsevier Science Ltd. All rights reserved Keywords: Vero cell; influenza vaccine: serum free
Efficient vaccine production requires the growth of large quantities of virus produced in high yields from a readily available, safe host system. Conventional methods for producing influenza vaccine have always involved the growth of the viruses in embryonated chicken eggs. This is a cumbersome process as each egg must be sterilized, candled, inoculated with virus and incubated before harvesting small volumes of allantoic fluid from each egg and pooling before puritication. In a typical chicken embryo operation, between one and two eggs are required for one dose of influenza vaccine. The lack of reliable supplies of high quality eggs results in limitations in the amount of vaccine which may be produced and creates fears that there could be major shortfalls in vaccine supply in case of a major pandemic’, when the demand for vaccine would be much higher. In addition to these logistic and supply problems, embryonated chicken eggs have other livitations as a host system for vaccine production. For example, there are increasing concerns about the presence of adventitious viruses, particularly retroviruses in eggs2, which would compromise the production of live, attenuated influenza virus vaccines. Also it has been reported that variant viruses are selected when human influenza virus is passaged in the egg. This host system induces a selective pressure for variants which grow well in the *Biomedical Research Center, Baxter-lmmuno, Uferstr. 15, Otth/Donau, Austria. tAuthor to whom all corre-
A-2304
spondence should be addressed. (Received 15 July 1997; revised version received 24 October 1997; accepted 28 October 1997)
960
Vaccine
1998 Volume
16 Number
9/l 0
embryonated egg, but which may not be re resentative 35 of the wild type virus circulating in humans . In contrast to influenza viruses grown in eggs, virus propagated exclusively in mammalian-derived tissue culture has been reported to be representative of the natural virus”. Studies in ferrets have also demonstrated that an inactivated influenza vaccine grown in Madin-Darby canine kidney cells (MDCK) induced higher mean serum hemagglutination inhibition (HI) and neutralizing antibody titres than did egg-grown vaccine and induced superior protection against subsequent challenge with infectious virus grown in either cell type’. These reports have demonstrated the possible superiority of a mammalian cell-derived vaccine and emphasize the necessity for a mammalian cell line that could be used to replace chicken eggs in the production of influenza vaccines. However, although influenza viruses can be propagated in several cell lines, none of these have been licensed for vaccine production”. This is because of concerns about tumorogenicity and presence of adventitious viruses in such continuous cell lines. The MDCK cell line appears to be a promising candidate for influenza vaccine production. A master cell bank has been generated, which was extensively characterized and demonstrated to be free of detectable infectious agents of viral or other microbial origin. Tumorogenicity studies also demonstrated that it is highly unlikely that MDCK cells arc able to grow or induce tumors in humans, but the cell line has not as yet been approved for use in human vaccine production. Clinical studies of a MDCK-derived influenza subunit vaccine have, however, been authorized. Such studies have demonstrated that the vaccine is equal
Vero a// derived influenza vaccine: 0. Kistner et al. with respect to immunogenicity and safety to a conventional egg-derived vaccine “.“‘. The continuous monkey kidney cell line, Vera, has however been licensed for use in the manufacture of human vaccines and is used by some manufacturers for the production of polio and rabies vaccines”. Vet-o cells are susceptible to a wide range of viruses, but initial attempts to grow influenza virus on these cells were unsuccessful’-.“. A recent report has however demonstrated that the Vero cell line is a suitable system for isolation and productive replication of influenza A and B viruseslJ. We report here on the development of a Vero cell system which is capable of producing high titre influenza virus at an industrial scale and on the development of a candidate inactivated influenza whole virus vaccine utilizing this system.
lished following a number of passages in this serum free medium and this cell bank was used to generate cell cultures, which were infected with influenza virus. All cultures had been passaged in serum free medium before infection with influenza virus. Vero cells were either propagated as monolayers in Roux or roller bottles or as microcarrier-based fermenter cultures under serum free conditions. Vero cell cultures were inoculated with the different influenza viruses at a multiplicity of infection (m.0.i.) of approximately 0.01 TCID5” cell and allowed to adsorb for 1 h. Porcine trypsin’ (Sigma) was then added with an activity of 20mU ml . Incubation was carried out at 32°C until development of maximum cytopathic effect or for a maximum of 72 h before harvesting. The virus yield was determined by hemagglutination (HA) assay. Growth of influenza virus in eggs
MATERIALS AND METHODS Virus strains The following influen.za viruses were kindly provided by the Center for Disea.se Control (CDC, Atlanta, GA, USA), by the Institute of Virology (University of GielJen, Germany) or by the National Institute for Biological Standards and Control (NIBSC, Potters Bar, London, UK): A/PR/‘8/34 (HlNl) = PR8, A/Brazil/ 11178 (HINl) = Brazil, A/Singapore/6/86 (HlNl) = Singapore 6, A/Taiwan/ l/86 (H 1N 1) = Taiwan, A/Texas/ 36/91 (H 1N 1) = Texas 36, X 113 (high-growth-reassortant Texas 36) A/Singapore /l/57 (H2N2) = A2 Singapore, A/Hongkong/l/68 (H3N2) = Hongkong, A/Hongkong/5/83 (H3N2) = Hongkong 5, A/Shanghai/l6/85 (H3N2) = Shanghai 16, A/Beijing/353/89 (H3N2) = Beijing 353, A/Johannesburg/33/94 (H3N2) = Johannesburg, NIB 34 and RESVIR 8 (high-growth-reassortants Johannesburg), AJWuhan/359/95 = Wuhan, A/ Nanchang/933/95 = Narrchang, RESVIR 9 (high-growthreassortant Nanchang), B/Massachusetts/71 = B/ Massachusetts, B/Panama/45/90 = B/Panama, BIHarbin/ 7194 = B/Harbin. Standard reagents The following reagents were purchased from NIBSC (Potters Bar, London, UK): purified hemagglutinin (HA) antigen from A/Texas 36/91 (HlNl, code 951510) Johannesburg/33/94 (H3N2, code 95/516), A/Nanchang/933/95 (H3N2, code 96/648), B/Harbin/ 7194 (B, code 95/52(l); specific anti-HA antisera directed against A/Texas 36/91 (HlNl, code 92/504), code A/Johannesburg/33/94 (H3N2, 951524) A/Nanchang/933/95 (H3N2, code 961724) BIHarbin/ 7/94 (B, code 95/530) and specific antisera directed against the neuraminidase from Nl, N2 and B strains: Nl (code 96/716), N2 (code 96/696), B (code 96/740). Growth of influenza virus in Vero cells The World Health Organisation (WHO)-certified Vero cell line was used for growth of influenza virus. This cell line has been fully characterized and fulfills all the requirements for (continuous cell lines used for manufacture of biological products. The Vero cells were adapted to growth in a DMEM-based medium which was totally serum free. A cell bank was estab-
Eleven-day-old embryonated hen eggs were inoculated with 0.2 ml of a virus suspension and incubated for 3 days at 33°C. Then, the eggs were cooled overnight at 4°C before harvesting the virus-containing allantoic fluid. Virus titre was determined by HA assay. Large-scale production of influenza virus in microcarrier fermenter culture A single ampoule of Vero cells with a defined passage number was thawed from liquid nitrogen and passaged in Roux and roller bottles to produce sufficient cells to inoculate a 1.5 I fermenter using Cytodex3 (Pharmacia) microcarriers. After a cell number of approximately 5 x 10” I ’ has been obtained, the microcarriers were trypsinized and cells with microcarriers were used as an inoculum for a 12 I fermenter. This in turn was used as an inoculum for a 150 I vessel which is then used to inoculate a 1200 I fermenter. When a cell density of approximately 5 x 10’ cells litre-’ has been achieved, 1.2 1 of virus inoculum was added. This was equivalent to a m.o.i. of 0.01 TCIDs,‘celll’. Incubation was carried out at 32°C for 2-3 days using trypsin at a concentration of 20 mU ml ‘. Hemagglutination (HA)-assay Influenza viruses were diluted in two-fold steps with phosphate buffered saline (PBS) and 50 ~1 of each dilution were added to 50 bll of a 1% suspension of fowl red blood cells in a V-shaped microtitre plate. After gentle agitation, the plates were left undisturbed for 30 min at room temperature and then read. The last dilution showing complete hemagglutination was taken as the end point and was expressed as hemagglutination units (HAU) per test volume (50 /II). Hemagglutination-inhibition assay (HI) Influenza viruses were diluted to give a preparation with 4 HAU per 50 ktl. The antibody preparations were diluted in two-fold steps and 50 cll of each dilution were added to 50 ltl of the virus dilution with 4 HAU in a V-shaped microtitre plate. After gentle agitation, the plates were incubated for 30 min at room temperature. Then 50 ~tl of a 1% suspension of fowl red blood cells were added to each well and the plates were left another 30 min before reading. The reciprocal value of
Vaccine 1998 Volume 16 Number 9/10
961
Vero all derived influenza vaccine: 0. Kistner et al the highest dilution of inhibited hemagglutination HI titre. Determination content
antibody which was determined
of the specific hemagglutinin
completely to hc the
antigen
The concentration of hemagglutinin in the different influenza virus antigen preparations was determined by the single radial immunodiffusion (SRD) as described by Wood et ~1.‘~ using the NIBSC standard reagents. Briefly, influenza virus preparations and reference antigen standards were lysed with Zwittergent, diluted, added to wells in agarose plates containing reference antisera against the HA proteins of the corresponding strains and incubated for 18 h at room temperature. The size of the precipitation zones, which were stained by Coomassie blue, were measured and the HA content of the virus preparations was antigen calculated by slope ratio analysis, plotting the values of the diameters of the precipitation zones against the dilutions of reference antigen and virus preparations. Neuraminidase assay The presence and identity of neuraminidase in virus preparations was determined by neuraminidase activity (NA) and neuraminidase inhibition assay (NI) as described by Aymard-Henry et af.‘” using the NIBSC standard reagents. Briefly, two-fold dilutions of virus preparations were prepared and incubated with fetuin as substrate for 18 hours at 37°C. Following sequential addition of periodate, arsenite, thiobarbituric acid and Warenoff reagents, colour development was measured by spectrophotometric analysis at 550 nm. A standard dose (which gave an extinction of 0.6-0.9) for use in the NI assay was then selected. This standard dose was incubated with serial dilutions of NIBSC anti-neuraminidase antisera and the NA assay was performed again. The antiserum dilution which resulted in a 50% inhibition of neuraminidase activity was thus determined. Preparation of mouse sera against Vero cell proteins Non-infected Vero cells were harvested, adjusted to a concentration of 2 x 10’ cells per ml and lysed in RIPA buffer (0.01 M phosphate buffer, pH 7.2+40 mM sodium fluoride +O.l% SDS +l% TritonX-100+2mM EDTA). Mice were immunized with the lysate equivalent to 2 x lo7 Vero cells in complete Freund’s adjuvant (CFA) and boostered twice at 4 week intervals with the same lysate in incomplete Freund’s adjuvant. Two weeks after the last booster, mice were bled and the derived sera were used for Westernblot analysis. PAGE and Westernblot analysis SDS-PAGE of influenza virus preparations was carried out according to Laemmli17. Westernblot was performed by electrophoretic transfer of the proteins from the gel to polyvinyl difluoride (PVDF) membrane (Millipore) for 90 minutes. After blotting, the membranes were incubated overnight with the specific NIBSC anti-HA antisera, diluted 1:lOOO in TBS/Tween or the mouse serum against Vero cell proteins, diluted
962
Vaccine
1998 Volume
16 Number
9/10
I:100 in TBS/Twcen. After intensive washing the membranes were incubated for 1 h with horseradish peroxidase conjugated rabbit anti-sheep IgG or goat anti-mouse IgG (Chemical Accurates), diluted I:5000 in TBS/Twecn. After washing the membranes again, the blot was developed by adding diaminobenzidinc and HJ02. Ten minutes later the reaction was stopped by transferring the membranes into water.
Virus purification
for antigenicity
studies
Pooled allantoic fluids from virus infected eggs or supernatants from infected Vero cell cultures were clarified by low speed centrifugation, concentrated by ultrafiltration (300 KD cut-off) before purification on a 20-60% continuous sucrose gradient. The peak fractions were pooled, diluted and inactivated with 0.025% (w/v) formalin for 24 h at 32°C. Following diafiltration to remove formalin the HA content was determined by SRD analysis.
Determination of the effective dose 50 (ED,,,) of vaccine preparations Vero cell-derived and egg-derived influenza virus vaccine preparations were diluted with PBS and adjuvanted with aluminium hydroxide [AI(,OH),] to and an give an HA-antigen content of 15 /lg ml AI concentration of 0.2%. These preparations were serially diluted 1:4 with PBS containing 0.2% AI( The HA-antigen concentrations u;ed for immunization were 15 /lg ml , 3.7: /lg ml (1:4), 0.234 /lg ml (164) and 0.938 /Lg ml (1:16), 0.059 /[g ml ’ (1:256). Groups of ten CD1 mice (6-9 weeks old) were injected subcutaneously (s.c.) with 1 ml of each dilution. Control groups were injected with PBS containing 0.2% Al(OH)3. Four weeks after the immunization the mice were boostered with the same antigen preparation and a blood sample was drawn from each mouse. Two weeks after the booster, the mice were bled by heart puncture. One part of the individual sera was used directly for antibody measurement in ELISA, another part was diluted 1:5 in PBS and heat-inactivated for one hour at 56°C before measurement of antibody titre in an HI assay using egg-derived wild type (wt) virus as antigen.
ELISA The ELISA was performed on influenza A virus coated and influenza B virus coated tiicrotitre plates (Genzyme Virotech, Russelsheim, Germany). Each serum sample was diluted 1:lO in buffer (PBS-goat serum, 0.5% Tween 20). One hundred microlitres of each sample were transferred to the wells of the microtitre plates and incubated at 37°C for 1 h. After five washing steps with PBS/Tween, each well of the plate was tilled with 100 itI of horseradish peroxidase conjugated anti-mouse IgG (Chemical Accurates, diluted 1:15 000 in dilution buffer) and incubated for 1 h at 37°C. Again the plates were washed five times using PBS/Tween. Subsequently, 200 111 of OPD substrate (10 mg o-phenylene-diamine hydrochloride) were added per well. After incubation at room temperature
Vero all derived influenza vaccine: 0. Kistner et al. for IO minutes in the dark, the reaction was stopped by addition of 50 111of 5 hl HzS04 and the optical density of each well was measured with a microplate reader (Dynatech) at 495 nm against a reference wavelength of 630 nm. All positions with an optical density of at least two times greater than that of the sera of the control animals, but with an extinction greater than or equal to 0. IO0 were considered as positive.
Statistical analysis The effective dose nlecessary to induce seroconversion in 50% of mice, measured either by ELISA or by HI assay was determined by logit analysis. The parameters of the model were estimated by the maximum likelihood method applying Newton algorithm’“.
RESULTS Vero cell line safety The Vero cell line was established in 1962 by Yasumura and Kawakita’” from the kidney of a normal adult African Green monkey. At present, the cell line is distributed by the American Type Tissue Collection (ATCC) at passage level 124 under the designation ATCC CCL 81. The WHO Vero IO-87 cell bank has been prepared by the Institut Merieux with passage level 134 and has been extensively characterized for absence of viral contamination and lack of tumorogenicity at this passage level”‘~‘“. The master cell bank (MCB) used for the production of influenza virus as described here was additionally fully characterized for the presence of a range of specific adventitious viruses. The data presented in 7”ahfr I demonstrate that the cell bank was found to be free of retroviruses, human papilloma virus, Epstein-Barr virus, cytomegalovirus and bovine viral contaminants. In addition the MCB did not contain any infectious agent of viral or other microbial origin as determined by microbiologic tests and tests in tissue culture, baby mice, adult mice, guinea pigs, rabbits and embryonated specific pathogen free (SPF) eggs. The working cell bank (WCB) and production cell bank (PCB) were similarly tested for bacterial and mycotic sterility, mycoplasma, mycobacterium tuberculosis, adventitious viruses and were found to be free of such contaminants. In addition Vero cells from the MCB did not produce palpable tumors following inoculation of athymic nude mice and observation for a 3-week period. Table 1
Virus yield in Vero cell culture The ability of a wide range of influenza A and B virus strains to grow in Vero cells under serum-free conditions was determined. Monolayer cultures in I75 cm’ Roux bottles were infected with 0.1 ml of egg-derived virus (m.o.i. 0.01 TCID,,, cell ‘) and after adsorption ,100 ml of serum free medium containing of trypsin were added. Incubation was 20 mU ml carried out under the conditions described previously and following harvesting the HA titre in cell culture medium was determined. A direct comparison was made with the virus yield obtained following infection of embryonated eggs with the same viruses. The data presented in Table 2 demonstrate that all strains tested could grow to high titre in Vero cells, with HA titres in the range of 128-256 being obtained for all strains. These titres were in almost all cases lower than those obtained in the embryonated egg, but a larger volume was obtained from the Vero cell culture (100 ml) than from the embryonated egg (circa 7 ml). The discrepancy in titre between the two systems was most evident for the high growth (HG) reassortant strains. The HG strains X 113, NIB 34, RESVIR 8 and RESVIR 9 gave HA titres between 1024 and 2048 in the embryonated egg whereas growth in Vero cell culture resulted in an HA titre of 256 for all four strains. The lowest m.o.i. required to achieve maximum virus titre in Vero culture was also investigated and determined to be approximately 0.001 TCIDS,, cellI’. With this m.o.i. the maximum titre was achieved 2-3 days post-infection. Infection with lower m.o.i.s resulted in maximum titres being achieved 5-6 days post-infection. Production of influenza virus vaccine strains in industrial scale fermenter systems Following the demonstration that a range of influenza strains could be grown to high titre in Vero cells, studies were carried out to develop a system for industrial scale vaccine production. Virus antigen production was investigated using a 1200 litre microcarrier culture fermenter system as described above. The growth of the influenza vaccine strains in use for the 1995/1996 and 1996/1997 seasons, i.e. Texas 36 (A/HlNl), Johannesburg (A/H3N2), Nanchang (A/H3N2) and BiHarbin (B), was investigated in this system. All four strains yielded HA titres of 256 in a total volume of 1200 I cell culture medium supernatant. These titres were equal to or higher than the titres obtained in monolayer cultures, demonstrating that the Vero cell technology
Analysis of the Vero master cell bank for adventitious viruses
Test
Result
Reverse transcriptase assay Human papilloma virus Epstein-Barr virus (EBV) detection Epstein-Barr nuclear antigen (EBNA) test Electron microscopy (negative stain) Thin-section electron microscopy In vitro assay for detection oi non-murine viral contaminants In viva assay for non-murine viral contaminants Detection of cytomegalovirus (CMV) by co-cultivation assay Induction of retroviral particles by Siododeoxyuridine Extended assay for bovine virus detection
Negative for Mn2+ and Mg*+ dependent reverse transcriptase assay Negative for HPV types 6, 11, 16, 18, 31, 33 and 35 EBV DNA was not detected Negative for EBNA Viral particles were not observed Viral particles were not detected No evidence of viral contamination was found No evidence of viral contamination was found CMV not detected No retroviral induction after treatment with Siododeoxyuridine No bovine viral contaminants were detected
Vaccine
1998 Volume
16 Number
9/10
963
Vero all derived influenza vaccine: 0. Kistner et al Table 2 Comparison of HA-titres obtained for different influenza virus strains grown in Vero cell cultures and in embryonated eggs Hemagglutinating Units (HAU)
Neuraminidase
Type/Subtype
Strain
Vero
Egg
A/HlNl
PR 8 Brazil Singapore 6 Taiwan Texas 36 x113 A2 Singapore Hongkong Hongkong 5 Shanghai 16 Beijing 353 Johannesburg NIB 34 RESVIR 8 Wuhan Nanchang RESVIR 9 B/Massachusetts B/Panama B/Harbin
256 128 128 128 128 256 128 128 128 256 256 256 256 256 256 256 256 128 128 256
1024 1024 128 512 256 2048 512 1024 256 128 256 128 1024 2048 512 512 2048 512 256 512
A/H2N2 A/H3N2
B
could be scaled virus yield.
up successfully
without
reduction
in
Comparison of the antigenicity of Vero cell- and egg-derived virus following immunization of mice For a comparison of the antigenicity of Vero celland egg-derived virus, mice were immunized with inactivated influenza virus preparations derived from both systems. The 1995/1996 vaccine strains Texas 36, Johannesburg and B/Harbin received as standard WHO egg isolates were passaged at least five times in the Vero cell line, and then purified by sucrose gradient centrifugation as described and inactivated by treatment with 0.025% (w/v) formalin at 32°C for 24 h. The corresponding strains X 113 (Texas 36, HG reassortant), NIB 34 (Johannesburg HG reassortant) and B/Harbin were subjected to the same number of passages in eggs and purified and inactivated identically. All preparations were adjusted to a specific HA content of 15 /fg, adjuvanted with A1(OH)3 and inoculated S.C. into groups of ten mice. Four weeks after primary immunization, the mice were boostered with the same formulation and 2 weeks later they were sacrificed. The sera from individual mice in each group were pooled and the HI titres from each pool were determined using both Vero cell-derived and egg-derived HA antigen in the assay. The data shown in Table 3 demonstrate that almost identical HI titres were obtained for the specific mouse sera against the corresponding Vero cell-derived and egg-derived influenza antigen, with little or no cross-reaction with antigen from the non-corresponding strains. For example, serum derived from mice immunized with Vero cell-derived Texas 36 had HI titres of 2560 when measured against both Vero cell and egg-derived Texas 36 antigen and had little reactivity against the Johannesburg and B/Harbin antigens. Similar results were obtained for the Johannesburg and B/Harbin strains,
964
Vaccine 1998 Volume 16 Number 9/l 0
demonstrating that limited passage not significantly alter the antigcnicity
in Veto cells did of thcsc strains.
characterization
Further characterization of the Vcro cell-derived virus was carried out by means of a comparison of the ncuraminidase antigenic properties with that of the egg-derived virus. The neuraminidasc was charactcrizcd by means of the ncuraminidase inhibition assay using specific anti-neuraminidasc scra as described above. These standard scra obtained from NIBSC, Potters Bar, were raised against ncuraminidasc from egg grown virus. The vaccine strains for the 1996/lY97 season, i.e. Texas 36, Nanchang and BiHarbin, were characterized in this manner. The data presented in Tuhle 4 demonstrated that no significant diffcrcnces could be detected between the Vero- and egg-dcrivcd virus strains in this assay. The same anti-N1 titrcs were measured for the specific set-a against both the Veto cell- and egg-derived HlNl strain and also for the N2 serum against the H3N2 strain. The anti-B serum, however, had a somewhat higher titrc against the Vcro cell-derived virus (10000) than against the egg-derived virus (3000). Also, the specific sera had little or no reactivity against the heterologous virus strains. Development of an inactivation and purification scheme for a whole virus vaccine Following the demonstration that the Vero ccl1 technology could be scaled up to a level required for industrial production, a purification scheme was established to provide a vaccine which fulfilled all the regulatory requirements for influenza whole virus vaccines. The following scheme was established for large scale routine production: the supernatants from the microcarrier cultures were clarified using a separator (Westfalia 4SA8, 5000 g, 280 I h ‘), before treatment with benzonase (1 000 units I ‘) to destroy Vero cell DNA, followed by viral inactivation with formalin (0.025% w/v end concentration) for 24 h at 32°C. The infectivity of Texas 36 and BiHarbin was reduced below the level of detection after 1 h, and that of Nanchang after 2 h formalin treatment. The inactivated material was then concentrated by ultrafiltration (200000 Da m.w. cut-off) and further clarified by protamine sulphate precipitation before purification by continuous flow zonal centrifugation over a O-50% sucrose gradient. The virus containing peak pool was then subjected to ultrafiltration and sterile filtration. Following measurement of the HA content and appropriate dilution, the vaccine was formulated with all three virus strains. The efficacy of this purification procedure in removing contaminating Vero cell protein was demonstrated by Western blot analysis of materials drawn from successive production and purification steps of the Nanchang strain. Western blot analysis using specific Nanchang anti-HA serum (NIBSC) illustrates the purification scheme used to obtain puritied material with well defined HA1 and HA2 bands (lane 5 and 6, Figure la). Analysis using an anti-Vero cell serum demonstrated that a variety of Vero specific bands were present until after the sucrose gradient purification (lane 5 and 6, Figure Ib). No Vero cell protein bands could be detected using this methodology, subsequent to this purification step.
Vero all derived influenza vaccine: 0. Kistner et al. Table 3 Hemagglutination-inhibition
(HI) titre of mouse sera immunized with different influenza virus vaccine preparations against the corresponding Vero cell-derived and egg-derived influenza viruses HI assay antigen Texas 36
Johannesburg
Type/subtype
Immunization antigen (serum)
Source
Vero
Egg
Vero
Egg
A/HlNl
Texas 36 Xl 13 (HG Texas 36) Johannesburg NIB 34 (HG Johannesburg) B/Harbin B/Harbin
Vero cells Egg Vero cells Egg Vero ceils Egg
2560 640 10 10 10 10
2560 640 40 40 10 40
10 10 5120 2560 10 10
40 80 5120 1280 40 80
A/H3N2 B
Comparison of the immunogenicity egg-derived vaccine strains
of Vero cell- and
The immunogenicity of Vero cell- and egg-derived vaccine strains for the lYY5/1996 season, i.e. Texas 36, Johannesburg and B/Ha&in, was compared by potency by studies in mice. Potency was determined immunizing mice with various dilutions of a vaccine strain and calculating the effective dose (ED,,,) which results in seroconversion in 50% of the mice. The EDS,, values calculated on the basis of the influenza ELISA titres and the HI titrcs are summarized in Tuhfr 5. In all cases the Vero cell-derived vaccine was more immunogenic than the #equivalent egg-derived vaccine strain. The EDSo values for all strains were between two to four-fold lower for the Vero-derived than those for the equivalent egg-derived strain, when calculated on the basis of induction of binding antibodies measured by ELISA. For example the values obtained for Vero- and egg-derived Texas 36 were 71 and 204 ng, respectively, i.e. a three-fold difference in potency. The potency values obtained on the basis of the HI assay determinations also demonstrate that the Vero cell-derived material was more immunogenic than the egg-derived vaccine. However, the differences varied between a slighl: advantage for the B/Harbin strain, i.e. 140 ng compared with 162 ng and a nine-fold difference for the Texas 36 strain.
B/Harbin Vero 10 10 10 10 160 160
Egg 10 10 40 40 320 320
consisting of 15 jig of HA antigen was determined (Tahlr 6). Further characterization vaccine
of the Vero cell-derived
Prior to the initiation of efficacy studies in humans, the vaccine was subjected to the standard tests required by the European Pharmacopoeia for egg-derived vaccinesZ4, to the tests required for products derived from continuous cell lines-’ and to a 1
2345678
1
2345678
(a) Influenza vaccine yield obtained using the serum free Vero cell technology
The vaccine strains in use for the 1995/1996 and 1996/1997 seasons were all grown in a 1200 I serumfree Vero cell microcarrier culture fermenter and the antigen yield was measured. All four virus strains achieved a HA titre of 256 under these conditions. The purification procedure ‘was carried out as previously described and the amount of doses of purified vaccine Table 4 Neuraminidase
inhibition (NI) titre of Vero cell-derived and egg-derived influenza virus vaccine strains against NIBSC neuraminidase specific antiserum NIBSC anti-neuraminidase antiserum
Virus
Source
Texas 36 (A/HlNl)
Egg Vero
Nanchang (A/H3N2)
Egg Vero
B/Harbin (B)
Egg Vero
Anti-N1
Anti-N2
300 300 30 <30 30 <30
30 <30 3000 3000 30 <30
Anti-NB 30 <30 30 <30 3000 10000
@I Figure 1 Westernblot analysis of the purification steps involved in the manufacture of Vero cell-derived Nanchang using (a) specific anti-HA serum and (b) specific anti-Vero cell serum. Lane 1: virus harvest, lane 2: after formalin inactivation, lane 3: after ultrafiltration, lane 4: after protamine sulphate precipitation, lanes 5, 6: purified peak fractions from sucrose gradient, lane 7: Vero cell lysate, lane 8: egg-derived Nanchang (NIBSC)
Vaccine 1998 Volume 16 Number 9/l 0
965
Vero all derived influenza vaccine: 0. Kistner et al. Table 5
Comparison of Vero cell-derived and egg-derived formalin inactivated influenza virus vaccine preparations dose (ED,,) required for seroconversion of mice, as determined by ELISA or by hemagglutination inhibition (HI) assay
in terms of effective
EDs0 (ng) Type/subtype
Antigen
Source
ELISA
HI
A/H1 Nl
Texas 36 Xl 13 (HG Texas 36) Johannesburg NIB 34 (HG Johannesburg) B/Harbin
Vero Egg Vero Egg Vero Egg
71 204 29 50 35 153
120 1092 38 89 140 162
A/H3N2 B
number of additional tests, such as pyrogenicity testing which are normally not required for influenza vaccines. The results of these tests are detailed in Tahfr 7 and demonstrate that the vaccine fulfills all of these requirements, e.g. the DNA content was below the level required for vaccines derived from continuous cell lines (100 pg dose ‘).
DISCUSSION Influenza viruses exhibit a strict species specificity and organ tropism, which is illustrated by their inability to replicate in a large number of different cell types’“. The exact mechanisms for this restriction in specific cell lines are not fully understood, but it is known that cleavage of the precursor hemagglutinin protein by an intracellular protease into the HA1 and HA2 subunits is essential for a productive infection27,2x. When a cell contain an appropriate enzyme, does not non-infectious virus with uncleaved hemagglutinin is produced. However, it has been reported that addition Table 6 Vaccine yield obtained from a 1200 I Vero cell fermenter
Virus strain
Maximum HA titre in cell medium
A/Texas 36 (HlNl) A/Johannesburg (H3N2) A/Nanchang (H3N2) B/Harbin
256 256 256 256
Table 7
Control
of trivalent
bulk
Total HA yield in purified vaccine (9)
Vaccine dose equivalents
2.80 3.07 3.15 4.98
186 660 204 660 210000 332 000
vaccine
and
final
container
product Test
Release criteria per dose
Bacterial and mycotic sterility Haemagglutinin content (SRD-assay) Protein content Sucrose content Residual protamine sulphate Residual formaldehyde Residual benzonase Residual DNA Endotoxin content &AL-assay) Pyrogenicity Abnormal toxicity Identity-haemagglutinin antigen Identity-neuraminidase antigen
Sterile 2 15 ,rg I 250 jig SlOmg 55 jrg < 5 /rg 55mg
966
Vaccine
1998 Volume
16 Number
9/10
of trypsin or a trypsin-like enzyme to the culture medium can result in extracellular cleavage and generation of infectious virus particles”‘.3”. Previously, the only classes of cultured cells used for the production of licensed human viral vaccines were primary cells and human diploid cells. Continuous cell lines were not used because of fears concerning the potential tumorogenicity and viral contamination of such cell lines. Vero cells, a continuous African Green monkey kidney cell line, have been extensively tested in this respect and a cell bank approved by WHO has been established”. This has been demonstrated to be free of viral adventitious agents and to have no tumorogenic potential at this passage level”‘-‘3. We have utilized this WHO approved cell stock to make a master cell bank. which was passaged exclusively under serum-free conditions. This cell bank has again been extensively tested and demonstrated to be free of viral adventitious agents (Tuhfr I). Testing in nude mice also demonstrated that the cell line had no tumorogenic potential at this passage level and was thus an acceptable substrate for the manufacture of human vaccines. It had, however, been previously reported that influenza A and B viruses were restricted in their growth in Vero cells”.‘3. Recent studies’“.“‘.“‘, however, have demonstrated that repeated addition of trypsin to the culture medium of influenza virus-infected Vero cells restores the multiple growth attern of influenza A and P B strains. Govorkova et al.’ demonstrated that of 72 egg-derived influenza A strains, 00% replicated to detectable HA levels in trypsin treated Vero cells after the first passage and 51% after the second passage, but no data was provided on the virus titres obtained for these strains. We report here on the ability to grow 100% of strains tested to high titre in a serum-free Vero cell culture. All of 20 strains tested gave HA titres of 128 or 256 in Vero cell culture (Tuhlr 2). Although these titres were somewhat lower than those obtained in the egg following infection with the same volume of virus, the total amount of antigen obtained in the Vero cell culture was greater due to the larger volume of medium obtained from cell culture (100 ml) compared to 7-8 ml per egg. The lowest m.o.i. required to generate maximum virus titre in the Vero system after 2-3 days incubation was determined to be in the order of 0.001 TCID,,‘cell ‘. It was not possible to make a direct comparison with the m.o.i. required for maximum virus production in the egg, due to difficulties in estimating the cell number in the egg tissue. It is, however, likely to be lower than 0.001 TCIDS,, cell ‘. i.e. the efficiency of virus production is likely to be higher in the egg system. The Vero cell system can, however, be very successfully utilized in
Vero all derived influenza vaccine: 0. Kistner et al.
large volume fermenters with subsequent advantages for industrial production. This system could he successfully scaled up to a 1200 I fermenter volume without loss in virus antigen yield (Trrhke 6) and permits for the first time the industrial scale production of influenza vaccine in an approved continuous cell line. This is of particular signiticancc in that it allows production of large quantities of vaccine using wild-type (wt) virus, i.e. without the necessity for generation of high growth reassortants. This will allow rapid production of vaccine should a pandemic strain of influenza appear, and could greatly contribute to the control of such a pandemic. Antigenic diffcrenccs have been frequently observed between the HAS of influenza viruses that had been isolated from the same clinical sample and cultivated exclusively in MDCK cells or adapted to egg growth. In addition the viruses could be clearly distinguished on the basis of their reaction with various monoclonal antibodies3’,34. We have compared the antigenicity of virus which was originally isolated in eggs and further passaged in Vero cells with that of virus which was exclusively passaged in eggs. No differences were seen in the titres of mOuse scra immunized with either eggor Vero-derived antigen, when tested in HI assays using Vera- and egg-derived antigen (7X& 3). These tindin s are in agreement with the data of Govorkova et al. b who reporteld no differences in the HA reactivity between a parental egg-derived HlNl strain and its serially Vero cell-passaged strain. Sequence analysis of the HAI by these authors also demonstrated identity of the amino acid sequence with the parental strain after 20 passages in Vero cells. Comparison of the potency of egg- and Vero cellderived antigen demonstrates, however, that the Vero cell-derived antigen has a two to four-fold increased immunogenicity on the basis of ELISA antibody measurements (Table 5). This could be due to an improved antigenicity when HA is presented associated with Vero cell membranes as opposed to chick cell membranes. It should, however, be considered that this apparent improvement could be artifactual. Determinations of HA antigen are carried out by the SRD assay using the standard antisera which are used for measurement of HA in egg-derived vaccines. These antisera are derived by immunization with egg-derived antigen and all calibration curves are constructed with these reagents. Thus it is feasible that the size of a precipitation zone generated by interaction of Veroderived antigen with specific sera raised against egg-derived antigen may not reflect the real HA antigen content of Vero-derived material. This possibility is now being investigated by the establishment of a SRD assay with antisera raised following immunization with Vero-derived HA antigen. The demonstration that the potency of the egg-derived antigen was three or ten-fold lower on the basis of the HI titre for the A strains may also not imply a real difference. This may be due to the fact that whereas immunization was carried out with the HG reassortant, the HI titre is always determined using the egg-derived wt virus antigen. Thus we see no major difference in potency for the B strain on the basis of the HI titre determinations, as immunization and HI
assay are carried out with the egg-derived wt virus antigen. A real improvement in immunogenicity may, however, be achieved when the original isolation of virus is carried out in Vero cells, thus preventing the generation of variants which are selected en the basis of their ability to grow well in eggs, but may not be representative of the circulating wt virus population. Prior to initiation of human trials, it was essential to demonstrate that the Vero cell-derived vaccine fulfilled all requirements as determined by the European the WHO and other regulatory Pharmacopoiea, organizations’4.‘5. The vaccine was demonstrated to fulfill all such requirements (Tahke 7) and clinical trials have now been initiated with a trivalent vaccine consisting of Amexas 36 (H 1N 1), A/Nanchang (H3N2) and BiHarbin in the UK and Austria.
ACKNOWLEDGEMENTS The authors are grateful to Dr J. Wood, NIBSC, Potters Bar, UK for provision of multiple virus strains, antisera and antigen reagents, for his generous assistance in establishing the SRD assay and for many fruitful discussions. The support of Mr Robert Newman, NIBSC, Potters Bar, UK in establishing the NA assay is also gratefully acknowledged. We would like to thank Dr Nancy Cox, CDC, Atlanta, USA and Dr Christoph Scholtissek, University of GieOen, Germany for the provision of many virus strains. The authors are grateful to Frau Stefanie Fabi for her support in preparing this manuscript.
REFERENCES 1 2
3
4
5
6 7
8
9
10
Rowe, P.M. Pandemic influenza (un)preparedness described. The Lancer 1995, 346, 1699. Petricciani, J.C. Regulatory philosophy and acceptability of cells for the production of biologicals. Dev. Biol. Stand. 1991, 75, 9-15. Schild, G.C., Oxford, J.S., De Jong, J.C. and Webster, R.G. Evidence for host-cell selection of influenza virus antigenic variants. Nature (London) 1983, 303, 706-709. Robertson, J.S., Bootman, J.S. and Newman, Ft. et al. Structural changes in the haemagglutinin which accompany egg adaptation of an influenza A (Hl Nl) virus. Virology 1987, 160, 31-37. Hardy, C.T., Young, S.A., Webster, R.G., Naeve. C.W. and Owens, R.F. Egg fluids and cells of the chorioallantoic membrane of embryonated chicken eggs can select different variants of Influenza A (H3N2) virus. Virology 1995, 211, 302-306. Robertson, J.S. Clinical influenza virus and the embryonated hen’s egg. Rev. Med. Viral. 1993, 3, 97-106. Katz, J.M. and Webster, R.G. Efficacy of inactivated influenza A virus (H3N2) vaccines grown in mammalian cells or embryonated eggs. J. Infect. Dis. 1989, 160, 191-198. Cell culture as a substrate for the production of influenza vaccines: memorandum from a WHO meeting. Bull. WHO 199573, 431-435. Brands, R., Palache, A.M. and van Scharrenburg, G.J.M. Madin Darby Canine Kidney (MDCK)-cells for the production of inactivated influenza subunit vaccine. Safety characteristics and clinical results in the elderly. In: Options for the Control of influenza III. Proceedings of the Third International Conference on Options for the Control of Influenza, Cairns, Australia, 4-9 May 1996 (Eds Brown, L.E., Hampson, A.W. and Webster, R.G.). Excerpta Medica, 1996, pp. 683-693. Palache, A.M., Brands, R. and van Scharrenburg, G.J.M. lmmunogenicity and reactogenicity of influenza subunit vaccines produced either in Madin Darby Kidney (MDCK)-
Vaccine 1998 Volume 16 Number 9/l 0
967
Vero all derived influenza vaccine: 0. Kistner et al.
11
12
13 14
15
16
17
18
19
20
21
968
cells or in fertilised chicken eggs. J. Infect. Dis. 1997, 176, (suppl. l), 20-23. Montagnon, B.J., Fanget, B. and Nicholas A.J. The largescale cultivation of Vero cells in microcarrier culture for virus vaccine production. Preliminary results for killed poliovirus vaccine. Dev. Biol. Stand. 1987, 47-55. Nakamura, K. and Homma, M. Protein synthesis in Vero cells abortively infected with Influenza B virus. J. Gen. Viral. 1981, 56,199-202. Lau, SC. and Scholtissek, C. Abortive infection of Vero cells by an Influenza A virus (FPV). Virology 1995, 212, 225231. Govorkova, E.A., Murk, G., Meignier, B., De Taisne, C. and Webster, R.G. African Green Monkey Kidney (Vero) cells provide an alternative host cell system for Influenza A and B virus. J. Viral. 1996, 70, 5519-5524. Wood, J.M., Schild, G.C., Newman, R.W. and Seagroatt, V. An improved single-radial-immunodiffusion technique for the assay of influenza haemagglutinin antigen: application for potency determinations of inactivated whole virus and subunit vaccines. J. Biol. Stand. 1977, 5, 237-247. Aymard-Henry, M., Coleman, M.T., Dowdle, W.R., Laver. W.G., Schild, G.C. and Webster, R.G. Influenza virus neuraminidase and neuraminidase-inhibition test procedures. Bull. WHO 1973,48,199-202. Lammli, U.K. Cleavage of structural proteins during the assembly of the head of Bacteriophage T4. Nature 1970, 227, 680-685. Cavalli Sforza, L. Biometric-Grundztige biologisch-medizinischer Statistik 3rd edition. Gustav Fischer, Stuttgart, 1972, pp. 159-186. Yasumura, Y. and Kawakita, Y. Studies on SV40 in tissue culture-Preliminary step for cancer research “in vitro” (translated from Nihon Rinsho 1963, 21, 1201-l 215 in Japanese). In: Vero Cells-Origin, Properties and Biomedical Applications (Eds Simizu, E. and Terasima, T.). Department of Microbiology, Chiba University, Japan, 1988, pp. 1-19. Horaud, F. Absence of viral sequences in the WHO-Vero cell bank: a collaborative study. Develop. Biol. Stand. 1992, 76, 43-46. Vincent-Falquet, J.C., Peyron, L., Souvras, M., Moulin, J.C., Tektoff, J. and Patet, J. Qualification of working cell banks for the Vero cell line to produce licensed human vaccines. Dev. Biol. Stand. 1989, 70, 153-l 56.
Vaccine 1998 Volume 16 Number 9/l 0
22 23
24 25
26
27
28
29
30
31
32
33
34
Levenbook, I.S., Petricciani, J.C. and Elisberg, B.L. Tumorogenicity of Vero cells. J. Biol. Stand. 1984, 12, 391-398. Furesz, J., Fanok, A., Contreras. G. and Becker, B. Tumorogenicity testing of various cell substrates for production of biologicals. Dev. Biol. Stand. 1989, 70, 233-243. European Pharmacopeia 3rd Edition, 1997, 0159 (l-4). Influenza Vaccine (whole virion, inactivated). Points to consider in the characterization of cell lines used to produce biologicals, Office of Biologics Research and Review, Centre for Drugs and Biologics, FDA (USA) 1993. Murphy, B.R. and Webster R.G. Orthomyxoviruses. In: Fields Virology Vol. 1, 3rd edition (Eds. Field, B.N., Knipe. D.M., Howl&, P.M., Chanock, R.M., Melnick, J.L.. Monath, T.P., Roizman. B. and Straus, SE.). Liooincott-Raven. Philadelphia, 1996, pp. 1397-1446. Lazarowitz, S.G. and Choppin, P.W. Enhancement of the infectivity of Influenza A and B viruses by proteolytic cleavage of the hemagglutinin polypeptide. Virology 1975, 68, 440-454. Rott, R., Reinacher, M., Orlich, M. and Klenk, H.-D. Cleavability of hemagglutinin determines spread of avian influenza viruses in the chorioallantoic membrane of chicken embryo. Arch. Viral. 1980, 65, 123-133. Klenk. H.-D.. Rott. R.. Orlich. M. and Bliidorn. J. Activation of influenza A ‘viruses by trypsin treatment. Vir&ogy 1975, 68, 4266439. Stienecke-Grober, A., Vey, M. and Angliker, H. et al. Influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin-like endoprotease. EMBO 1992, 11, 2407-2414. Govorkova, E.A., Kaverin, N.V., Gubareva, L.V., Meignier, B. and Webster, R.G. Replication of influenza A viruses in a green monkey kidney continuous cell line (Vera). J. Infect. Dis. 1995,172,250-253. Kaverin, N.V. and Webster, R.G. Impairment of multicycle influenza virus growth in Vero (WHO) cells by loss of trypsin activity. J. Viral. 1995, 89, 2700-2703. Oxford, J.S., Corcoran, T. and Knott, R. et a/. Serological studies with influenza A(H1 Nl) viruses cultivated in eggs or in a canine kidnev cell line (MDCK). Bull. WHO 1987. 65, 181-187. ’ Katz, J.M., Naeve, C.W. and Webster, R.G. Host cell-mediated variation in H3N2 influenza viruses. Virology 1987, 156, 386-395.