Journal of the Royal Society of Medicine Volume 85 May 1992
Progress with
W N Burnette PhD
a
recombinant whooping cough vaccine:
V L Mar MS
D W Whiteley MS
a
285
review
T D Bartley PhD
Amgen Inc, Amgen Centre, Thousand Oaks, CA 91320, USA Keywords: whooping cough; recombinant vaccine
The problems of pertussis vaccine The causative agent of whooping cough, Bordetella pertussis, was first identified in 19061. Nearly 40 years later, the first vaccine to prevent this devastating paediatric disease became widely available in the United States2. Introduction of the chemicallyinactivated whole bacterial cell vaccine. led to a dramatic reduction in the incidence of pertussis3. Side effects associated with immunization had been clearly recognized during development of the vaccine4'5, but the relative benefits of vaccination in the face of a disease of epidemic proportions far outweighed the estimated risks. However, as pertussis vaccination became universal and the perceived threat of disease diminished, the risks of untoward serious reactions to vaccine components became unacceptable in the public mind. The small but perceptible erosion in vaccine acceptance in the United States has resulted in a concomitant increase in disease frequency3'6. Loss of public confidence in vaccine safety has had perhaps a more striking effect in other developed nations, such as the United Kingdom where vaccine acceptance has plummeted7, and Japan and Sweden where government authorities suspended compulsory pertussis immunization either temporarily or
pertussis toxin had been molecularly cloned by J M Keith and colleagues'6"17. Based on their work and earlier structural analyses of the molecule, it was understood that pertussis toxin was a hexameric protein containing five distinct subunits in an A-B arrangement'8. The A protomer, or'Sl subunit, exhibits an ADP-ribosyltransferase activity'9 capable of modifying guanine-nucleotide regulatory (G) proteins of eukaryotic cell membranes, resulting in the toxic effects. The B oligomer, comprised of the S5 subunit and dimers of subunits S2 and S4 and of S3 and S4, possesses the cell receptor recognition properties of the toxin and is necessary for transport of the Si subunit20. Our goal was to identify and eliminate, by genetic techniques, the enzyme-related toxicity of the Si subunit without abrogating its known protective immunogenic potential2' and to produce the individual subunits by recombinant DNA methods in a host other than B. pertussis so as to avoid contamination of product by other toxic components ofthis pathogen. We then intended to assemble the complete toxin (holotoxin) in vitro for use as a fully-defined vaccine free of intrinsic side effects.
Paper read to Royal Society of Medicine Anglo-American Meeting on
Applications of
Therapeuticgynd Preventive Medicine, 4-6 December 1989
permanently7'8.
In order to improve-pertussis vaccine safety without compromising its efficacy, considerable effort has lately been expended in the development of new acellular vaccine products. These vaccines are the result of attempts to fractionate B. pertussis cells and to identify immunoprotective components lacking significant adverse reactogenicity. Clinical trials of various acellular vaccine preparations9-1" have convincingly demonstrated that the major exotoxin of B. pertussis, commonly called pertussis toxin, is the necessary and probably sufficient component required for immunization. Unfortunately, this toxin is also a major virulence factor in pertussis disease12 and is believed to be a contributing factor -in severe vaccine reactions'3. Although such acellular vaccines are treated with inactivating chemical agents, such as formalin and glutaraldehyde, these procedures reduce immunogenicity and permit recovery of measurable pertussis toxin activity on storage. Further, even exhaustive purificationcannot eliminate the possibility of contamination by other well-known toxins of B. pertussis14. The challenge of new vaccine development We therefore sought a new approach to the development of an inactivated. toxin ('toxoid') for pertussis that relied upon advances in recombinant DNA technology'5. The operon encoding the complex
Recombinant expression of pertussis toxin subunit proteins The individual cistronic elements encoding each of the pertussis toxin subunits were subcloned from the operon into plasmid vectors optimized for expression of heterologous proteins in Escherichia coli22. Initially, the subunits were separately expressed with their native leader peptide sequences intact. We found that leader processing could be efficiently obtained only with the Si subunit22. For this reason, the remaining subunits were subsequently expressed in recombinant E. coli with a methionine initiation codon substituting for each of their leader sequences; with the exception of S4, the amino-terminal methionine residue is efficiently removed from each of the subunits by E. coli methionine aminopeptidase, resulting in native 'mature' protein sequences22. All the subunits were produced as large, insoluble inclusion bodies in the recombinant host at levels ranging from 1.7% to 43.8% of total cell protein22. The recombinant Si subunit retained the ADPribosyltran,sferase activity of the native protein. Without the contribution of sub-immunogenic doses of active toxin, none of the individual recombinant subunits was capable of eliciting a significant neutralizing or protective immune response in mice22. This reinforced the need to achieve reconstitution,of the more immunologically.potent-holotoxin molecule or of other multimeric structures comprised
0141:0768/92/ 050285-03/$02.OO/O
© 1992 The Royal Society of Medicine
286
Journal of the Royal Society of Medicine Volume 85 May 1992
of the appropriate protective epitopes displayed in their authentic antigenic conformations. Elimination of Si enzyme activity The next challenge to our programme was the abrogation of toxin-related ADP-ribosyltransferase activity without destruction of the toxin-dominant protective antigenic determinant or the ability of the Si subunit to associate effectively with the subunits of the B oligomer to form the holotoxin. We solved this problem by applying a rational approach to site-directed mutagenesis. Preliminary experiments indicated that a region critical for both the enzymatic and immunogenic properties resided near the amino terminus of the Si polypeptide. By controlled exonucleolytic digestion, a series of sequentially deleted DNA molecules representing the Si gene were produced and each expressed in E.- coli resulting in defined amino terminally-truncated proteins23. In collaboration with J M Keith and W Cieplak of the National Institutes of Health, these proteins were analysed for enzyme activity and ability to bind a monoclonal antibody defining the protective epitope. We were thus able to identify a domain of the Si subunit, delimited by amino acid residues 8 and 15, that was essential for both properties23. The sequence of this region is one of a number shared in part by the pertussis toxin Si subunit and the A subunits of cholera toxin and E. coli heat-labile enterotoxin 7, both of which also exert their toxic effects through ADP-ribosyltransferase modification of eukaryotic membrane G proteins. The spatial linkage of enzyme activity and the protective determinant introduced a unique level of complexity to our problem. It necessitated a site-specific approach to mutagenesis that would permit us to uncouple this relationship without damaging the capability of the Si molecule to acquire a conformation consistent with restoration of the epitope and association with the B oligomer. Employing synthetic oligonucleotide linkers that spanned the sequence of the critical region, single and double codon changes were substituted into this domain24. These resulted in a series of site-specific mutant analogues ofthe Si subunit having one or two amino acid substitutions between residues 8 and 15 of the mature protein sequence. Only a single analogue of this series, the substitution of lysine for the native arginine residue at position 9, manifested the properties we sought24. This mutated protein retained its ability to form the protective epitope while reducing the ADP-ribosyltransferase activity to 0.02% of that for native Si. In continuing studies25'26, we have identified additional residues that participate in catalysis; however, no other single residue has yet been shown to have such a significant impact on enzyme activity as arginine 925,27.
Assembly of pertussis holotoxin As important as the mutation in the Si subunit was for substantial elimination of toxicity, it would be of only pedagogical value if it interfered with the ability of Si to fold into a conformation compatible with holotoxin assembly. Likewise, it would not be feasible to assess the effect of the Si mutation on toxicity without the cell targeting capabilities provided by the B oligomer subunits. We were therefore obliged to achieve reconstitution of the holotoxin in order to evaluate the potential reduction in toxic reactivity
and its protective immunogenicity. As a prelude to complete reconstitution with recombinant subunits, we initially attempted to assemble holotoxin moieties by combining recombinant Si subunits, either of native amino acid sequence or of the Arg9-Lys analogue form, with B oligomer isolated from native pertussis toxin by D L Burns of the United States Food and Drug Administration28. After purifying the recombinant Si species and permitting them to undergo controlled reoxidation and folding (Si contains two cysteine residues involved in an intramolecular disulphide linkage), they were individually mixed with natural B oligomer in vitro. Pioneering work by Tamura and colleagues18 gave us cause to believe that association would occur under these conditions to form holotoxin. We evaluated holotoxin formation by electrophoresis in non-denaturing, non-reducing polyacrylamide gels and found that both the enzymatically-active and inactive analogue forms of Si were capable of specifically associating with B oligomer to form holotoxin-like macromolecules28. Pertussis toxin had been shown to elicit a morphologic 'clustering' of Chinese hamster ovary cells in culture29, directly related to the activity of the Si subunit3O and correlated with its in vivo toxicity. When applied to cultured Chinese hamstr ovary cells, the holotoxin species containing the enzymaticallyactive recombinant Si subunit produced a morphological response indistinguishable from that of native toxin . In contrast, holotoxin comprised of B oligomer and the Arge-Lys analogue of Si evoked little or no cytopathic effect, indicating the formation of a 'genetic holotoxoid'28. Future of pertussis holotoxoid vaccine development Recent investigations in our laboratory have revealed that holotoxin and holotoxoid can be assembled in vitro, albeit with relatively low efficiency, employing all five of the recombinant pertussis toxin subunits (S1, S2, S3, S4, and S5) under carefully controlled conditions with chaotropic agents; in addition, a B oligomer-like multimer can likewise be assembled in the absence of the Si subunit (N Burnette et al., submitted). Toxin containing the mutated Si subunit3' and the completely recombinant B oligomer are both currently being examined for residual toxicity and for their ability to stimulate protective immune responses in animals. In this regard, Rappuoli and colleagues32 have demonstrated that Si mutations, such as those we have described24, can be introduced directly into the pertussis toxin operon in Bordetella by homologous recombination. The authentic host then assembles and secretes inactive holotoxin which is capable of invoking immunologic protection in mice. Although these results confirm the validity of our approach to the production of a genetically modified toxoid vaccine, the relatively modest level of pertussis toxin synthesis32 and the presence of other potentially pathogenic toxins found in Bordetella'4 may eventually limit the use of this organism in pertussis vaccine manufacturing. Nevertheless, a vaccine containing recombinant toxoid purified from B. pertussis has recently been introduced into human clinical trials33. A recombinant means of holotoxoid vaccine production in a heterologous host22-2A'" will enable us to prevent genetic reversions that might encode an active toxin,
Journal of the Royal Society of Medicine Volume 85 May 1992
to manufacture significantly greater quantities of material in vastly purer form, and to control effectively the composition of the- final vaccine;
product. References 1 Bordet J, Gengou 0. Le microbe de la coqueluche. Ann Inst Pasteur 1906;20:731-41 2 Felton HM, Willard CY. Current status of prophylaxis by Hemophilus pertussis vaccine. JAMA 1944;126: 294-9 3 Cherry JD. The epidemiology of pertussis and pertussis immunization in the United Kingdom and the United States: a comparative study. Curr Probl Pediatr 1984;14:1-78 4 Madsen T. Vaccination against whooping cough. JAMA 1933;101:187-8 5 Kendrick PL, Eldering G. A study in active immnunization against pertussis. Am J Hyg 1939;29:133-53 6 Bass JW, Stephenson SR. The return of pertussis. Pediatr Infect Dis 1987;6:141-4 7 Cherry JD, Brunell PA, Golden GS, Karzon DT. Report of the task force on pertussis and pertussis immunization - 1988. Pediatrics 1988;81(suppl):939-84 8 Romanus V, Jonsell R, Bergquist S-O. Pertussis in Sweden after cessation of general immunizationmin 1979. Pediatr Infect Dis 1987;6:364-71 9 Kimura M, Kuno-Sakai H. Acellular pertussis vaccines and fatal infections. Lancet 1988;i:881-2 10 Ad Hoc Group for the Study of Pertussis Vaccines.Placebo-controlled trial of two acellular pertussis vaccines in Sweden -protective efficacy and adverse events. Lancet 1988i:955-60 11 Olin P, Storsaeter J, Romranus V. The efficacy of acellular pertussis vaccine. JAMA 1989;261:560 12 Pittman M. The concept of pertiis as a toxin-mediated disease. Pediatr Infect Dis 1984 3:467-86 13 Pittman M. Neurotoxicity of Bordetella peHtussis. Neurotoxicology 1986;7:53-68 14 Weiss AA, Hewlett EL. Virulence factors of Bordetella pertussis. Ann Rev Microbiol 1986;40:661-86 15 Burnette WN. The advent of recombinant pertussis vaccines. BiolTechnology 1990;8:1002-5 16 Locht C, Barstad PA, Coligan JE, et aL Molecular cloning of pertussis toxin genes. Nucl Acids Res 1986;14:3251-61 17 Locht C, Keith JM. Pertussis toxin gene: nucleotide sequence and genetic organization. Science 1986; 232:1258-64 18 Tamura M, Nogimori K, Murai S, et aL Subunit structure of the islet-activating protein, pertussis toxin, in conformity with the A-B model. Biochemistry 1982;21:5516-22 19 Katada T, Tamura M, Ui M. The A protomer of isletactivating protein, pertussis tpxin, as an active peptide catalyzing ADP-ribosylation of a membrane protein. Arch Biochem Biophys 1983,224:290-8
20 Tamura M, Nogimori K, Yajima M, Ase K, Ui M. A role of the B-oligomer moiety of islet-activating protein, pertusis toxin, ip development of the bi;iogical effects on intact cells. J Biol Chem 1983;258:6756-61 21 Sato H, Ito A, Chiba J, Sato Y. Monoclonal antibody against pertussis toxin: effect on toxin activity and pertussis infections. Infect Immun 1984;46:422-8 22 Burnette WN, Mar VL, Cieplak W, et al. Direct expression of Bordetellapertussis toxin subunits to high levels in Escherichia coli. Bio/Technology 1988;6: 699-706 23 Cieplak W, Burnette WN, Mar VL, et aL Identification of a region in the S1 subunit of pertussis toxin that is required for enzymatic activity and that contributes to the formation of a neutralizing antigenic determinant. Proc Nati Acad Sci USA 1988;85:4667-71 24 Burnette WN, Cieplak W, Mar VL, Kaljot KT, Sato H, Keith JM. Pertussis toxin S1 mutant with reduced enzyme activity and a conserved protective epitope. Science 1988;242:72-4 25 Kaslow HR, Schlotterbeck JD, Mar VL, Burnette WN. Alkylation ofcysteine 41, but not cysteine 2Q0, decreases the ADP-ribosyltransferase activity of the Si subunit of pertussis toxin. J Biol Chem 1989;264:6386-90 26 Cieplak W, Jr., Mar VL, Burnette WN, Keith JM, Locht C. Photolabelling of mutant forms of the S1 subunit of pertussis toxin with NAD+. Biochem J
1990;,28:547-51 27 Lobet Y, Cieplak W, Jr., Smith SG, Keith JM. Effects of mutations on the enzyme activity and immunoreactivity of the Si subunit of pertussis toxin. Infect Immun 1989;57:3660-2 28 Bartley TD, Whiteley DW, Mar VL, Burns DL, Burnette WN. Pertussis holotoxoid formed in vitro with a genetically deactivated Si subunit. Proc Nati Acad Sci USA 1989;86:8353-7 29 Hewlett EL, Sauer KT, Myers GA, Cowell JL, Guerrant RL. Induction of a novel morphological response in Chinewe hamster ovary cells by pertussis toxin. Infect Immun 1983;40:1198-203 30- Burns DL, Kenimer JG, Manclark CR. Role of the A subunit of pertussis toxin in alteration of Chinese hamster ovary cell morphology. Infect Immun 1987; 55:24-8 31 Arciniega JL, Shahin RD, Burnette WN, et aL Contribution of the B oligon5er to the protective activity of genetically attenuated pertussis toxin. Infect Immun 1991;59:3407-10 32 Pizza M, Covacci A, Bartoloni A, et aL Mutants of pertusis toxin suitable for vaccine development. Science
1989;246:497-500 33 Podda A, Nencioni L, De Magistris MT, et aL Metabolic, humorail, and cellular responses in adult volunteers immunized with genetically inactivated pertussis toxin mutant PT-9K/129G. J Exp Med 1990;172:861-8
(Accepted 5 November 1991)
287
Progress with
W N Burnette PhD
a
recombinant whooping cough vaccine:
V L Mar MS
D W Whiteley MS
a
285
review
T D Bartley PhD
Amgen Inc, Amgen Centre, Thousand Oaks, CA 91320, USA Keywords: whooping cough; recombinant vaccine
The problems of pertussis vaccine The causative agent of whooping cough, Bordetella pertussis, was first identified in 19061. Nearly 40 years later, the first vaccine to prevent this devastating paediatric disease became widely available in the United States2. Introduction of the chemicallyinactivated whole bacterial cell vaccine. led to a dramatic reduction in the incidence of pertussis3. Side effects associated with immunization had been clearly recognized during development of the vaccine4'5, but the relative benefits of vaccination in the face of a disease of epidemic proportions far outweighed the estimated risks. However, as pertussis vaccination became universal and the perceived threat of disease diminished, the risks of untoward serious reactions to vaccine components became unacceptable in the public mind. The small but perceptible erosion in vaccine acceptance in the United States has resulted in a concomitant increase in disease frequency3'6. Loss of public confidence in vaccine safety has had perhaps a more striking effect in other developed nations, such as the United Kingdom where vaccine acceptance has plummeted7, and Japan and Sweden where government authorities suspended compulsory pertussis immunization either temporarily or
pertussis toxin had been molecularly cloned by J M Keith and colleagues'6"17. Based on their work and earlier structural analyses of the molecule, it was understood that pertussis toxin was a hexameric protein containing five distinct subunits in an A-B arrangement'8. The A protomer, or'Sl subunit, exhibits an ADP-ribosyltransferase activity'9 capable of modifying guanine-nucleotide regulatory (G) proteins of eukaryotic cell membranes, resulting in the toxic effects. The B oligomer, comprised of the S5 subunit and dimers of subunits S2 and S4 and of S3 and S4, possesses the cell receptor recognition properties of the toxin and is necessary for transport of the Si subunit20. Our goal was to identify and eliminate, by genetic techniques, the enzyme-related toxicity of the Si subunit without abrogating its known protective immunogenic potential2' and to produce the individual subunits by recombinant DNA methods in a host other than B. pertussis so as to avoid contamination of product by other toxic components ofthis pathogen. We then intended to assemble the complete toxin (holotoxin) in vitro for use as a fully-defined vaccine free of intrinsic side effects.
Paper read to Royal Society of Medicine Anglo-American Meeting on
Applications of
Therapeuticgynd Preventive Medicine, 4-6 December 1989
permanently7'8.
In order to improve-pertussis vaccine safety without compromising its efficacy, considerable effort has lately been expended in the development of new acellular vaccine products. These vaccines are the result of attempts to fractionate B. pertussis cells and to identify immunoprotective components lacking significant adverse reactogenicity. Clinical trials of various acellular vaccine preparations9-1" have convincingly demonstrated that the major exotoxin of B. pertussis, commonly called pertussis toxin, is the necessary and probably sufficient component required for immunization. Unfortunately, this toxin is also a major virulence factor in pertussis disease12 and is believed to be a contributing factor -in severe vaccine reactions'3. Although such acellular vaccines are treated with inactivating chemical agents, such as formalin and glutaraldehyde, these procedures reduce immunogenicity and permit recovery of measurable pertussis toxin activity on storage. Further, even exhaustive purificationcannot eliminate the possibility of contamination by other well-known toxins of B. pertussis14. The challenge of new vaccine development We therefore sought a new approach to the development of an inactivated. toxin ('toxoid') for pertussis that relied upon advances in recombinant DNA technology'5. The operon encoding the complex
Recombinant expression of pertussis toxin subunit proteins The individual cistronic elements encoding each of the pertussis toxin subunits were subcloned from the operon into plasmid vectors optimized for expression of heterologous proteins in Escherichia coli22. Initially, the subunits were separately expressed with their native leader peptide sequences intact. We found that leader processing could be efficiently obtained only with the Si subunit22. For this reason, the remaining subunits were subsequently expressed in recombinant E. coli with a methionine initiation codon substituting for each of their leader sequences; with the exception of S4, the amino-terminal methionine residue is efficiently removed from each of the subunits by E. coli methionine aminopeptidase, resulting in native 'mature' protein sequences22. All the subunits were produced as large, insoluble inclusion bodies in the recombinant host at levels ranging from 1.7% to 43.8% of total cell protein22. The recombinant Si subunit retained the ADPribosyltran,sferase activity of the native protein. Without the contribution of sub-immunogenic doses of active toxin, none of the individual recombinant subunits was capable of eliciting a significant neutralizing or protective immune response in mice22. This reinforced the need to achieve reconstitution,of the more immunologically.potent-holotoxin molecule or of other multimeric structures comprised
0141:0768/92/ 050285-03/$02.OO/O
© 1992 The Royal Society of Medicine
286
Journal of the Royal Society of Medicine Volume 85 May 1992
of the appropriate protective epitopes displayed in their authentic antigenic conformations. Elimination of Si enzyme activity The next challenge to our programme was the abrogation of toxin-related ADP-ribosyltransferase activity without destruction of the toxin-dominant protective antigenic determinant or the ability of the Si subunit to associate effectively with the subunits of the B oligomer to form the holotoxin. We solved this problem by applying a rational approach to site-directed mutagenesis. Preliminary experiments indicated that a region critical for both the enzymatic and immunogenic properties resided near the amino terminus of the Si polypeptide. By controlled exonucleolytic digestion, a series of sequentially deleted DNA molecules representing the Si gene were produced and each expressed in E.- coli resulting in defined amino terminally-truncated proteins23. In collaboration with J M Keith and W Cieplak of the National Institutes of Health, these proteins were analysed for enzyme activity and ability to bind a monoclonal antibody defining the protective epitope. We were thus able to identify a domain of the Si subunit, delimited by amino acid residues 8 and 15, that was essential for both properties23. The sequence of this region is one of a number shared in part by the pertussis toxin Si subunit and the A subunits of cholera toxin and E. coli heat-labile enterotoxin 7, both of which also exert their toxic effects through ADP-ribosyltransferase modification of eukaryotic membrane G proteins. The spatial linkage of enzyme activity and the protective determinant introduced a unique level of complexity to our problem. It necessitated a site-specific approach to mutagenesis that would permit us to uncouple this relationship without damaging the capability of the Si molecule to acquire a conformation consistent with restoration of the epitope and association with the B oligomer. Employing synthetic oligonucleotide linkers that spanned the sequence of the critical region, single and double codon changes were substituted into this domain24. These resulted in a series of site-specific mutant analogues ofthe Si subunit having one or two amino acid substitutions between residues 8 and 15 of the mature protein sequence. Only a single analogue of this series, the substitution of lysine for the native arginine residue at position 9, manifested the properties we sought24. This mutated protein retained its ability to form the protective epitope while reducing the ADP-ribosyltransferase activity to 0.02% of that for native Si. In continuing studies25'26, we have identified additional residues that participate in catalysis; however, no other single residue has yet been shown to have such a significant impact on enzyme activity as arginine 925,27.
Assembly of pertussis holotoxin As important as the mutation in the Si subunit was for substantial elimination of toxicity, it would be of only pedagogical value if it interfered with the ability of Si to fold into a conformation compatible with holotoxin assembly. Likewise, it would not be feasible to assess the effect of the Si mutation on toxicity without the cell targeting capabilities provided by the B oligomer subunits. We were therefore obliged to achieve reconstitution of the holotoxin in order to evaluate the potential reduction in toxic reactivity
and its protective immunogenicity. As a prelude to complete reconstitution with recombinant subunits, we initially attempted to assemble holotoxin moieties by combining recombinant Si subunits, either of native amino acid sequence or of the Arg9-Lys analogue form, with B oligomer isolated from native pertussis toxin by D L Burns of the United States Food and Drug Administration28. After purifying the recombinant Si species and permitting them to undergo controlled reoxidation and folding (Si contains two cysteine residues involved in an intramolecular disulphide linkage), they were individually mixed with natural B oligomer in vitro. Pioneering work by Tamura and colleagues18 gave us cause to believe that association would occur under these conditions to form holotoxin. We evaluated holotoxin formation by electrophoresis in non-denaturing, non-reducing polyacrylamide gels and found that both the enzymatically-active and inactive analogue forms of Si were capable of specifically associating with B oligomer to form holotoxin-like macromolecules28. Pertussis toxin had been shown to elicit a morphologic 'clustering' of Chinese hamster ovary cells in culture29, directly related to the activity of the Si subunit3O and correlated with its in vivo toxicity. When applied to cultured Chinese hamstr ovary cells, the holotoxin species containing the enzymaticallyactive recombinant Si subunit produced a morphological response indistinguishable from that of native toxin . In contrast, holotoxin comprised of B oligomer and the Arge-Lys analogue of Si evoked little or no cytopathic effect, indicating the formation of a 'genetic holotoxoid'28. Future of pertussis holotoxoid vaccine development Recent investigations in our laboratory have revealed that holotoxin and holotoxoid can be assembled in vitro, albeit with relatively low efficiency, employing all five of the recombinant pertussis toxin subunits (S1, S2, S3, S4, and S5) under carefully controlled conditions with chaotropic agents; in addition, a B oligomer-like multimer can likewise be assembled in the absence of the Si subunit (N Burnette et al., submitted). Toxin containing the mutated Si subunit3' and the completely recombinant B oligomer are both currently being examined for residual toxicity and for their ability to stimulate protective immune responses in animals. In this regard, Rappuoli and colleagues32 have demonstrated that Si mutations, such as those we have described24, can be introduced directly into the pertussis toxin operon in Bordetella by homologous recombination. The authentic host then assembles and secretes inactive holotoxin which is capable of invoking immunologic protection in mice. Although these results confirm the validity of our approach to the production of a genetically modified toxoid vaccine, the relatively modest level of pertussis toxin synthesis32 and the presence of other potentially pathogenic toxins found in Bordetella'4 may eventually limit the use of this organism in pertussis vaccine manufacturing. Nevertheless, a vaccine containing recombinant toxoid purified from B. pertussis has recently been introduced into human clinical trials33. A recombinant means of holotoxoid vaccine production in a heterologous host22-2A'" will enable us to prevent genetic reversions that might encode an active toxin,
Journal of the Royal Society of Medicine Volume 85 May 1992
to manufacture significantly greater quantities of material in vastly purer form, and to control effectively the composition of the- final vaccine;
product. References 1 Bordet J, Gengou 0. Le microbe de la coqueluche. Ann Inst Pasteur 1906;20:731-41 2 Felton HM, Willard CY. Current status of prophylaxis by Hemophilus pertussis vaccine. JAMA 1944;126: 294-9 3 Cherry JD. The epidemiology of pertussis and pertussis immunization in the United Kingdom and the United States: a comparative study. Curr Probl Pediatr 1984;14:1-78 4 Madsen T. Vaccination against whooping cough. JAMA 1933;101:187-8 5 Kendrick PL, Eldering G. A study in active immnunization against pertussis. Am J Hyg 1939;29:133-53 6 Bass JW, Stephenson SR. The return of pertussis. Pediatr Infect Dis 1987;6:141-4 7 Cherry JD, Brunell PA, Golden GS, Karzon DT. Report of the task force on pertussis and pertussis immunization - 1988. Pediatrics 1988;81(suppl):939-84 8 Romanus V, Jonsell R, Bergquist S-O. Pertussis in Sweden after cessation of general immunizationmin 1979. Pediatr Infect Dis 1987;6:364-71 9 Kimura M, Kuno-Sakai H. Acellular pertussis vaccines and fatal infections. Lancet 1988;i:881-2 10 Ad Hoc Group for the Study of Pertussis Vaccines.Placebo-controlled trial of two acellular pertussis vaccines in Sweden -protective efficacy and adverse events. Lancet 1988i:955-60 11 Olin P, Storsaeter J, Romranus V. The efficacy of acellular pertussis vaccine. JAMA 1989;261:560 12 Pittman M. The concept of pertiis as a toxin-mediated disease. Pediatr Infect Dis 1984 3:467-86 13 Pittman M. Neurotoxicity of Bordetella peHtussis. Neurotoxicology 1986;7:53-68 14 Weiss AA, Hewlett EL. Virulence factors of Bordetella pertussis. Ann Rev Microbiol 1986;40:661-86 15 Burnette WN. The advent of recombinant pertussis vaccines. BiolTechnology 1990;8:1002-5 16 Locht C, Barstad PA, Coligan JE, et aL Molecular cloning of pertussis toxin genes. Nucl Acids Res 1986;14:3251-61 17 Locht C, Keith JM. Pertussis toxin gene: nucleotide sequence and genetic organization. Science 1986; 232:1258-64 18 Tamura M, Nogimori K, Murai S, et aL Subunit structure of the islet-activating protein, pertussis toxin, in conformity with the A-B model. Biochemistry 1982;21:5516-22 19 Katada T, Tamura M, Ui M. The A protomer of isletactivating protein, pertussis tpxin, as an active peptide catalyzing ADP-ribosylation of a membrane protein. Arch Biochem Biophys 1983,224:290-8
20 Tamura M, Nogimori K, Yajima M, Ase K, Ui M. A role of the B-oligomer moiety of islet-activating protein, pertusis toxin, ip development of the bi;iogical effects on intact cells. J Biol Chem 1983;258:6756-61 21 Sato H, Ito A, Chiba J, Sato Y. Monoclonal antibody against pertussis toxin: effect on toxin activity and pertussis infections. Infect Immun 1984;46:422-8 22 Burnette WN, Mar VL, Cieplak W, et al. Direct expression of Bordetellapertussis toxin subunits to high levels in Escherichia coli. Bio/Technology 1988;6: 699-706 23 Cieplak W, Burnette WN, Mar VL, et aL Identification of a region in the S1 subunit of pertussis toxin that is required for enzymatic activity and that contributes to the formation of a neutralizing antigenic determinant. Proc Nati Acad Sci USA 1988;85:4667-71 24 Burnette WN, Cieplak W, Mar VL, Kaljot KT, Sato H, Keith JM. Pertussis toxin S1 mutant with reduced enzyme activity and a conserved protective epitope. Science 1988;242:72-4 25 Kaslow HR, Schlotterbeck JD, Mar VL, Burnette WN. Alkylation ofcysteine 41, but not cysteine 2Q0, decreases the ADP-ribosyltransferase activity of the Si subunit of pertussis toxin. J Biol Chem 1989;264:6386-90 26 Cieplak W, Jr., Mar VL, Burnette WN, Keith JM, Locht C. Photolabelling of mutant forms of the S1 subunit of pertussis toxin with NAD+. Biochem J
1990;,28:547-51 27 Lobet Y, Cieplak W, Jr., Smith SG, Keith JM. Effects of mutations on the enzyme activity and immunoreactivity of the Si subunit of pertussis toxin. Infect Immun 1989;57:3660-2 28 Bartley TD, Whiteley DW, Mar VL, Burns DL, Burnette WN. Pertussis holotoxoid formed in vitro with a genetically deactivated Si subunit. Proc Nati Acad Sci USA 1989;86:8353-7 29 Hewlett EL, Sauer KT, Myers GA, Cowell JL, Guerrant RL. Induction of a novel morphological response in Chinewe hamster ovary cells by pertussis toxin. Infect Immun 1983;40:1198-203 30- Burns DL, Kenimer JG, Manclark CR. Role of the A subunit of pertussis toxin in alteration of Chinese hamster ovary cell morphology. Infect Immun 1987; 55:24-8 31 Arciniega JL, Shahin RD, Burnette WN, et aL Contribution of the B oligon5er to the protective activity of genetically attenuated pertussis toxin. Infect Immun 1991;59:3407-10 32 Pizza M, Covacci A, Bartoloni A, et aL Mutants of pertusis toxin suitable for vaccine development. Science
1989;246:497-500 33 Podda A, Nencioni L, De Magistris MT, et aL Metabolic, humorail, and cellular responses in adult volunteers immunized with genetically inactivated pertussis toxin mutant PT-9K/129G. J Exp Med 1990;172:861-8
(Accepted 5 November 1991)
287
