Biozogicuzs
(1990)
18, 305-308
The Effect of Amphiphilic Polymers on Growth of Berm/a pertussis and Production of 6. pertussis Haemagglutinins H. E. Nlkolajewski,* H. Liepert,* G. Nikoiajewski,t U. Pfiiller,* S. Swidsinski* and G. Wettig* *Staatliches lnstitut ftir Immunpriiparate und Nahrmedien, Klement-Gottwald-A/lee 317-321, Berlin, 1120, L? 0. R. tZentra/institut fijr physikalische Chemie der Akademie der Wissenschaften der D. D. R., Rudower Chaussee 5, Berlin, 1199, D. D. R.
Abstract. The effect, upon addition to a culture medium of amphiphilic polymers such as partly hydrolysed polyvinyl acetate (PVAc) or partly methylated cellulose (MeCel) on growth and production of haemagglutinins-the filamentous haemagglutinin (FHA) and the pertussis toxin (PT)-has been investigated. As a result, an increase of HA-titer and PT could be achieved. Besides these effects the addition of the amphiphilic polymers stimulated growth of B. pertussis. The above results are comparable to, or even better than, those obtained by addition of Heptakis (2,6-O-dimethyl) /3-cyclodextrin (Me+CD) or polyvinyul alcohols (PVA) which have already been described as compounds to enhance growth and yield of haemagglutinins.
Introduction
haemagglutinins. 5 Starting from this discovery the Japanese manufacturers were then able to produce both haemagglutinins more conveniently in stirred tank cultures.‘j A drawback of the use of Me-&CD however is its price which may increase the cost of vaccine production. The primary purpose of the study presented here has been to look for substances which are highly effective in stimulating formation of haemagglutinins, yet available at a reasonable price. The mechanism which leads to the stimulating effect of Me-P-CD is not yet fully understood. The following interpretation however may serve as a basis to aid the search for other substances giving rise to a similar effect. Thus, Me-@-CD may capture within its hydrophobic cavity substances which are inhibitory to growth of B. pertussis and formation of PT and FHA or alternatively carry and transfer substances supporting growth and antigen yield. The stabilization of the suspension of B. pertussis during cultivation may also play an important role in enhancing growth and antigen yield. Amphiphilic polymers with their folded structure in aqueous solution are also likely to trap substances in their hydrophobic pockets and should likewise stabilize bacterial suspensions.
Pertussis vaccines have been used for more than 50 years. By far the most widely used type of pertussis vaccine consists of whole bacterial cells which are killed by either heat or chemical treatment. Since, however, the cellular pertussis vaccine may occasionally give rise to adverse reactions, the benefits of the vaccine in comparison with the risk accompanying vaccination have come into question. Due to this problem-especially in Japan-considerable efforts have been undertaken in recent years to produce a safer vaccine consisting of purified antigenic components.’ For the acellular vaccine the two structurally different haemagglutinins-the filamentous haemagglutinin (FHA) and the pertussis toxin (PT)-play an important role as components.23 The early methods to isolate both haemagglutinins suffered from the very ineffective method of their production by means of static cultures.4 Consequently, for economic reasons, particular attention was paid by Japanese manufacturers to produce PT and FHA with reasonable yields. Towards a large scale production major progress was achieved by introducing Heptakis (2,&Odimethyl) @yclodextrin (Me-D-CD) to promote cell growth and synthesis of 10461056/90/040305+04
$03.00/O
0
1990 The
International
Association
of Biological
Standardization
306
H. E. Nikolajewski
Much of this paper describes shaker flask cultivations of B. pertussis in the presence of amphiphilic polymers such as methyl cellulose (MeCel) and partly hydrolysed polyvinyl acetate (PVAc) to investigate their capability to enhance growth ofB. pertussis and production of haemagglutinins.7 MeCel and PVAc are commercially available and find widespread use as emulsifiers and stabilizers of suspensions in the pharmaceutical, paint and food industries. For comparative studies Me-p-CD and polyvinyl alcohol (PVA) were used in some sets of experiments, because the latter has proven to be enhansive with respect to cell and antigen yield.’ Materials
and methods
Culture medium
Bordetella pertussis strain Tohama phase I was grown in a Cohen-Wheeler medium with the addition of O-75 g glutamic acid per 100 ml medium. In order to avoid growth limitation after the depletion of the carbon source during cultivation, 05 g glutamic acid was fed to a 100 ml culture medium when cell density reached approximately 140-150 IOU/ml. Either amphiphilic polymers or Me-b-CD were then added to the culture medium. Partly hydrolysed polyvinyl acetate (PVAc) ‘Mowio118-88’ and pure polyvinyl alcohol (PVA) were obtained from Hoechst (Frankfurt am Main, F.R.G.). Methyl cellulose (MeCel) ‘Methocel K 100’ was obtained from Dow Chemical (Frankfurt am Main, F.R.G.) and Heptakis (2,6-O&methyl) /3-cyclodextrin was from the SIGMA Chemical Company (St. Louis, U.S.A.). Method of cultivation
A one litre Erlenmeyer flask containing a 100 ml culture medium was used for cultivation. A seed of B. pertussis was added to give a final cell concentration of 8 x 10’ cells/ml being equivalent to 8 IOU/ml. Cultivation was carried out on a reciprocal shaker at 100 strokes per minute at 35°C. Cell concentration
The cell concentration was determined trophotometer (Specol 11, Carl-Zeiss-Jena, 530 nm
by a specG.D.R.) at
Total PT content
After solubilization of the B. pertussis suspension, by treatment with lysozym, the cellbound, together with excreted PT, was measured.
et sl.
The total PT content was determined by the HpElisa-technique developed by Sato et ~1.’ As a reference, purified PT was used which was a generous gift from Yuji Sato (National Institute of Health, Tokyo, Japan). Haemagglutinin test
After removing cells by centrifugation, the HAactivity of the supernatant was assayed by means of fresh, washed, sheep erythrocytes. Two-fold serial dilutions of each sample were prepared in 0.05 ml PBS volumes in round bottomed microtiter plates. An equal volume of a 0.5% suspension of red blood cells was added and the mixture was incubated at room temperature for 3-4 h. The HAtiter of the samples was defined as the reciprocal of that dilution which caused complete agglutination. Results and discussion The data in Table 1 demonstrate strikingly that the use of PVAc and MeCel assists the growth of B. pertussis and the formation of haemagglutinins hence, a stimulating effect has been found independent of the particular cultural conditions in each set of experiments. The same is also true of the time course of cultivation (Table 2) where the stimulating effect of amphiphilic polymers remained throughout the growth of B. pertussis irrespective of a steady change of the cultural state naturally occurring during batch cultivation. Furthermore, no significant differences between the stimulating effect of PVAc, M&e1 and Me-P-CD have so far been detected. A further comparative study including PVA (Table 1, experiment C and D) showed that the partly acylated polyvinyl alcohol PVAc is superior to PVA in enhancing the formation of haemagglutinins. Interestingly, the latter finding is, in a certain way, consistent with results obtained by Imaizumi,” who found that non-methylated cyclodextrins are less effective than Me-&CD. The same applies to polysaccharides MeCel and starch. We found that while MeCel stimulated growth of B. pertussis and formation of haemagglutinins, the use of starch did not produce this effect.” Obviously, the enhanced hydrophobic properties of Me-PCD, PVAc and MeCel due to alkyl or acyl groups render them more effective than cyclodextrin, polyvinyl alcohol and starch. It is the authors hope that results presented here give rise to a more economic production of FHA and PT. Furthermore, the results and likewise the considerations with respect to structure-efficiency relationships may make further search for highly effective substances easier.
Effecf of amphiphilic
Table 1. The effects of addition of amphiphihc polymers of B. pertussis and production of haemagglutinins
Experiment
A
Experiment
B
Experiment
C
Experiment
D
or Me-P-CD
Added substance (15 g per litre culture medium)
Cultivation time (h) 32 32 32 32 24 24 24 28 28 28 28
None
PVA
PVAc
MeCel
medium
on growth
PT 512 4096 4096 4096 192 4096 4096 384 192 4096 4096 128 64 4096 2048 256
Table 2. The effects of addition of amphiphilic polymers tration, pertussis toxin production and haemagglutinin pertussis Added substance (1.5 g per litre culture medium)
to Cohen-Wheeler
HA-titer
None PVAc MeCel Me-p-CD None PVAc MeCel None PVA PVAc Me-P-CD None PVA PVAc MeCel Me-/J-CD
24 24 24 24 24
307
polymers
bg/l)
IOU
8.1 13.1 11.3 9.2 2.7 9.7 9.8 3.3 6.0 5.6 4.4
146 189 207 180 102 139 144 147 169 193 207 114 145 154 157 160
2.6 5.4 5.6 4.9 7.4
on time course of cell concentiter during cultivation of B.
Cultivation time
PT
(h)
HA-titer
(mg/l)
22 26 30 22 26 30 22 26 30 22 26 30
64 192 192 80 320 160 1024 2048 4096 768 2048 4096
2.0 2.7 6.8 7.4 5.8 1.7 3.0 9.7 1.3 2.7 9.8
References 1. Sato Y, Kimura M, Fukumi H. Development of a pertussis component vaccine in Japan. Lancet 1984; i: 122-126. 2. Arai H, Sato Y. Separation and characterization of two distinct haemagglutinins contained in purified leuCOCyf.4MiS-promoting factor from Bordetella pertussis. Biochim Biophys Acta 1976; 444: 765-782.
1.0
IOU 58 80 101
105 140 158 67 104 139 77 115 144
Sato Y, Sato H, Izumiya K, Cowell JL, Manclark CR. Role of antibody to filamentous haemagglutinin and to leucocytosis promoting factor-haemagglutinin in immunity to pertussis. In: Robbins JB, Hill JC and Sadoff JC eds, Seminars in Infectious Disease,vol. IV, Bacterial Vaccines. New York: Thieme-Stratton. Sato Y, Cowell JL, Burstyn DG, Manclark CR. Separation and purification of the haemagglutinins from Bor,detella pertussis. Infect Immun 1983; 41: 313-320.
308
H. E. Nikolajewski
5. Imaizumi A, Suzuki Y, Ono S, Sato H, Sato Y. Heptakis (2,6-O-dimethyl) &cyclodextrin: a Novel Growth Stimulant for Bordetella pertussis Phase I. J Clin Microbial 1983; 17: 781-786. 6. Ginnaga A, Koba H, Sakuma S, Kitagawa H, Yamada A, Suzuki Y. Method for the production of HA fraction containing protective antigens of Bordetella pertussis and pertussis vaccine. European Patent Application Number: 84103504.1. 7. Nikolajewski HE, Liepert H, Pfuller U. Nikolajewski G, Swidsinski S, Wettig G. Verfahren zur Kultivation von Mikroorganismen. DD Patent Application WP C 12 N 0 3278631.
et al.
8. Greenspan GN. Stimulation of antigen production by Bordetella pertussis. US Patent 4 551 429 (1985). 9. Sato H, Sato Y, Ito A. Affinity of pertussis toxin produced by Bordetella pertussis for human haptoglobin: application to the in vitro assayof the toxin. J Microbial Meth 1983; 1: 99-109. 10. Imaizumi A, Suzuki Y, Ginnaga A, Sakuma S, Sato Y. A new culturing method for production of filamentous haemagglutinin of Bordetella pertussis. J Microbial Meth 1984; 2: 339347.
Received for publication 20 March accepted 2 August 1990.
1990;
(1990)
18, 305-308
The Effect of Amphiphilic Polymers on Growth of Berm/a pertussis and Production of 6. pertussis Haemagglutinins H. E. Nlkolajewski,* H. Liepert,* G. Nikoiajewski,t U. Pfiiller,* S. Swidsinski* and G. Wettig* *Staatliches lnstitut ftir Immunpriiparate und Nahrmedien, Klement-Gottwald-A/lee 317-321, Berlin, 1120, L? 0. R. tZentra/institut fijr physikalische Chemie der Akademie der Wissenschaften der D. D. R., Rudower Chaussee 5, Berlin, 1199, D. D. R.
Abstract. The effect, upon addition to a culture medium of amphiphilic polymers such as partly hydrolysed polyvinyl acetate (PVAc) or partly methylated cellulose (MeCel) on growth and production of haemagglutinins-the filamentous haemagglutinin (FHA) and the pertussis toxin (PT)-has been investigated. As a result, an increase of HA-titer and PT could be achieved. Besides these effects the addition of the amphiphilic polymers stimulated growth of B. pertussis. The above results are comparable to, or even better than, those obtained by addition of Heptakis (2,6-O-dimethyl) /3-cyclodextrin (Me+CD) or polyvinyul alcohols (PVA) which have already been described as compounds to enhance growth and yield of haemagglutinins.
Introduction
haemagglutinins. 5 Starting from this discovery the Japanese manufacturers were then able to produce both haemagglutinins more conveniently in stirred tank cultures.‘j A drawback of the use of Me-&CD however is its price which may increase the cost of vaccine production. The primary purpose of the study presented here has been to look for substances which are highly effective in stimulating formation of haemagglutinins, yet available at a reasonable price. The mechanism which leads to the stimulating effect of Me-P-CD is not yet fully understood. The following interpretation however may serve as a basis to aid the search for other substances giving rise to a similar effect. Thus, Me-@-CD may capture within its hydrophobic cavity substances which are inhibitory to growth of B. pertussis and formation of PT and FHA or alternatively carry and transfer substances supporting growth and antigen yield. The stabilization of the suspension of B. pertussis during cultivation may also play an important role in enhancing growth and antigen yield. Amphiphilic polymers with their folded structure in aqueous solution are also likely to trap substances in their hydrophobic pockets and should likewise stabilize bacterial suspensions.
Pertussis vaccines have been used for more than 50 years. By far the most widely used type of pertussis vaccine consists of whole bacterial cells which are killed by either heat or chemical treatment. Since, however, the cellular pertussis vaccine may occasionally give rise to adverse reactions, the benefits of the vaccine in comparison with the risk accompanying vaccination have come into question. Due to this problem-especially in Japan-considerable efforts have been undertaken in recent years to produce a safer vaccine consisting of purified antigenic components.’ For the acellular vaccine the two structurally different haemagglutinins-the filamentous haemagglutinin (FHA) and the pertussis toxin (PT)-play an important role as components.23 The early methods to isolate both haemagglutinins suffered from the very ineffective method of their production by means of static cultures.4 Consequently, for economic reasons, particular attention was paid by Japanese manufacturers to produce PT and FHA with reasonable yields. Towards a large scale production major progress was achieved by introducing Heptakis (2,&Odimethyl) @yclodextrin (Me-D-CD) to promote cell growth and synthesis of 10461056/90/040305+04
$03.00/O
0
1990 The
International
Association
of Biological
Standardization
306
H. E. Nikolajewski
Much of this paper describes shaker flask cultivations of B. pertussis in the presence of amphiphilic polymers such as methyl cellulose (MeCel) and partly hydrolysed polyvinyl acetate (PVAc) to investigate their capability to enhance growth ofB. pertussis and production of haemagglutinins.7 MeCel and PVAc are commercially available and find widespread use as emulsifiers and stabilizers of suspensions in the pharmaceutical, paint and food industries. For comparative studies Me-p-CD and polyvinyl alcohol (PVA) were used in some sets of experiments, because the latter has proven to be enhansive with respect to cell and antigen yield.’ Materials
and methods
Culture medium
Bordetella pertussis strain Tohama phase I was grown in a Cohen-Wheeler medium with the addition of O-75 g glutamic acid per 100 ml medium. In order to avoid growth limitation after the depletion of the carbon source during cultivation, 05 g glutamic acid was fed to a 100 ml culture medium when cell density reached approximately 140-150 IOU/ml. Either amphiphilic polymers or Me-b-CD were then added to the culture medium. Partly hydrolysed polyvinyl acetate (PVAc) ‘Mowio118-88’ and pure polyvinyl alcohol (PVA) were obtained from Hoechst (Frankfurt am Main, F.R.G.). Methyl cellulose (MeCel) ‘Methocel K 100’ was obtained from Dow Chemical (Frankfurt am Main, F.R.G.) and Heptakis (2,6-O&methyl) /3-cyclodextrin was from the SIGMA Chemical Company (St. Louis, U.S.A.). Method of cultivation
A one litre Erlenmeyer flask containing a 100 ml culture medium was used for cultivation. A seed of B. pertussis was added to give a final cell concentration of 8 x 10’ cells/ml being equivalent to 8 IOU/ml. Cultivation was carried out on a reciprocal shaker at 100 strokes per minute at 35°C. Cell concentration
The cell concentration was determined trophotometer (Specol 11, Carl-Zeiss-Jena, 530 nm
by a specG.D.R.) at
Total PT content
After solubilization of the B. pertussis suspension, by treatment with lysozym, the cellbound, together with excreted PT, was measured.
et sl.
The total PT content was determined by the HpElisa-technique developed by Sato et ~1.’ As a reference, purified PT was used which was a generous gift from Yuji Sato (National Institute of Health, Tokyo, Japan). Haemagglutinin test
After removing cells by centrifugation, the HAactivity of the supernatant was assayed by means of fresh, washed, sheep erythrocytes. Two-fold serial dilutions of each sample were prepared in 0.05 ml PBS volumes in round bottomed microtiter plates. An equal volume of a 0.5% suspension of red blood cells was added and the mixture was incubated at room temperature for 3-4 h. The HAtiter of the samples was defined as the reciprocal of that dilution which caused complete agglutination. Results and discussion The data in Table 1 demonstrate strikingly that the use of PVAc and MeCel assists the growth of B. pertussis and the formation of haemagglutinins hence, a stimulating effect has been found independent of the particular cultural conditions in each set of experiments. The same is also true of the time course of cultivation (Table 2) where the stimulating effect of amphiphilic polymers remained throughout the growth of B. pertussis irrespective of a steady change of the cultural state naturally occurring during batch cultivation. Furthermore, no significant differences between the stimulating effect of PVAc, M&e1 and Me-P-CD have so far been detected. A further comparative study including PVA (Table 1, experiment C and D) showed that the partly acylated polyvinyl alcohol PVAc is superior to PVA in enhancing the formation of haemagglutinins. Interestingly, the latter finding is, in a certain way, consistent with results obtained by Imaizumi,” who found that non-methylated cyclodextrins are less effective than Me-&CD. The same applies to polysaccharides MeCel and starch. We found that while MeCel stimulated growth of B. pertussis and formation of haemagglutinins, the use of starch did not produce this effect.” Obviously, the enhanced hydrophobic properties of Me-PCD, PVAc and MeCel due to alkyl or acyl groups render them more effective than cyclodextrin, polyvinyl alcohol and starch. It is the authors hope that results presented here give rise to a more economic production of FHA and PT. Furthermore, the results and likewise the considerations with respect to structure-efficiency relationships may make further search for highly effective substances easier.
Effecf of amphiphilic
Table 1. The effects of addition of amphiphihc polymers of B. pertussis and production of haemagglutinins
Experiment
A
Experiment
B
Experiment
C
Experiment
D
or Me-P-CD
Added substance (15 g per litre culture medium)
Cultivation time (h) 32 32 32 32 24 24 24 28 28 28 28
None
PVA
PVAc
MeCel
medium
on growth
PT 512 4096 4096 4096 192 4096 4096 384 192 4096 4096 128 64 4096 2048 256
Table 2. The effects of addition of amphiphilic polymers tration, pertussis toxin production and haemagglutinin pertussis Added substance (1.5 g per litre culture medium)
to Cohen-Wheeler
HA-titer
None PVAc MeCel Me-p-CD None PVAc MeCel None PVA PVAc Me-P-CD None PVA PVAc MeCel Me-/J-CD
24 24 24 24 24
307
polymers
bg/l)
IOU
8.1 13.1 11.3 9.2 2.7 9.7 9.8 3.3 6.0 5.6 4.4
146 189 207 180 102 139 144 147 169 193 207 114 145 154 157 160
2.6 5.4 5.6 4.9 7.4
on time course of cell concentiter during cultivation of B.
Cultivation time
PT
(h)
HA-titer
(mg/l)
22 26 30 22 26 30 22 26 30 22 26 30
64 192 192 80 320 160 1024 2048 4096 768 2048 4096
2.0 2.7 6.8 7.4 5.8 1.7 3.0 9.7 1.3 2.7 9.8
References 1. Sato Y, Kimura M, Fukumi H. Development of a pertussis component vaccine in Japan. Lancet 1984; i: 122-126. 2. Arai H, Sato Y. Separation and characterization of two distinct haemagglutinins contained in purified leuCOCyf.4MiS-promoting factor from Bordetella pertussis. Biochim Biophys Acta 1976; 444: 765-782.
1.0
IOU 58 80 101
105 140 158 67 104 139 77 115 144
Sato Y, Sato H, Izumiya K, Cowell JL, Manclark CR. Role of antibody to filamentous haemagglutinin and to leucocytosis promoting factor-haemagglutinin in immunity to pertussis. In: Robbins JB, Hill JC and Sadoff JC eds, Seminars in Infectious Disease,vol. IV, Bacterial Vaccines. New York: Thieme-Stratton. Sato Y, Cowell JL, Burstyn DG, Manclark CR. Separation and purification of the haemagglutinins from Bor,detella pertussis. Infect Immun 1983; 41: 313-320.
308
H. E. Nikolajewski
5. Imaizumi A, Suzuki Y, Ono S, Sato H, Sato Y. Heptakis (2,6-O-dimethyl) &cyclodextrin: a Novel Growth Stimulant for Bordetella pertussis Phase I. J Clin Microbial 1983; 17: 781-786. 6. Ginnaga A, Koba H, Sakuma S, Kitagawa H, Yamada A, Suzuki Y. Method for the production of HA fraction containing protective antigens of Bordetella pertussis and pertussis vaccine. European Patent Application Number: 84103504.1. 7. Nikolajewski HE, Liepert H, Pfuller U. Nikolajewski G, Swidsinski S, Wettig G. Verfahren zur Kultivation von Mikroorganismen. DD Patent Application WP C 12 N 0 3278631.
et al.
8. Greenspan GN. Stimulation of antigen production by Bordetella pertussis. US Patent 4 551 429 (1985). 9. Sato H, Sato Y, Ito A. Affinity of pertussis toxin produced by Bordetella pertussis for human haptoglobin: application to the in vitro assayof the toxin. J Microbial Meth 1983; 1: 99-109. 10. Imaizumi A, Suzuki Y, Ginnaga A, Sakuma S, Sato Y. A new culturing method for production of filamentous haemagglutinin of Bordetella pertussis. J Microbial Meth 1984; 2: 339347.
Received for publication 20 March accepted 2 August 1990.
1990;
