INFECTION AND IMMUNITY, May 1990, p. 1308-1315 0019-9567/90/051308-08$02.00/0 Copyright © 1990, American Society for Microbiology

Vol. 58, No. 5

Characterization of Genetically Inactivated Pertussis Toxin Mutants: Candidates for a New Vaccine against Whooping Cough LUCIANO NENCIONI,* MARIAGRAZIA PIZZA,' MASSIMO BUGNOLI,1 TERESA DE MAGISTRIS,1 ANNALISA DI TOMMASO,l FRANCO GIOVANNONI,1 ROBERTO MANETTI,1 ILIO MARSILI,1 GIACOMO MATTEUCCI,1 DANIELE NUCCI,1 ROBERTO OLIVIERI,1 PIERO PILERI,1 RIVO PRESENTINI,1 LUIGI VILLA,' JOHAN GERRIT KREEFTENBERG,2 SERGIO SILVESTRI,' ALDO TAGLIABUE,1 AND RINO RAPPUOLI' Sclavo Research Center, Via Fiorentina 1, 53100 Siena, Italy,' and Rijksinstituut Voor Volksgezondheid En Milieuhygiene, Bilthoven, The Netherlands2 Received 27 October 1989/Accepted 26 January 1990

The introduction of two amino acid substitutions within the enzymatically active subunit Si of pertussis toxin (PT) abolishes its ADP-ribosyltransferase activity and toxicity on CHO cells (Pizza et al., Science 246:497-500, 1989). These genetically inactivated molecules are also devoid of other in vivo adverse reactions typical of PT, such as induction of leukocytosis, potentiation of anaphylaxis, stimulation of insulin secretion, and histamine sensitivity. However, the mutant PT molecules are indistinguishable from wild-type PT in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and maintain all the physical and chemical properties of PT, including affinity for toxin-neutralizing poly- and monoclonal antibodies. Either alone or stabilized with formaldehyde, PT mutants are able to induce high levels of neutralizing antibodies and to protect mice in a dose-dependent fashion against intracerebral challenge with virulent B. pertussis. These results clearly show that these genetically inactivated PT molecules are nontoxic but still immunogenic and justify their development as a component of a new, safer aceliular vaccine against whooping cough.

The introduction of mass vaccination with inactivated Bordetella pertussis cells reduced the incidence of and mortality from whooping cough (53). However, the local and systemic adverse reactions associated with the whole-cell vaccine administration (9) discouraged its use in infants and children, with a consequent increase of pertussis cases. In particular, rare cases of permanent brain damage and death (25) were responsible for most of the worries and stimulated the research for safer vaccines (46). Among the factors mediating pathogenesis of B. pertussis (31, 51), pertussis toxin (PT), after chemical detoxification, was found to induce protective immunity in animal models (26) and to protect children from severe disease during a phase III clinical trial carried out in Sweden (1, 30). This suggested that PT either alone or combined with other antigens could be used for effective prevention of whooping cough, provided that complete and irreversible detoxification is achieved. However, the chemical methods so far used for the detoxification of PT may yield a product with reduced immunogenicity or with residual toxin activity (45), a feature which has been suggested to be responsible for the severe side-effects of the cellular pertussis vaccine (44). To overcome the problems associated to the chemical detoxification of PT, we used a genetic approach to obtain nontoxic PT molecules suitable for vaccine development. PT is a protein of 105 kilodaltons composed of five subunits, named S1 to S5. Subunit Si (A promoter) is an enzyme which has a toxic effect on eucaryotic cells by ADP ribosylating a family of GTP-binding proteins involved in transmembrane signaling (10, 18). Subunits S2, S3, S4, and S5 are present in a 1:1:2:1 ratio and form a nontoxic oligomer (B oligomer) which binds receptors on the surface of eucaryotic cells and delivers the toxic subunit S1 across the cell membrane so that it can reach the target GTP-binding *

proteins. The cloning of the genes coding for the five subunits of PT from the chromosome of B. pertussis and the determination of their nucleotide sequences (22, 29) allowed the identification of a number of amino acids essential for the enzymatic activity of the S1 subunit (3, 7, 32). We replaced the codons of some of these amino acids in the chromosome of B. pertussis and obtained new strains which produced mutated forms of PT. Some of them produced PT molecules with reduced or undetectable toxicity which were able to induce protective immunity in mice (33). The most promising toxoids for vaccine production were the PT double mutants 9K/129G, 13L/129G, and 261/129G, all containing the substitution Glu-129 -> Gly in addition to Arg-9 -- Lys, Arg-13 -* Leu, or Trp-26 -> Ile, respectively (33). The aim of the present study was to investigate in animal models the safety and immunogenicity of PT double mutants in order to justify their further evaluation in humans as candidates for the development of new acellular pertussis vaccines. MATERIALS AND METHODS Strains and media. B. pertussis W28-9K/129G, containing Arg-9 -> Lys and Glu-129 -* Gly, and B. pertussis W2813L/129G, containing Arg-13 -+ Leu and Glu-129 -* Gly, have been described by Pizza et al. (33). B. pertussis W28-261/129G containing Trp-26 -- Ile and Glu-129 -* Gly was obtained by the same procedure (33; M. Pizza and R. Rappuoli, unpublished data). The strains were grown in a 20-liter fermentor using Stainer Scholte medium (43) containing 1.0 mg of dimethyl (2, 6-O-)P-cyclodextrin per ml. Animals. Inbred BALB/c female mice (5 to 7 weeks of age) and CD1 male mice (3 to 4 weeks of age) were obtained from Charles River, Calco, Italy. Outbred self white Hartley guinea pigs were bred in our animal facility in Sclavo, Italy. Wistar rats were purchased from Charles River and used at RBM Institute, Ivrea, Italy, for acute toxicity experiments. Preparation of PT and PT-9K/129G. Both antigens were

Corresponding author. 1308

VOL. 58, 1990

purified from the culture supernatants of the wild-type strain B. pertussis W28 and the recombinant strain B. pertussis W28 expressing the S1 double mutant PT-9K/129G by AffiGel blue absorption and by successive column affinity chromatography with Fetuin-Sepharose (2, 22, 29). Purified PT and PT-9K/129G were routinely characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), protein content, ADP-ribosyltransferase activity, and antibody recognition as previously described (33). In addition, quality control has focused on ensuring the absence of traces of dimethyl (2, 6-O-)P-cyclodextrin (5) and cibacron blue (49). The absence of fetuin, the 69-kilodalton protein, and filamentous hemagglutinin was verified by Western (immuno-) blot analysis (50). The absence of traces of dermonecrotic toxin was verified in the guinea pig model as described previously (20). Purified PT was frozen in 50% glycerol and stored at -70°C. Stabilization of PT-9K/129G. Purified PT-9K/129G was dialyzed for 24 h at 4°C against phosphate-buffered saline (PBS), pH 7.4, containing 0.025 M lysine (Ajinomoto, Tokyo, Japan) and 0.01% thimerosal. After protein determination (23), formaldehyde (4% solution in PBS, pH 7.4) was added to the mixture so that the final ratio (wt/wt) between protein and Formalin was 0.3. The mixture was incubated at 37°C for 48 h and dialyzed exhaustively against saline. The free Formalin content was less than 0.01% (wt/vol). The final toxoid preparation (PTF-9K/129G) was again analyzed for the protein content (23) and electrophoresed on an SDSpolyacrylamide gel (21). Amino acid composition. The analysis of residues in the toxoid preparations before and after treatment with formaldehyde was performed as described previously (41), after acid hydrolysis of proteins in 6 N HCI at 110°C for 24 h in vials sealed under high vacuum, with a Chromakon 500 amino acid analyzer (Kontron, Zurich, Switzerland). During the acid hydrolysis, tryptophan residues were destroyed and therefore were not determined. After the same treatment, because of deamidation, asparagine and glutamine could not be distinguished from aspartate and glutamate. Stability. PT and PT mutants before or after Formalin treatment were stored at 4, 20, and 37°C. At various times the antigens were assayed for their toxicities on CHO cells, for their electrophoretic profiles by the method of Laemmli (21), and for their affinities for toxin-neutralizing poly- and monoclonal (4) antibodies by radioimmunoassay as previously described in detail (33). Hemagglutination. The hemagglutination assay was performed as previously described (37) by using glutaraldehydefixed chicken erythrocytes as target cells. Results were expressed as doses of hemagglutinin causing complete agglutination of erythrocytes. Human T-cell mitogenicity. The mitogenic activity of the double mutant PT-9K/129G was assayed as the proliferative response of human peripheral blood mononuclear cells (PBMC) from normal adults. Briefly, Fycoll-Hypaque (Pharmacia Fine Chemicals AB, Uppsala, Sweden)-separated PBMC were plated in 96-well flat-bottomed tissue culture plates (Costar, Cambridge, Mass.) at 105 cells per well in 0.2 ml of RPMI 1640 (Gibco Laboratories, Paisley, Scotland) supplemented with 2 mM L-glutamine, 1% nonessential amino acids, 5 x 10-5 M 2-mercaptoethanol, and 10% human AB serum. Wild-type PT and PT-9K/129G were then added to the wells at final concentrations of 0.1, 0.3, 1.0, and 3.0 pug/ml. After 96 h of incubation, 1 pRCi of [3H]thymidine (specific activity, 185 GBq/mmol; Amersham International, Amersham, United Kingdom) was added to wells. Cells

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were harvested 16 h later on glass-fiber filters with a cell harvester (Skatron, Lier, Norway), and incorporated radioactivity was determined by liquid scintillation. Results are expressed as the mean counts per minute of duplicate cultures. ELISA. The enzyme-linked immunosorbent assay (ELISA) was a modified version of that described by Engvall and Perlmann (12). Wells of each flat-bottomed polystyrene microtest plate (Dynatech Laboratories Inc., Alexandria, Va.) were coated with PBS, pH 7.4, containing 1 ,ug of purified PT. The coating was performed for 2 h at 37°C and overnight at 4°C in a humidified chamber. After minimization of nonspecific absorption of serum proteins to the plastic with 1% bovine serum albumin (BSA) in PBS, dilutions of serum samples from guinea pigs were incubated for 2 h at 37°C. Incubation of specific alkaline phosphatase-conjugated immunoglobulin G antibodies (Miles, Yeda, Israel) was done at 37°C for 2 h. Volumes of 100 ,ll were used in all steps, and washing of the plates was performed three times between incubations by using PBS containing 0.05% Tween 20 and 0.02% NaN3. The enzyme substrate reaction that developed at room temperature was measured at 405 nm on a Titertek Multiskan (Flow Laboratories, McLean, Va.). Controls for each plate included wells with serum samples but no antigen and wells with antigen but no serum samples. Each serum sample was tested in duplicate, and absorbance values were averaged. Immunoglobulin G titers were evaluated by graphing the five serum dilutions against the average of the respective absorbances. The intercept through the flex point with the axis of absorbances represents the absorbance value of the undiluted serum (ABSMAX). The averages of the ABS-MAX obtained for the group of immunized guinea pigs were then compared after every bleeding with that of preimmune sera. The increase of ABS-MAX was considered statistically significant when it was at least four times higher than the prevaccination value. Chinese hamster ovary (CHO) cells assay. The clustering activity of PT or PT mutants was detected as described by Hewlett et al. (17). Briefly, CHO cells were incubated for 48 h with different doses of the double mutants (0.01 to 5 ,ug/ml) or wild-type PT (0.3 pg/ml to 90 ng/ml), and the minimum clustering dose was determined. Toxin neutralization. The method used to evaluate the level of toxin-neutralizing antibodies induced by vaccination was essentially the same as that described elsewhere (14, 15). Briefly, sera from guinea pigs obtained after vaccination with one or two doses of PTF-9K/129G were diluted directly in the wells of flat-bottomed microplates (Costar) in 25 ,ul of Dulbecco modified Eagle medium (Flow Laboratories, McLean, Va.). Purified wild-type PT (120 pg) in 25 plI of Dulbecco modified Eagle medium was added to each well, and the plates were incubated for 3 h at 37°C. After the incubation period, 0.2 ml of Dulbecco modified Eagle medium containing 104 CHO cells previously treated with 1 mg of trypsin per ml were added to each well and incubated for 48 h at 37°C in a 5% CO2 atmosphere. As a positive control, the clustering effect of PT alone was titered in each plate. Neutralizing titers were expressed as the reciprocal of the highest serum dilution causing complete inhibition of the clustering activity induced by the native toxin. Leukocytosis-promoting activity. Leukocytosis was assayed by injecting female BALB/c mice intraperitoneally (i.p.) on day 0 with 0.5 ml of saline either alone or containing 0.004, 0.02, 0.04, 0.1, 0.5, or 1 p,g of PT or 2.5, 12.5, 25, or 50 ,ug of PT-9K/129G. On day +3, blood samples were

1310

NENCIONI ET AL.

drawn at the orbital plexus and leukocytosis was measured in a Coulter counter (Coulter Electronics Ltd., Luton, England). Results were expressed as the mean + standard deviation of leukocyte counts from five animals tested individually. Histamine-sensitizing activity. The histamine sensitization was assayed by injecting groups of five female BALB/c mice i.p. on day 0 as described for leukocytosis evaluation. On day +6, mice were challenged i.p. with 4 mg of histamine dichlorohydrate (Sigma Chemical Co., St. Louis, Mo.) in 0.5 ml of saline, and 24 h later deaths were registered. Potentiation of anaphylaxis. Induction of anaphylactic sensitivity was evaluated by the method described by Steinman et al. (44). BALB/c mice (H-2d genotype) were injected intravenously in the tail vein on days 0 and +2 with 0.2 ml of saline containing either 0.04, 0.1, or 0.5 ,ug of PT or2.5 or 7.5 ,ug of PT-9K/129G. On days -1, + 1, and +6, the same mice received 1 mg of bovine serum albumin (BSA; Sigma) i.p. in 0.2 ml of saline. Deaths were recorded 2 h after the last administration of BSA. Islet-activating protein activity. Islet-activating protein activity was assayed as described elsewhere (19). Briefly, groups of five mice were injected i.p. with either 25 ,ug of PT-9K/129G, 1 ,ug of PT (lot no. JNIH-5), or saline. Four days later, insulin levels were determined in the mouse sera. Acute toxicity. The study of i.p. and subcutaneous toxicity was performed by injecting mice and rats with 1,500 ,ug of PT mutants per kg of body weight before and after Formalin treatment. Animals were observed throughout 14 days, and changes in body weight or any other symptoms of local and systemic adverse reactions after the vaccine administration were registered. Immunogenicity. Groups of self white guinea pigs were inoculated subcutaneously with 3, 10, 25, or 50 ,ug of PTF9K/129G adsorbed onto Al(OH)3 (0.5 mg per dose). An additional group of guinea pigs was injected subcutaneously with 0.75 ml of DPT whole-cell vaccine corresponding to 1.5 human doses. Four weeks later, animals were bled and boosted with the same doses of Al(OH)3-adsorbed PTF9K/129G and DPT vaccine as the first immunization. Two weeks after the boost, sera were collected and tested by ELISA for antitoxin titers and toxin-neutralizing activity by the CHO cell assay. Vaccine potency. Groups of 16 male CD1 mice were vaccinated i.p. according to the World Health Organization recommendations with 0.24, 1.20, 6, or 30 ,ug of PTF9K/129G adsorbed onto A1(OH)3 (1 mg/ml) or with 0.24, 1.20, 1.92, 4.80, 12, and 30 ,ug of fluid PT-9K/129G. As a positive control, groups of mice were injected through the same route with 0.00032, 0.001, 0.008, or 0.04 ml of a standard cellular pertussis vaccine (lot no. 9) provided by the National Institutes of Health (Bethesda, Md.). Fourteen days after vaccination, mice were challenged intracerebrally (IC) with a suspension of virulent B. pertussis 18323 (Sclavo, Italy) containing 300 median lethal doses. Mice were observed throughout 14 days, and deaths were registered. Statistical analysis. Unless otherwise stated, differences in the mean values of the experimental systems were assessed by Student's t test. RESULTS Production of PT mutants. The strains of B. pertussis W28, whose gene encoding the subunit S1 of PT had been modified to produce the nontoxic forms of PT (9K/129G, 13L/129G, and 261/129G), were grown in a 20-liter fermentor with

INFECT. IMMUN.

1

SI-

2

3

4

5

6

._

S2-.-

83

S5 S4

FIG. 1. Electrophoretic pattern of PT (lanes 1 and 4), PT9K/129G (lanes 2 and 5) and formaldehyde-treated PT-9K/129G (PTF-9K/129G) (lanes 3 and 6) on day 0 (lanes 1 through 3) and after storage for 1 month at 37°C (lanes 4 through 6). Positions of subunit bands are indicated.

Stainer and Scholte modified medium for 36 to 48 h. When the cultures had reached an optical density at 590 nm of 14.0 to 18.0, before the late logarithmic phase, the cells were removed by centrifugation and the mutant PT molecules were recovered after sterile filtration of the supernatant by Affi-Gel blue absorption and Fetuin-Sepharose affinity chromatography as described by Sekura et al. (39). Although the preparations described in this paper were obtained with routine yields of approximately 2 mg of purified PT mutant per liter of cultures, lately the yields have been increased three- to fivefold. Most of the tests described below were performed with each of the three double mutants. However, as we have never detected any significant differences between them, we will describe in this paper only the results obtained with the double mutant PT-9K/129G. Physicochemical and in vitro properties. When tested in SDS-PAGE stained with Coomassie blue, the purified PT9K/129G proved to be free of any detectable protein contaminant and showed a pattern undistinguishable from that of wild-type PT (Fig. 1). The absence of Fetuin, filamentous hemagglutinin, and 69-kilodalton protein, which are possible contaminants of acellular pertussis vaccines (40), was also confirmed in a Western blot using antibodies specific for these proteins. The absence of dermonecrotic toxin activity was confirmed by a skin test with guinea pigs (20). The presence of cyclodextrin, which is a major component of the growth medium, was excluded by thin-layer chromatography (5) (data not shown). The lipopolysaccharide content measured in the Limulus amebocyte lysate test was usually below 1 to 2 endotoxin units per ml, where 1 endotoxin unit corresponds to 0.1 ng of endotoxin. However, some of the lots (for instance, the one used for the islet-activating protein assay) showed higher endotoxin contamination. The amino acid composition (Table 1) was in agreement with the values predicted from the amino acid sequences. As previously reported (33), PT-9K/129G was devoid of ADP-ribosyltransferase activity. In marked contrast, its affinity for protective mono- and polyclonal antibodies to PT (33) (Table 2) and its hemagglutinating activity (100 ng per well) were similar to that of wild-type PT. Similarly, the double mutant PT-9K/129G retained a mito-

ACELLULAR PERTUSSIS VACCINE

VOL. 58, 1990 TABLE 1. Amino acid compositions of PT, PT-9K/129G, and PTF-9K/129G

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40

Amino acid/protein molar ratio of:

Amino acid

Asx Thr Ser Glx Pro

Gly Ala

Cys Val Met

Ile Leu Tyr Phe Lys His Arg Trp

pFra

PT-9K/129G

PTF-9K/129G

65 70 67 82 55 80 87 26 67 29 40 74 62 32 32 16 62 6

61.2 70.1 60.1 NDb 81.3 79.8 ND 72.9 28.8 40.0 75.8 63.5 30.5

67.0 65.6 61.4 93.5 ND 85.5 90.0 ND 67.9 26.7 38.0 77.3 61.2 31.0

39.3 16.7 63.5 ND

164.9c 14.6 65.0 ND

70.5

Theoretical values deduced from the primary structure of the protein. ND, Not determined. c Note the increase in lysine residues after Formalin treatment in the presence of 0.025 M lysine. a

b

genic activity for human T lymphocytes comparable with that of native PT (13) (Fig. 2). This is in agreement with the observation that the B oligomer by itself is responsible for the mitogenic activity of PT (16, 47). Formaldehyde stabilization. Before further testing of the PT double mutant, part of it was treated with formaldehyde at 37°C for 48 h in the presence of 0.025 M lysine. This was done on the basis of our previous observations that Formalin stabilization of the nontoxic diphtheria mutant CRM197 was necessary for the induction of protective immunity (34). In SDS-PAGE, the Formalin-treated. PT mutant (PTF9K/129G) showed the same basic pattern as the wild-type PT, with the addition of few extra bands: one migrating below subunits S2 and S3, and the others with molecular weights higher than that of S1 (Fig. 1, lane 3). The amino acid composition of PTF-9K/129G was still in agreement with the theoretical values, the only exception being a fourfold increase of lysine residues deriving from the formaldehyde treatment in the presence of lysine (Table 1). Formalin treatment reduced the hemagglutinating activity by a factor of two but did not significantly change the mitogenic properties of PT-9K/129G (not shown). Stability. Storage of all molecules at 4 or 20°C for up to 4 months did not result in changes of their electrophoretic patterns (not shown) or of their affinity constants (Table 2). However, storage at 37°C for 1 month or longer resulted in a

30

20

M -

PT-9K/129G

-

10

0

medium

0.1

0.3

1.0

3.0

,ug/mi FIG. 2. Mitogenic response of PBMC to wild-type PT and to double mutant PT-9K/129G. PBMC used in this assay showed no significant antigen-specific response to heat-inactivated PT. Standard deviations were below 15%. kcpm, Thousands of counts per minute.

progressive decrease in the intensity of the band corresponding to the Si subunit of PT and PT-9K/129G (Fig. 1). In contrast, the Formalin-treated mutant did not show any alteration of the SDS pattern after 1 month of storage at 37°C (Fig. 1). In vivo biological activities. The biological activities were evaluated only on the mutant PT-9K/129G, assuming that the PTF-9K/129G can only have less activity than the molecule not treated with formaldehyde in these tests. As previously reported, PT-9K/129G was unable to promote leukocytosis in mice up to a dose of 50 ,ug per mouse (33) (Table 3). Similarly, the double mutant protein, when inoculated i.p., did not stimulate any sensitization of mice to a lethal challenge performed 6 days later with the vasoactive amine histamine. In contrast, native PT when used at the dose of 0.5 ,ug caused 100% deaths in groups of five animals (Table 3). The ability of PT to potentiate BSA anaphylaxis, a phenomenon which has been associated with pertussis vaccine encephalopathy (45), was also absent in the PT-9K/129G mutant. This pertussis-dependent murine anaphylaxis involves repeated immunizations of mice with PT or its double mutant and sensitization and challenge with BSA. Anaphylaxis occurred in 80% of mice given 40 ng of PT, while no deaths were recovered 24 h after the last challenge with BSA in groups of animals receiving up to 7.5 ,ug of PT-9K/129G (Table 3).

TABLE 2. Affinity of the PT preparations for toxin-neutralizing y-globulins or for the anti-Si monoclonal antibody 1B7 Affinity of PT fora: Affinity of PT-9K/129G fore: Affinity of PTF-9K/129G for=: Days stored (temp [°C]) y-Globulin

0 (4) 120 (4) 120 (20)

2.0

x

1010

NDb

1B7

y-Globulin

1B7

y-Globulin

1B7

2.4 x 108 ND

9.8 x 109 9.5 x 109

6.1 x 108 5.7 x 108 2.9 x 108

1.7 x 1010 1.4 x 1010 1.5 x 1010

3.2 x 108 6.2 x 108 2.1 x 108

ND ND 3.1 x 1010 a Data were evaluated by nonlinear regression analysis (24), are expressed as Ka (liters/mol), and are Standard deviation values never exceeded 15%. b ND, Not determined.

geometric mean values of samples tested in triplicate.

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INFECT. IMMUN.

TABLE 3. In vivo biological activities of PT-9K/129G compared with purified native PT No. of deaths/total no. tested

Expt group

Dose (,ug/mouse)

Saline PT

PT-9K/129G

Leukocytosisa (PBMC/ml) tSD (106)

after challenge with: BSA

0/5

0/5

8.3

0/5 0/5 0/5 1/5 5/5 5/5

NDb ND 4/5 4/5 5/5 ND

ND ND ND ND ND 19.6

0/5

0/5 0/5

5.23 ± 0.51

0.004 0.020 0.040 0.100 0.500 1.000 2.500 7.500 12.500 25.000 50.000

5.94 10.33 14.34 17.73 45.73 55.19

Insuline secretion (mU/liter)

Histamine

± 0.41 ± 0.82 ± 0.83

+ 1.12 ± 3.76 ± 6.62

4.45 + 0.41 ND 4.39 ± 0.32 4.79 ± 0.44 4.71 + 0.35

ND 0/5 0/5

ND ND 5.0 5.0 ND

ND ND ND

0/5

a Results are expressed as mean number of PBMC ± standard deviation from five mice assayed individually. b ND, Not determined.

In agreement with the above results, 1 ,ug of PT injected i.p. induced, as expected, a significant increase of the insulin secretion by pancreatic islets, whereas mice treated with 25-fold-larger amounts of PT-9K/129G had insulin levels in blood comparable with those of control mice (Table 3). When tested for acute toxicity in animal models according to the World Health Organization guidelines, (52), 1,500 ,ug of PT-9K/129G per kg (approximately 1,000 times the foreseen per-kilogram human dose, before or after Formalin treatment) did not alter the body weight nor induce any detectable symptom of local or systemic adverse reaction (data not shown). Immunogenicity. To investigate the immunogenicity of the genetically engineered PT, we treated groups of six guinea pigs with one or two injections, 4 weeks apart, of 3, 10, 25, or 50 ,ug of Al(OH)3-adsorbed PTF-L9K/129G and with 1.5 human dose of DPT whole-cell vaccine. When tested for antitoxin titers in the ELISA, undiluted sera from animals bled 4 weeks after the first immunization showed A405s (ABS-MAX) which were from 32- to 40-fold higher than those before immunization. A further 50% increase of the ELISA titers could be observed in sera obtained 2 weeks after the booster injection (Fig. 3). The increase in antibody titers after immunization with PTF-9K/129G correlated with the neutralizing activity in the CHO cell assay. In fact, the sera resulting from a single injection of 3, 10, 25, and 50 ,ug of PTF-9K/129G were able to neutralize the clustering effect on CHO induced by 120 pg of wild-type PT at the dilution of 1/20, 1/20, 1/20, and 1/80, respectively. The toxin-neutralizing titers increased dramatically after the second immunization. In fact, antibodies obtained from animals given as little as 3 ,ug of the double mutant were able to neutralize the toxin activity up to a dilution of 1/1,280 (Fig. 3). Vaccine potency. We next studied the ability of the Formalin-treated and aluminum-adsorbed double mutant to protect mice from a lethal IC infection with virulent B. pertussis, a test which correlates with the protective activity of the cellular vaccine. Mice were immunized i.p. with various amounts of a standard cellular vaccine as a positive control and various dilutions of fluid PT-9K/129G or Al(OH)3-adsorbed PTF-9K/129G. Immunizations were followed 2 weeks later by IC challenge with the standard virulent strain, B. pertussis 18323. The AI(OH)3-adsorbed PTF-9K/129G

induced a dose-dependent protection of mice from paralysis or death with a median protective dose (see Table 4, footnote a) of 1.2 ,ug per mouse (Table 4). Interestingly, the results obtained with fluid PT-9K/129G not treated with formaldehyde were slightly better than those obtained with the Formalin-treated, Al(OH)3-adsorbed molecules. In fact, 100% protection was achieved after IC challenge any time we used PT-9K/129G as the immunogen (33) (Table 4). However, the median protective dose (1.1 ,ug per mouse) did not change significantly. DISCUSSION Enzymatically inactive, nontoxic forms of PT which are indistinguishable from wild-type PT in SDS-PAGE and with 5

5

4

4

X &-4

Eu

z

0

3 -,

x

C.;

3

2

2

O.

D

0

1

0

0 PRE IMMUNE

0.75 m1

DPT

3

10

25

50

PTF-9K/129G (gg)

FIG. 3. ELISA levels and toxin-neutralizing titers of antibodies raised in guinea pigs by one and two subcutaneous injections of DPT vaccine or different doses of PTF-9K/129G adsorbed onto A1(OH)3. Antitoxin antibody levels are expressed as ABS-MAX. Neutralizing titers (NT) are expressed as reciprocals of the highest serum dilutions resulting in 100% inhibition of the clustering effect on CHO cells induced by 120 pg of wild-type PT tested in triplicate.

ACELLULAR PERTUSSIS VACCINE

VOL. 58, 1990 TABLE 4. Mouse protective activity of A1(OH)3adsorbed-Formalin-treated PT-9K/129G (PTF-9K/129G) and fluid PT-9K/129G against IC infection No. of survivors/ total no. tested

NIH standard cellular vaccine (lot no. 9)"

PTF-9K/129G (adsorbed)

PT-9K/129G (fluid)

0.04 0.008 0.0010 0.00032

16/16 13/16 9/16 1/16

30 6 1.20 0.24

14/16 14/16

30 12 4.80 1.92 1.20 0.24

16/16

8/16 2/16 16/16

12/16 10/16 7/16 3/16

a Doses of the NIH vaccine are in milliliters; doses of the other two vaccines are in micrograms per mouse. Doses which protect 50% of mice from a lethal IC challenge with live B. pertussis 18323 are 1.2 and 1.1 ,ug per mouse, respectively, for PTF-9K/129G and PT-9K/129G. b Vaccine contains 8 international protective units per ml. NIH, National Institutes of Health.

conserved B- and T-cell epitopes (11, 35) have been previously proposed as candidates for new acellular vaccines against whooping cough (33). In this paper, we have characterized the physicochemical, biological, and immunological properties of these mutants, and we show that they are indeed good candidates for new pertussis vaccines. The absence of toxicity has been shown in a variety of systems. In the most sensitive test (the CHO cell assay), PT-9K/129G has been shown to be at least 106-fold less toxic than wild-type PT (33). In all the other tests, we have never observed any toxicity at the maximal dose used. In fact, the genetically produced PT toxoid failed to induce pathologic disorders typical of PT, such as leukocytosis, stimulation of histamine sensitivity, potentiation of anaphylaxis, and hyperinsulinemia, in mice. Although this study was primarily started for vaccine purposes, the data reported in this paper allow us to conclude in a definitive way that all the toxic biological activities of PT are due to the ADP-ribosyltransferase activity of the Si subunit (42, 48). The only activity not affected by the genetic manipulation of the Si subunit is the mitogenicity for T cells, which is maintained by the PT-9K/129G and the other nontoxic double mutants. In agreement with this observation, it has been previously reported that the purified B oligomer alone is mitogenic for T cells (16, 47). We have been able to further confirm this observation by using a B oligomer purified from a culture supernatant of a B. pertussis strain engineered to secrete only the B oligomer (M. Pizza et al., manuscript in preparation). While showing that the toxic biological properties are due to the enzymatic activity of the Si subunit, we also give conclusive evidence that the immunogenicity of PT (including its ability to protect mice in the IC challenge assay) is independent from enzymatic activity. This observation merits particular attention because the general adjuvant effect of the enzymatically active PT (6, 27) has led to speculation that this property would be an absolute requirement to obtain protection in the IC challenge and has led to doubt about the usefulness of enzymatically inactivated PT in a vaccine. Here, for the first time, we have been able to dissect

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these two activities and to show that enzymatically inactive PT molecules are excellent candidates for pertussis vaccines, even if the abolishment of the enzymatic activity may affect the adjuvant properties of PT (6). To assess the best formulations to be included in a vaccine, we have tested the stability and the immunogenic properties of PT-9K/129G before or after stabilization with formaldehyde. In contrast with our previous experience with the CRM197 mutant of diphtheria toxin, for which formaldehyde stabilization of the molecule was necessary to obtain protective immunity (34), we have found that PT-9K/129G is as good as its Formalin-treated analog in inducing neutralizing antibodies and in protection against IC challenge with virulent B. pertussis. Similarly, both formulations were equally stable when stored at 4 or 20°C. However, after long storage at 37°C, progressive loss of the S1 subunit was observed in the sample not treated with formaldehyde by using SDS-PAGE. This suggests that a mild stabilization with formaldehyde, although not necessary, might improve the stability of a PT-9K/129G-based vaccine. This vaccine would have obvious advantages over all the others so far proposed, which contain PT detoxified by a variety of chemical reagents such as formaldehyde (36), glutaraldehyde (35), tetranitromethane (Siber et al. and Winberry et al., Int. Workshop Bordetella pertussis, Hamilton, Mont., 1988), trinitrobenzenesulfonic acid (13), ethyl acetimidate (28), ethyl dimethylaminopropyl carbodiimide (8), or hydrogen peroxide (38). In fact, PT toxoid produced by the above methods has either one or both of the following problems: it can revert to toxicity or have a reduced immunogenicity caused by the harsh conditions required for complete detoxification and prevention of reversion. Additional problems encountered in the development of a chemically detoxified PT vaccine are batch-to-batch reproducibility, long and expensive tests for assessment of reversion of each lot, and, finally, the handling of large amounts of toxic material by the personnel involved in vaccine production. None of these are problems with PT-9K/129G. The only biological activity of the double mutant is the in vitro mitogenicity for T lymphocytes. Although the in vivo role of this activity is not known, it can be predicted that it is either minimal or absent because of the relatively high concentrations (0.3 to 1.0 ,ug/ml) required to have a detectable mitogenic effect in vitro. These concentrations will only be present at the site of injection in humans. The safety of this product is further supported by the evidence that treatment of mice and rats with 1,500 ,g per kg, which is 1,000 times the foreseen per-kilogram human dose, did not result in any sign of local or systemic toxicity. We therefore conclude that, in view of the disadvantages met with PT toxoid produced by chemical reagents, the molecules that we have described, being immunogenic and nontoxic, are the antigens of choice for a new pertussis vaccine and deserve further evaluation in humans. Indeed, phase I clinical trials in human adult volunteers are under way. ACKNOWLEDGMENTS We thank Roy Gross and Nicholas Carbonetti for critical reading of the manuscript, Antonella Mori and Catherine Mallia for secretarial work, Giorgio Corsi for graphic work, Sclavo Quality Control Center for some of the in vitro tests, and Fabrizio Zappalorto for some of the animal studies. Part of this work was supported by a grant from ENI (Ente Nazionale Idrocarburi).

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NENCIONI ET AL.

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