Microbial Pathogenesis 1991 ; 11 : 423-431

Protective activity of Bordetella adenylate cyclase-hemolysin against bacterial colonization Nicole Guiso, Marek Szatanik and Murielle Rocancourt Unite de Bacteriologie Moleculaire et Medicale, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France

(Received July 6, 1991 ; accepted in revised form August 31, 1991)

Guiso, N . (Unite de Bacteriologie Moleculaire et Medicale, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France), M . Szatanik and M . Rocancourt . Protective activity of Bordetella adenylate cyclase-hemolysin against bacterial colonization . Microbial Pathogenesis 1991 ; 11 : 423-431 . Bordetella pertussis synthesizes several factors . It has been suggested that one of these

factors, the adenylate cyclase-hemolysin (AC-Hly), directly penetrates target cells and impairs their normal functions by elevating intracellular cAMP . In the present study, we show that active immunization with purified B . pertussis AC-Hly or AC (a fragment of the AC-Hly molecule carrying only the adenylate cyclase activity but no toxin activity in vitro) protects mice against B. pertussis intranasal infection . Immunization with AC-Hly or AC significantly shortens the period of bacterial colonization of the mouse respiratory tract . Furthermore, B . parapertussis AC-Hly or AC are also protective antigens against B . parapertussis colonization ; their protective activities are equivalent to that of the whole-cell vaccine . These results suggest that AC-Hly may play an important role in Bordetella pathogenesis, in a murine model . If this factor plays a similar role in the human disease, its use as a protective antigen could reduce not only the incidence of the disease, but also the asymptomatic human reservoir by limiting bacterial carriage . Key words : Bordetella pertussis ; Bordetella parapertussis ; pathogenicity ; adenylate cyclase-

hemolysin ; protective antigen ; murine model .

Introduction Bordetella pertussis, the causative agent of whooping cough, has been effectively

controlled by the use of heat-killed whole-cell pertussis vaccines . Large-scale vaccination in various countries for about 40 years provide children with substantial protection against the disease, but this protection is incomplete ; B . pertussis still seems to be widely prevalent in immunized populations .' Moreover, adverse effects are claimed to be associated with this vaccine although this evidence is disputed .` These two reasons encouraged us to develop a new acellular vaccine . This new vaccine needs to be devoid of side effects, to have similar or better protective efficiency than the present vaccine, i .e . to protect against the disease, but also against infection in order to minimize carriage of B . pertussis and B . parapertussis, the other human pathogen . Important steps toward this goal are the identification and isolation of virulence factors and potential protective antigens . The course of the infection can be divided into two major stages, adherence and disease .' First, the bacteria adhere to the ciliated 0882-4010/91/120423+09 503 .00/0

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cells of the respiratory tract by synthesizing and secreting factors such as filamentous hemagglutinin (FHA) and pertussis toxin (PTX) where they persist, grow and multiply . During a second stage, the bacteria induce local disease (ciliostasis) and systemic disease (lymphocytosis and alterations in blood chemistry) by synthesizing toxins such as PTX, dermonecrotic toxin, tracheal cytotoxin and adenylate cyclase-hemolysin (AC-Hly) . 4 Two of these factors, FHA and PTX, have received considerable attention as potential candidates for acellular vaccines .` Numerous studies have demonstrated their ability to protect mice against B . pertussis, by the use of respiratory and intracerebral lethal challenges . 6 Data from clinical trials demonstrated that acellular vaccines containing PTX alone or PTX plus FHA, are safer than whole-cell vaccines and provide some protection against typical whooping cough . However, those vaccines induce less protection against mild or asymptomatic pertussis infection ." Furthermore, no or low protection will be induced by these vaccines against B . parapertussis, bacteria eliciting pertussis-like diseases without expressing active PTX . 10 '" It is tempting to think that the efficacy of these vaccines might be increased by adding other protective antigens such as agglutinogens, 12 pertactin (69 kDa) 13, ' 4 or the bifunctional protein adenylate cyclase-hemolysin (AC-Hly) . 15 By generating a panel of mutants, A . Weiss et al. showed, 15,17 using a murine respiratory model, that not only PTX but also AC-Hly are required for lethal infection . Moreover, we recently showed that passive and active immunization with AC (a fragment of the AC-Hly molecule carrying only the adenylate cyclase catalytic activity) protects mice against pulmonary and intracerebral lesions induced by highly virulent variants of B . pertussis, suggesting a role as a protective antigen for this molecule . 18 " 9 In the present investigation, B . pertussis and B . parapertussis AC-Hly and AC fragments were purified and their role as protective antigens, against bacterial colonization, were examined and compared, using the adult mouse respiratory model . We show that active immunization with AC-Hly or AC can significantly reduce B . pertussis and B . parapertussis colonization in the lungs of mice . Furthermore, the protective efficiency of these antigens is similar or equivalent to that of whole-cell vaccines . Results AC-Hly and AC purification from B . pertussis and B . parapertussis AC-Hly was purified from the bacteria and AC fragments from culture supernatants . As shown in Fig . 1 (a) (lanes 2 and 4) we could obtain, from both B . pertussis and B . parapertussis culture supernatants, purified preparations containing polypeptides between 40 and 43 kDa . We know that the three polypeptides purified from B . pertussis culture supernatant are derived one from the other by proteolysis . 21 A monoclonal antibody specific for the 43 kDa peptide of B . pertussis24 recognizes the different polypeptides purified from B . parapertussis [Fig . 1(b), lanes 2 and 4] confirming the cross reactivity between these enzymes' $ and suggesting that, as in the case of B . pertussis, B. parapertussis enzyme is also proteolysed, using these purification procedures . However, the patterns of fragments obtained from both culture supernatants are different . The purified AC-Hly preparations obtained from both bacteria contain a major polypeptide of 200 or 210 kDa and some peptides of lower molecular weight [Fig . 1 (a), lanes 1 and 3] . Anti-B . pertussis AC monoclonal antibody recognizes both enzymes [Fig . 1(b), lanes 1 and 3] and many of the peptides, suggesting a proteolysis of the enzymes during purification processes . Our results confirm data from Bellalou



Protective activity of

Bordetella adenylate cyclase

42 5

(a)

(b)

200

200

40

43

43 1 1

2

3

2

3

4

4

Fig . 1 . (a) Purification of AC-Hly and AC . B . pertussis 18323 AC-Hly (lane 1), B . pertussis 18323 AC (lane 2), B . parapertussis 63 .2 AC-Hly (lane 3) and B . parapertussis 63 .2 AC (lane 4) were purified as described in Materials and methods and subjected to 4-15% SDS-PAGE . The gels were stained with Coomassie Blue . (b) Immunoblots of purified AC-Hly and AC fragments . Purified preparations were obtained as described in Materials and methods and subjected to 8-25% SDS-PAGE . The proteins were then transferred to HYBOND C-Super and incubated with monoclonal antibody specific to B . pertussis AC . The immunodetection was performed with peroxydase labelled rabbit anti-mouse antibodies . Lane 1, B . pertussis 18323 AC-Hly; lane 2, B . pertussis 18323 AC ; lane 3, B. parapertussis 63 .2 AC-Hly; lane 4, B . parapertussis 63 .2 AC .

et al., 22 but the fact that our preparations are more proteolysed may be due to the absence of BSA in our culture medium . These purified preparations are negative for FHA, PTX and pertactin when screened by Western immunoblotting with polyclonal anti-FHA, anti-PTX and anti-pertactin antibodies (data not shown) . After immunization of mice with AC-Hly or AC, the antiserums obtained recognize both AC-Hly and AC antigens . All anti-B . pertussis AC-Hly or AC recognize B . parapertussis AC-Hly or AC and vice-versa . Furthermore, these antiserums do not recognize FHA or PTX indicating again the absence of these factors in the purified preparations of AC-Hly or AC (data not shown) . Comparative protective efficacy of B . pertussis AC and AC-Hly against B . pertussis 18323 colonization in the murine intranasal model The role of AC-Hly and AC as protective antigens against colonization of the mouse respiratory tract was evaluated . We showed previously that active immunization twice with 4 yg of AC fragments protects mice against lethal respiratory or intracerebral challenge with B . pertussis 18323 strain ." ," Furthermore, we showed that active immunization with AC protects against bacterial colonization after sublethal respiratory or intracerebral challenge with B. pertussis 18323 . 19 In the present study, we examined the protective activity of the entire AC-Hly molecule and we compared it with that of the AC fragments or that of the whole-cell vaccine . As can be seen in Fig . 2, the protective efficacy of AC-Hly is similar to that of the AC preparation . In control mice immunized only with aluminium hydroxide, bacterial counts in the lungs increased from 5x10' cfu immediately after challenge to 6x10' cfu on days 3 and 6 after challenge . Mice immunized with AC-Hly or AC show no multiplication of the bacteria . In addition, a 1 log difference at day 2, and a 2 .5 log difference at day 8, were observed between the control and immunized animals . Protective efficacy of B . pertussis AC against B . pertussis Tohama .colonization in the murine intranasal model The B . pertussis 18323 strain is used as the reference strain in the mouse potency test of pertussis vaccines because it is more virulent than the other B . pertussis strains . It



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Fig . 2 . Bordetella pertussis 18323 colonization of the lungs of BALB/c mice actively immunized with purified B. pertussis AC-Hly or AC . Mice were immunized twice with a 2-week interval, with 25 pg of purified AC-Hly or 4 ,ug AC or 5 .10 8 killed B . pertussis 18323 whole-cell vaccine, adsorbed on aluminium hydroxide . The sublethal challenge, 7 .10 8 cfu of B . pertussis 18323, was given intranasally, 14 days after the last injection . Control mice received aluminium hydroxide alone . The plots show the geometric mean +standard deviation for six mice per time point . A, controls ; 0, B . pertussis AC-Hly; •, B . pertussis AC; x, whole-cell B . pertussis 18323 vaccine .

was shown, based on nucleotide sequence data, that this strain is 'intermediate' 10 between B . pertussis, B . parapertussis and B . bronchiseptica, the animal pathogen, and this could explain its increased virulence for mice ." As it is shown in Table 1, B . pertussis strain 18323 has three times more adenylate cyclase activity (and expresses three times more protein, data not shown) and is more virulent than B . pertussis Tohama strain, in both intracerebral and intranasal lethal models . 19 The fact that AC is highly immunoprotective against B . pertussis 18323 strain could in particular be due to this . Furthermore, it was shown that B . pertussis 18323 strain expresses less PTX activity than B . pertussis Tohama strain . For these two reasons, we decided to test the protective efficacy of AC fragments against Tohama strain infection . As can be seen in Fig . 3, AC is a protective antigen against colonization by B . pertussis Tohama strain in lungs . Again, this protection is observed very early after the infection ; at day 2 a 2 log difference is observed in the bacterial count number between AC immunized mice and control mice .

Table 1 Adenylate cyclase activity and LD 50 of B . pertussis 18323 and B . pertussis Tohama LD,, Respiratory infection

B P 18323 BP Tohama

Intracerebral infection

Adenylate cyclase activity'

Suckling mice

Adult mice

Adult mice

160 50

103 5x10°

3 x 106 2x10'

104 5x10 5

'Adenylate cyclase activities (U/ml) were measured in wholecell suspensions as described in Materials and methods .



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.` 0' C

N

CI

0' 0

0

5

10 Days after challenge

15

20

Fig . 3 . Bordetella pertussis Tohama colonization of the lungs of BALB/c mice actively immunized with purified B . pertussis AC . Mice were immunized twice with a 2-week interval, with 4,ug of AC or 5 .10 8 killed B. pertussis Tohama whole-cell vaccine adsorbed on aluminium hydroxide . The sublethal challenge, 8 .10 8 cfu of B . pertussis Tohama, was given intranasally, 14 days after the last injection . Control mice received aluminium hydroxide alone . A, controls; 0, B . pertussis AC ; x, whole-cell B . pertussis Tohama vaccine .

Protective efficacy of B . parapertussis AC-Hly and AC against B . parapertussis colonization in the murine intranasal model As we showed earlier,' $ B . parapertussis, which does not express PTX, induces an acute hemorrhagic edematous alveolitis, in the murine respiratory model, similar to that observed with B . pertussis . Furthermore, the ability to induce lethal pulmonary lesions is associated with enhanced in vitro adenylate cyclase expression ." For this reason AC-Hly and AC fragments were purified from B . parapertussis and their protective efficacy in the murine respiratory model was examined . As can be seen in Fig . 4, AC-Hly and AC are protective antigens against B . parapertussis colonization of the lungs . Moreover, their efficacy is similar to that of whole-cell vaccine .

0' C 7

N 0 4

u 0• 0

0

5

10 Days after challenge

15

Fig . 4 . Bordetella parapertussis 63 .2 colonization of the lungs of BALB/c mice actively immunized with purified B. parapertussis AC-Hly or AC . Mice were immunized twice with a 2-week interval, with 25 pg of AC-Hly or 4 pg of AC or 5 .108 killed B . parapertussis 63 .2 whole-cell vaccine, adsorbed on aluminium hydroxide, at 2 weeks interval . The sublethal challenge, 8 .108 cfu of B . parapertussis 63 .2, was given intranasally, 14 days after the last injection . Control mice received aluminium hydroxide alone . A, controls ; 0, B. parapertussis AC-Hly; •, B. parapertussis AC; x, whole-cell B . parapertussis 63 .2 vaccine .



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Discussion A pertussis acellular vaccine should not only protect the individuals against disease, as well as the present whole-cell vaccines do, but also reduce the asymptomatic carriage of B . pertussis . The inclusion of a particular antigen in this acellular vaccine will then depend on its role in the pathogenesis and in the demonstration of its protective potency in a relevant animal model for pertussis infection . The main candidates are at present FHA and PTX . FHA has been chosen, by several groups, because it is devoid of toxic properties and because of its prominent role in adhesion of B . pertussis to the ciliated cells of the host respiratory tract .""" PTX was chosen because of its important toxic role during pertussis disease ." However, pertussis-like diseases in humans and mice occur in the absence of production of PTX by B . parapertussis .'"' Among factors expressed by all three Bordetella species that could contribute to the disease, the AC-Hly molecule is a good candidate since (i) mutants deficient in the expression of this protein are severely impaired in their ability to cause a lethal infection in mice, (ii) purified AC-Hly is able to penetrate mammalian cells in vitro and disturb their cellular functions, and (iii) highly virulent derivatives of B . pertussis and B . parapertussis express higher levels of AC-Hly than parental strains ."' , 8' 28 For all these reasons we examined its role as a protective antigen . The same procedures were used to purify AC-Hly and AC from B . parapertussis as from B . pertussis. B. parapertussis AC-Hly and AC are very similar to the B . pertussis ones and cross-react with them . Two murine models are generally used to test protective efficacy of Bordetella factors: the respiratory and the intracerebral models . The respiratory infection is more adapted to such studies because it mimics more closely some parameters of the human disease . However, it remains that standard studies on protective efficacy of vaccines or purified antigens are conducted with the intracerebral model . We demonstrated previously that AC, as PTX, is a protective antigen against B . pertussis induced diseases in both animal models ." ," In the present study, using the respiratory model, we show that immunizations with purified AC-Hly or AC induce a protection against bacterial colonization . The protective efficiency obtained after immunizations with 2x25 Ug of AC-Hly is equivalent to that obtained after immunizations with 2x4 µg of AC . Very low protection was obtained after active immunizations with 2x5 Ug or 2x10 µg of AC-Hly (data not shown) . These results indicate that the protection induced by ACHly is equivalent to that of AC if mice are immunized with about the same number of molecules of AC-Hly or AC . This could be explained if the major protective epitopes are localized on the AC part of the AC-Hly . However, another explanation could be that, contrary to AC, AC-Hly may have toxin activity and may induce an immunosuppressed state . Experiments are in progress to test these hypotheses . The B . pertussis reference strain 18323 is derived from the same ancestor as B . parapertussis and is different from all other isolates of B . pertussis, which seem to be genotypically identical ." The protective efficacy of AC against colonization of another B . pertussis strain, the Tohama strain, was therefore evaluated . The protection against colonization by the Tohama strain is similar to that obtained against bacterial colonization of the 18323 strain . These results show that AC-H ly and AC are protective antigens against B . pertussis colonization in the murine model . The efficacies of protection of AC-Hly or AC are lower than the protective efficacy of the whole-cell vaccine (Figs . 2 and 3) . This suggests that AC-Hly protein is not the only factor involved in bacterial colonization . Other factors including FHA, PTX and pertactin may play a role . Protective effects of AC-Hly or AC occur very early during the infection by B .

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pertussis . Since AC-Hly protein has no demonstrated role in the adherence of the bacteria," our results could be explained by AC-Hly helping B . pertussis to survive and to multiply better on the surface of the respiratory epithelium, by causing a localised depression of the immune system as suggested by in vitro studies . 28 Our results suggest that anti-AC-Hly antibodies inhibit the interaction of AC-Hly with target cells and its invasivity . Bordetella parapertussis AC-Hly or AC can protect against bacterial colonization by B . parapertussis (Fig . 4) . The protective efficacy was equivalent to the protective efficacy of the whole-cell vaccine . B . parapertussis strains do not express PTX . Our results suggest that in B . parapertussis infections, AC-Hly could be the main factor responsible for bacterial colonization, in the murine model . Studies are now underway to determine the target cell of AC-Hly and to fully understand its role in the virulence and survival of B . pertussis, in vivo . These results, taken together with our results (N . Khelef et al., in preparation) and those of A . Weiss et al., 29 using adenylate cyclase mutants, strongly suggest that immunization with AC-Hly, preventing colonization, could reduce asymptomatic carriage, not only of B . pertussis, but also of other Bordetella pathogens for humans . All these results suggest that the acellular vaccine needs to contain more than one component and that AC-Hly should be seriously considered as one of these components .

Materials and methods Bacterial strains and culture conditions . The strains used in this study were lung-passaged derivatives of B . pertussis 18323 (Type strain ATCC 9797), B . pertussis Tohama (CIP 8132) and B . parapertussis (CIP 63-2) isolated as described by Guiso et a/. 18 . Bacteria were grown on Bordet Gengou agar-supplemented with 15% defibrinated sheep blood (BGS plate) at 36°C for 48 h . Subcultures in liquid medium were performed on Stainer-Scholte medium 21 for 20 h at 36°C, until absorbance measured at 650 nm reached 1 .0. Bacteria were harvested for subsequent purification of the intracellular AC-Hly protein . The supernatant was concentrated as previously described 21 for purification of the AC fragments .

B . pertussis and B . parapertussis AC-Hly purification . B . pertussis 18323 or B . parapertussis 63-2 AC-Hly were purified, from the bacteria, after urea extraction using a calmodulin affinity column, as described 22 except that BSA was neither added during growth of the bacteria nor in the final step of the purification . Our preparations consisted of a major polypeptide of 200 kDa and various peptides of lower molecular weight [Figure 1 (a), lanes 1 and 3] . B . pertussis and B . parapertussis AC purification . B . pertussis 18323 AC fragments were purified from culture supernatants using a calmodulin affinity column, as described previously . 21 Our preparation consisted of polypeptides that are structurally related as described, 21 [Figure 1 (a), Lane 2] . Bordetella parapertussis 63-2 AC fragments were purified using the same procedure [Fig . 1(a), lane 4] . Adenylate cyclase assay. Adenylate cyclase activity was measured as described previously . 27 One unit corresponds to one nmol of cAMP formed per min at 30°C and pH 8 .

Protein determination . Protein concentrations were measured according to the method of Bradford .23 Electrophoresis and immunoblotting . All sodium dodecyl -sulfate- polyacrylamide gel electrophoresis were performed using ready to use 4-15% or 8-25% gels from Pharmacia and a PhastSystem (Pharmacia) . For immunoblotting experiments proteins were transferred from polyacrylamide gels to Hybond C-Super membranes (Amersham) and incubated with monoclonal anti-AC antibodies . 24 The immunochemical detection was performed with peroxydaselabelled anti-mouse immunoglobulins using the Amersham ECL detection reagents .



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Determination of the LD 5O . Bordetella pertussis or B. parapertussis were grown on StainerScholte broth for 20 h till A650 = 1 .0 was reached . The bacterial suspensions were then serially diluted to provide challenge innoculum dilutions to evaluate the LD 50 . For the respiratory infection, 10 or 50 pl of bacterial suspension were injected intranasally, respectively, to groups of 10, 6-day-old suckling mice or 4-week-old female BALB/c mice . For the intracerebral infection, 20 yl of bacterial suspension were injected intracerebrally, to groups of 10, 4-weekold female BALB/c mice (CERJ, St Berthevin, France) . The LD 50 for the challenges innocula were determine by recording daily the number of dead mice . Vaccines. The lung-passaged derivatives were grown on Stainer-Scholte broth until an adsorbance of 1 .0 at 650 nm was reached and heat-killed (20 min at 56°C) . Whole-cell vaccine suspensions were diluted in saline to 5x 10 $ colony forming units (cfu) per dose, and adsorbed on aluminium hydroxide (1 mg/ml, final concentration) prior to use . Active immunizations. For active immunizations, groups of 4-week-old female BALB/c mice were injected subcutaneously with either 25 µg of purified B . pertussis or B. parapertussis ACHly or 4 pg of purified AC or 5x10 $ killed B . pertussis or B . parapertussis, adsorbed on aluminium hydroxide, twice at a 2 weeks interval . Controls were injected with aluminium hydroxide alone. The mice were bled 7 days after the last injection in order to assess the presence of circulating antibodies . The sublethal challenges were performed 14 days after the second injection . Challenges . B pertussis or B . parapertussis were grown on Stainer-Scholte for 20 h till A650 - 1 was reached . The bacterial suspensions were then diluted and sublethal challenges were performed by intranasal injection of 7x10' cfu of B . pertussis 18323 or 8x10' cfu of B. pertussis Tohama or 8x10' cfu B . parapertussis 63 .2 . Infected mice were sacrificed by cervical dislocation 1 h after exposure (at time designated day 0) and at various days thereafter (six mice per time point) . The lungs were removed and homogenized in saline with tissue grinders . Dilutions of lungs homogenates were sampled on BGS plates and cfu were counted after 3 to 4 days of incubation at 36°C . Statistical analysis . Data were tested for statistical significance by Student's t-test .

This work was supported by funds from the Pasteur Institute Fondation and Pasteur Merieux Serum Vaccins . We are grateful to M . J . Quentin-Millet for the gift of FHA and PTX used to obtain specific polyclonal antiserums and to C . Capiau for the anti-pertactin antibodies .

Note added in proof Adenylate toxin activity was assayed by measuring the internalized adenylate cyclase activity as described ." Our preparations have no detectable toxin activity ; since the toxin activity is very unstable, this result is not surprising and is probably due to the fact that BSA was not added during purification of AC-Hly .

References 1 . Griffiths E. Efficacy of whole-cell pertussis vaccine . In : Wardlaw AC, Parton R, eds . Pathogenesis and immunity . J Wiley 1988 ; 353-74 . 2 . Cherry JD . 'Pertussis vaccine encephalopathy', it is time to recognize it as the myth that it is . JAMA 1990 ;263 :1679-80 . 3. 4. 5. 6. 7.

Griffin MR, Ray WA, Mortimer EA, Fenichel GM, Schaffner W . Risk of seizures and encephalopathy after immunization with the diphteria-tetanus-pertussis vaccine . JAMA 1990 ; 263 : 1641-5. Robinson A, Ashworth LAE . Acellular and defined-component vaccines against pertussis . In : Wardlaw AC, Parton R, eds . Pathogenesis and immunity . J Wiley, 1988; 399-417 . Nencioni L, Pizza M, Bugnoli Metal. Characterization of genetically inactivated pertussis toxin mutants : Candidates for a new vaccine against whooping cough . Infect Immun 1990; 58 : 1308-15 . Sato Y, Sato H . Animal models of pertussis . In : Wardlaw AC, Parton R, eds . J Wiley, 1988 ; 309-25 . 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 1990 ; 955-9 .



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8 . Olin P, Storsaeter J . The efficacy of accelular pertussis vaccine . J Am Med Assoc 1989 ; 261 : 560 . 9 . Storsaeter J, Hallender H, Farrington CP, Olin P, Moliby R, Miller E . Secondary analyses of the efficacy of two acellular pertussis vaccines evaluated in a Swedish phase III trial . Vaccine 1990 ; 8 : 457-61 . 10 . Aric6 B, Gross R, Smida J, Rappuoli R . Evolutionary relationships in the genus Bordetella . Mol Microbiol 1987 ; 1 : 301-8 . 11 . Musser JM, Hewlett EL, Peppier MS, Selander RK . Genetic diversity and relationships in population of Bordetella spp . J Bacteriol 1986 ; 166 : 230-7 . 12 . Tuomanen E . Bordetella pertussis adhesins . In : Wardlaw AC, Parton R, eds . Pathogenesis and immunity . J Wiley, 1988 ; 75-94 . 13 . Charles IG, Dougan G, Pickard D et a/. Molecular cloning and characterization of protective outer membrane protein P .69 from Bordetella pertussis . Proc Nati Acad Sci USA 1989 ; 86 : 3554-8 . 14 . Shahin RD, Brennan M, Li ZM, Meade BD, Manclark CR . Characterization of the protective activity and immunogenicity of the 69 kD outer membrane protein of Bordetella pertussis. J Exp Med 1990 ; 171 : 63-8 . 15 . Glaser P, Danchin A, Ladant D, Barzu 0, Ullmann A . Bordete/la adenylate cyclase : the gene and the protein . Tokai J Exp Clin Med 1988 ; 13 : 239-52 . 16 . Weiss AA, Hewlett EL, Myers GA, Falkow S . Transposon Tn5-induced mutations affecting virulence factors of Bordetella pertussis. Infect Immun 1988; 42 : 33-45 . 17 . Weiss AA, Goodwin MM . Lethal infection by B . pertussis mutants in the infant mouse model . Infect Immun 1989; 57 : 3757-64 . 18 . Guiso N, Rocancourt M, Szatanik M, Alonso JM . Bordetella adenylate cyclase is a virulence associated factor and an immunoprotective antigen . Microb Pathogen 1989 ; 7 : 373-80 . 19 . Guiso N, Szatanik M, Rocancourt M . Bordetella adenylate cyclase : a protective antigen against lethality and bacterial colonization in murine intranasal and intracerebral models . Proceedings of the Sixth International Symposium on Pertussis . 1990 ; 207-11 . 20 . Stainer DW, Scholte MJ . A simple chemically defined medium for the production of Phase I Bordetella pertussis . J Gen Microbiol 1971 ; 63 : 211-20 . 21 . Ladant D, Brezin C, Alonso JM, Crenon i, Guiso N . Bordetella pertussis adenylate cyclase : purification, characterization and radioimmunoassay . J Biol Chem 1987 ; 261 : 16264-9 . 22 . Bellalou J, Ladant D, Sakamoto H . Synthesis and secretion of Bordete/la pertussis adenylate cyclase as a 200-Kilodalton protein . Infect Immun 1990 ; 58 : 1195-200 . 23 . Bradford M . A rapid and sensitive method for the quantitation of micrograms of protein, utilizing the principle of protein-dye binding . Anal Biochem 1976 ; 72 : 248-57 . 24 . Brezin C, Guiso N, Ladant D et al. Protective effects of anti-Bordetella pertussis adenylate cyclase antibodies against lethal respiratory infection of the mouse . FEMS 1987 ; 42 : 75-9 . 25 . Kimura A, Mountzouros KT, Relman DA, Falkow S, Cowell JL . Bordetella pertussis filamentous hemagglutinin : evaluation as a protective antigen and colonization factor in a mouse respiratory infection model . Infect Immun 1990 ; 58 : 7-16 . 26 . Relman D, Tuomanen E, Falkow S, Golenbock DT, Saukkonen K, Wright SD . Recognition of a bacterial adhesin by an integrin : macrophage CR3 (a,, /32, CD11 b/CD18) binds filamentous hemagglutinin of Bordetella pertussis . Cell 1990 ; 61 : 1375-82 . 27 . Ui M . The multiple biological activities of pertussis toxin . In : Wardlaw AC, Parton R, eds . Pathogenesis and immunity . J Wiley 1988; 121-45 . 28 . Hanski E . Invasive adenylate cyclase toxin of Bordetella pertussis . TIBS 1989 ; 14 : 459-63 . 29 . Goodwin M St M, Weiss AA . Adenylate cyclase toxin is critical for colonization and pertussis toxin is critical for lethal infection by Bordetella pertussis in infant mice . Infect Immun 1990; 58 : 3445-7 .

Protective activity of Bordetella adenylate cyclase-hemolysin against bacterial colonization.

Bordetella pertussis synthesizes several factors. It has been suggested that one of these factors, the adenylate cyclase-hemolysin (AC-Hly), directly ...
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