Recombinant P.69/pertactin: immunogenicity and protection of mice against Bordetella pertussis infection M . R o b e r t s * , J . P . Tite, N . F . F a i r w e a t h e r , G . D o u g a n a n d I . G . C h a r l e s
The immunogenicity of recombinant (r-) pertactin was examined. Parenteral immunization of mice with natural or r-pertactin produced a similar increase in serum anti-pertactin antibodies and a decrease in Bordetella pertussis lung counts following aerosol challenge. Study of the kinetics of B. pertussis growth in the respiratory tract of immunized and control mice revealed that immunization with r-pertactin halted the multiplication of B. pertussis in the lungs and facilitated the early onset of bacterial clearance. In the trachea, bacterial numbers declined sharply in immunized animals during the first 3 days after challenge but thereafter B. pertussis numbers remained fairly constant throughout the rest of the experiment. Very low doses (0.1 1~9) of r-pertactin were immunogenic and protective but only if the antigen was absorbed to alhydrogel. In vitro proliferation assays with lymphocytes from mice primed with either natural or r-pertactin indicated that the major T-cell epitopes of pertactin are conserved in the recombinant protein. Keywords: Pertactin; Bordetellapertussis; natural; recombinant
INTRODUCTION
Bordetella pertussis is the main aetiological agent of whooping cough, a severe respiratory disease that mainly affects infants and young children, although adults can also become infected 1. In Britain 2 and the USA 3 the currently available vaccine consisting of killed whole cell pertussis organisms has considerably reduced the incidence of the disease. However, the vaccine can cause local and systemic side effects4 and the immunity induced is thought to be relatively short-lived and protects against the disease rather than preventing infection5. As a consequence, considerable effort has been devoted to the development of efficacious acellular vaccines that are devoid of deleterious B. pertussis components. A number of protein antigens of B. pertussis, particularly those whose presence is regulated by the vir-locus6 (also called bvg 7), have been shown to induce protection against B. pertussis infection in various animal models 2. Filamentous haemagglutinin (FHA) and toxoided pertussis toxin (PTX) have received most attention 2'8. However, in a recent field trial in Sweden disappointing levels of protection against whooping cough were seen in infants receiving vaccines composed of FHA and PTX toxoid or PTX toxoid alone 9. Therefore additional immunogens are being sought which can be combined with PTX toxoid and/or FHA to provide satisfactory levels of protection. One possible candidate for inclusion in future acellular pertussis vaccines is the P.69 outer membrane protein. Department of Cell Biology, Wellcome Research Labs, Langley Court, Beckenham, Kent BR3 3BS, UK. *To whom correspondence should be addressed. (Received 29 May 1991; revised 31 July 1991; accepted 21 August 1991)
This protein is thought to be involved in the pathogenesis of pertussis, playing a role in the attachment to and/or invasion of eucaryotic cells by B. pertussis ~°'~. For this reason, P.69 has been named pertactin x°. Immunization with purified preparation of pertactin, or the passive administration of monoclonal antibodies (mAbs) which recognize pertactin, can protect infant mice from lethal respiratory challenge with B. pertussis ~2. Also, infection of humans with B. pertussis or administration of whole cell vaccine elicits both B-cell and T-cell responses against P.69 x3-15. Moreover, antibodies directed against the closely related P.68 protein of Bordetella bronchiseptica can provide protection against atrophic rhinitis in B. bronchiseptica-challenged piglets and mice t6'xT. We have cloned the pertaetin gene (prn) from B. pertussis xs and expressed pertactin to very high levels intracellularly in Escherichia coil~9. The recombinant protein forms insoluble inclusion bodies which can be readily solubilized and refolded using guanadinium hydrochloridC 9. This allows pertactin to be studied in the absence of other B. pertussis components. In this paper we examine the immunogenic and protective properties of recombinant P.69 in an adult mouse respiratory infection model. MATERIALS AND METHODS Bacterial strains and culture of bacteria B. pertussis strain BBC2611, a streptomycin-resistant strain of CN2992, was used in aerosol challenge experiments. Bacteria were cultured for 48 h on CohenWheeler agar 2° containing 10% (v/v) defibrinated horse blood and 100/~g ml- 1 streptomycin (Sigma, Poole, Dorset, UK ).
o26~lox/g2/oloo4~o6 © 1992 Butterworth-HeinemannLtd
Vaccine, Vol. 10, Issue 1, 1992
43
Immunogenicity of recombinant pertactin: M. Roberts e t a!
Isolation and purification of natural and recombinant pertactin Pertactin was isolated and purified from culture of B. pertussis CN2992 as previously described 21,2z. Inclusion bodies of recombinant pertactin were isolated from E. coli MM294 pPERtac36, solubilized and refolded using guanadinium hydrochloride as previously described 19. Traces of LPS were removed by ion-exchange chromatography using Q Sepharose Fast Flow resin (Pharmacia, Milton Keynes, UK). The samples were loaded on the column in 50 mM Tris HC1, pH 8.0, and eluted with a 0-0.4 M NaCI gradient. The LPS content of the eluted material was estimated to be < 1 ng/~g-1 of protein by Limulus amoebocyte lysate assay. Aerosol protection test Female Balb/c mice (3-4 weeks old; Charles River, Margate, UK) were immunized (intraperitoneally [i.p.], intramuscularly [i.m.] or subcutaneously [s.c.])with pertactin diluted in PBS with or without alhydrogel (25%v/v Superfos Biosector, Vedbaek, Denmark). Control mice received PBS/alhydrogel alone. Mice received one or two doses as indicated. One to four weeks after the final immunization mice were challenged by exposure to aerosols of B. pertussis BBC26 and the levels of B. pertussis in the respiratory tract of mice were determined as previously described a3. Anti-B. pertussis antibodies Serum anti-pertussis antibodies were measured using an enzyme-linked immunosorbent assay (ELISA) using r-pertactin as antigen, as preliminary experiments established that responses were the same if r-pertactin or n-pertactin were used as coating antigen, r-Pertactin (50 #1 ; 1/~g/ml- 1 in phosphate-buffered saline, pH 7.2) was absorbed onto 96-well microtitre plates (EIA 'Costar' NBL, Northumbria, UK) by incubation at 4°C overnight. Wells were aspirated and washed three times with PBS containing 0.05% (v/v) Tween 20 (PBS-T; Sigma), and then blocked with 3% (w/v) bovine serum albumin (BSA; Sigma) in PBS. After washing, 50 #1 of test serum appropriately diluted in PBS-T-0.1% BSA was added to the wells and the plates were covered and incubated at 37°C for 2 h. After washing, 50/A of rabbit anti-mouse immunoglobulins horseradish peroxidase conjugate (Dako, High Wycombe, Herts, UK) diluted 1 in 1000 in PBS-T-0.1% BSA was added per well. After incubation at 37°C for 2 h, the wells were washed and incubated at room temperature with 50 #1 of substrate (0.04% o-phenylenediamine hydrochloride ; Sigma) dissolved in phosphate citrate buffer (pH 5.0 [24mM citrate, 64mM disodium hydrogen phosphate] containing 40/~1 hydrogen peroxide). The reaction was terminated by the addition of 50/d 1 M sulphuric acid. Plates were read in a Titertek Multiscan MCC ELISA reader at 492 nm. The titre was expressed as the reciprocal of the highest dilution of test serum that gave an absorbance reading twice that of the similarly diluted pre-bleed serum. Absorbance values below 0.1 were discarded. T-cell proliferation assays Eight-week-old Balb/c mice were immunized s.c. with PBS/complete Freund's adjuvant (CFA ; 1 : 1 ) containing 10#g recombinant or natural pertactin or with
44
V a c c i n e , Vol. 10, I s s u e 1, 1992
PBS/CFA alone. Draining lymph nodes were removed 10 days later and lymph node proliferation assays were performed as previously described 24.
Statistics Analysis of statistical significance was performed using the Student's t test. RESULTS
Comparison of the immunogenicity of natural and recombinant pertactin Groups of adult Balb/c mice were immunized i.p. with 20/~g of alhydrogel-absorbed natural (n) or recombinant (r) pertactin on days 0 and 12 and were challenged with an aerosol of virulent B. pertussis on day 30. Pulmonary B. pertussis infection in adult mice is not usually lethal unless very large infectious doses are administered. The model assesses the ability of B. pertussis to colonize, survive and proliferate in the murine respiratory tract. The extent of the infection is determined by performing viable counts on lungs removed from infected mice. The numbers of B. pertussis in the lungs of immunized and control mice 14 days after challenge are shown in Table 1 along with the serum anti-pertactin titres prior to and after aerosol challenge. Mice immunized with either n- or r-pertactin had 300-500-fold fewer B. pertussis in their lungs than control mice. Both groups of immunized mice had high titres of anti-pertactin antibodies in their serum prior to aerosol challenge. Titres increased after aerosol challenge, but it is not known if this ~vas due to boosting of the serum response by the challenge organism, or if the titres would have reached this level in the absence of challenge. It is known that aerosol administration of B. pertussis can boost circulating anti-pertussis antibodies 2a. Serum anti-pertactin antibodies were not present in control mice even after aerosol challenge. This is consistent with earlier studies which found that antibodies to pertussis antigens do not appear in the serum of aerosol-challenged mice until ~ 3-4 weeks after challenge23. Thirty individual colonies of B. pertussis isolated from the lungs of immunized and control mice were screened to determine if they still produced pertactin by Western blotting using a pertactin-specific mAb, BB05. This was performed because it has been reported that immunization of mice with one type of fimbriae leads to the
Table 1 (~omparison of the effect of immunization with naturai or recombinant pertactin on B. pertussis lung carriage and serum anti-pertactin titres
Serum anti-pertactin titre
B. pertussis in lungs Immunogen 8
(c.f.u./lungs) b ( % of control)
Prior to challenge °
After challenge °
n-Pertactin r-Pertactin -
3.2 x 10a (0.32) 2.0 x 103 (0.2) 1.0 x 106
9000 9000
The immunogenicity of recombinant (r-) pertactin was examined. Parenteral immunization of mice with natural or r-pertactin produced a similar increase in serum anti-pertactin antibodies and a decrease in Bordetella pertussis lung counts following aerosol challenge. Study of the kinetics of B. pertussis growth in the respiratory tract of immunized and control mice revealed that immunization with r-pertactin halted the multiplication of B. pertussis in the lungs and facilitated the early onset of bacterial clearance. In the trachea, bacterial numbers declined sharply in immunized animals during the first 3 days after challenge but thereafter B. pertussis numbers remained fairly constant throughout the rest of the experiment. Very low doses (0.1 1~9) of r-pertactin were immunogenic and protective but only if the antigen was absorbed to alhydrogel. In vitro proliferation assays with lymphocytes from mice primed with either natural or r-pertactin indicated that the major T-cell epitopes of pertactin are conserved in the recombinant protein. Keywords: Pertactin; Bordetellapertussis; natural; recombinant
INTRODUCTION
Bordetella pertussis is the main aetiological agent of whooping cough, a severe respiratory disease that mainly affects infants and young children, although adults can also become infected 1. In Britain 2 and the USA 3 the currently available vaccine consisting of killed whole cell pertussis organisms has considerably reduced the incidence of the disease. However, the vaccine can cause local and systemic side effects4 and the immunity induced is thought to be relatively short-lived and protects against the disease rather than preventing infection5. As a consequence, considerable effort has been devoted to the development of efficacious acellular vaccines that are devoid of deleterious B. pertussis components. A number of protein antigens of B. pertussis, particularly those whose presence is regulated by the vir-locus6 (also called bvg 7), have been shown to induce protection against B. pertussis infection in various animal models 2. Filamentous haemagglutinin (FHA) and toxoided pertussis toxin (PTX) have received most attention 2'8. However, in a recent field trial in Sweden disappointing levels of protection against whooping cough were seen in infants receiving vaccines composed of FHA and PTX toxoid or PTX toxoid alone 9. Therefore additional immunogens are being sought which can be combined with PTX toxoid and/or FHA to provide satisfactory levels of protection. One possible candidate for inclusion in future acellular pertussis vaccines is the P.69 outer membrane protein. Department of Cell Biology, Wellcome Research Labs, Langley Court, Beckenham, Kent BR3 3BS, UK. *To whom correspondence should be addressed. (Received 29 May 1991; revised 31 July 1991; accepted 21 August 1991)
This protein is thought to be involved in the pathogenesis of pertussis, playing a role in the attachment to and/or invasion of eucaryotic cells by B. pertussis ~°'~. For this reason, P.69 has been named pertactin x°. Immunization with purified preparation of pertactin, or the passive administration of monoclonal antibodies (mAbs) which recognize pertactin, can protect infant mice from lethal respiratory challenge with B. pertussis ~2. Also, infection of humans with B. pertussis or administration of whole cell vaccine elicits both B-cell and T-cell responses against P.69 x3-15. Moreover, antibodies directed against the closely related P.68 protein of Bordetella bronchiseptica can provide protection against atrophic rhinitis in B. bronchiseptica-challenged piglets and mice t6'xT. We have cloned the pertaetin gene (prn) from B. pertussis xs and expressed pertactin to very high levels intracellularly in Escherichia coil~9. The recombinant protein forms insoluble inclusion bodies which can be readily solubilized and refolded using guanadinium hydrochloridC 9. This allows pertactin to be studied in the absence of other B. pertussis components. In this paper we examine the immunogenic and protective properties of recombinant P.69 in an adult mouse respiratory infection model. MATERIALS AND METHODS Bacterial strains and culture of bacteria B. pertussis strain BBC2611, a streptomycin-resistant strain of CN2992, was used in aerosol challenge experiments. Bacteria were cultured for 48 h on CohenWheeler agar 2° containing 10% (v/v) defibrinated horse blood and 100/~g ml- 1 streptomycin (Sigma, Poole, Dorset, UK ).
o26~lox/g2/oloo4~o6 © 1992 Butterworth-HeinemannLtd
Vaccine, Vol. 10, Issue 1, 1992
43
Immunogenicity of recombinant pertactin: M. Roberts e t a!
Isolation and purification of natural and recombinant pertactin Pertactin was isolated and purified from culture of B. pertussis CN2992 as previously described 21,2z. Inclusion bodies of recombinant pertactin were isolated from E. coli MM294 pPERtac36, solubilized and refolded using guanadinium hydrochloride as previously described 19. Traces of LPS were removed by ion-exchange chromatography using Q Sepharose Fast Flow resin (Pharmacia, Milton Keynes, UK). The samples were loaded on the column in 50 mM Tris HC1, pH 8.0, and eluted with a 0-0.4 M NaCI gradient. The LPS content of the eluted material was estimated to be < 1 ng/~g-1 of protein by Limulus amoebocyte lysate assay. Aerosol protection test Female Balb/c mice (3-4 weeks old; Charles River, Margate, UK) were immunized (intraperitoneally [i.p.], intramuscularly [i.m.] or subcutaneously [s.c.])with pertactin diluted in PBS with or without alhydrogel (25%v/v Superfos Biosector, Vedbaek, Denmark). Control mice received PBS/alhydrogel alone. Mice received one or two doses as indicated. One to four weeks after the final immunization mice were challenged by exposure to aerosols of B. pertussis BBC26 and the levels of B. pertussis in the respiratory tract of mice were determined as previously described a3. Anti-B. pertussis antibodies Serum anti-pertussis antibodies were measured using an enzyme-linked immunosorbent assay (ELISA) using r-pertactin as antigen, as preliminary experiments established that responses were the same if r-pertactin or n-pertactin were used as coating antigen, r-Pertactin (50 #1 ; 1/~g/ml- 1 in phosphate-buffered saline, pH 7.2) was absorbed onto 96-well microtitre plates (EIA 'Costar' NBL, Northumbria, UK) by incubation at 4°C overnight. Wells were aspirated and washed three times with PBS containing 0.05% (v/v) Tween 20 (PBS-T; Sigma), and then blocked with 3% (w/v) bovine serum albumin (BSA; Sigma) in PBS. After washing, 50 #1 of test serum appropriately diluted in PBS-T-0.1% BSA was added to the wells and the plates were covered and incubated at 37°C for 2 h. After washing, 50/A of rabbit anti-mouse immunoglobulins horseradish peroxidase conjugate (Dako, High Wycombe, Herts, UK) diluted 1 in 1000 in PBS-T-0.1% BSA was added per well. After incubation at 37°C for 2 h, the wells were washed and incubated at room temperature with 50 #1 of substrate (0.04% o-phenylenediamine hydrochloride ; Sigma) dissolved in phosphate citrate buffer (pH 5.0 [24mM citrate, 64mM disodium hydrogen phosphate] containing 40/~1 hydrogen peroxide). The reaction was terminated by the addition of 50/d 1 M sulphuric acid. Plates were read in a Titertek Multiscan MCC ELISA reader at 492 nm. The titre was expressed as the reciprocal of the highest dilution of test serum that gave an absorbance reading twice that of the similarly diluted pre-bleed serum. Absorbance values below 0.1 were discarded. T-cell proliferation assays Eight-week-old Balb/c mice were immunized s.c. with PBS/complete Freund's adjuvant (CFA ; 1 : 1 ) containing 10#g recombinant or natural pertactin or with
44
V a c c i n e , Vol. 10, I s s u e 1, 1992
PBS/CFA alone. Draining lymph nodes were removed 10 days later and lymph node proliferation assays were performed as previously described 24.
Statistics Analysis of statistical significance was performed using the Student's t test. RESULTS
Comparison of the immunogenicity of natural and recombinant pertactin Groups of adult Balb/c mice were immunized i.p. with 20/~g of alhydrogel-absorbed natural (n) or recombinant (r) pertactin on days 0 and 12 and were challenged with an aerosol of virulent B. pertussis on day 30. Pulmonary B. pertussis infection in adult mice is not usually lethal unless very large infectious doses are administered. The model assesses the ability of B. pertussis to colonize, survive and proliferate in the murine respiratory tract. The extent of the infection is determined by performing viable counts on lungs removed from infected mice. The numbers of B. pertussis in the lungs of immunized and control mice 14 days after challenge are shown in Table 1 along with the serum anti-pertactin titres prior to and after aerosol challenge. Mice immunized with either n- or r-pertactin had 300-500-fold fewer B. pertussis in their lungs than control mice. Both groups of immunized mice had high titres of anti-pertactin antibodies in their serum prior to aerosol challenge. Titres increased after aerosol challenge, but it is not known if this ~vas due to boosting of the serum response by the challenge organism, or if the titres would have reached this level in the absence of challenge. It is known that aerosol administration of B. pertussis can boost circulating anti-pertussis antibodies 2a. Serum anti-pertactin antibodies were not present in control mice even after aerosol challenge. This is consistent with earlier studies which found that antibodies to pertussis antigens do not appear in the serum of aerosol-challenged mice until ~ 3-4 weeks after challenge23. Thirty individual colonies of B. pertussis isolated from the lungs of immunized and control mice were screened to determine if they still produced pertactin by Western blotting using a pertactin-specific mAb, BB05. This was performed because it has been reported that immunization of mice with one type of fimbriae leads to the
Table 1 (~omparison of the effect of immunization with naturai or recombinant pertactin on B. pertussis lung carriage and serum anti-pertactin titres
Serum anti-pertactin titre
B. pertussis in lungs Immunogen 8
(c.f.u./lungs) b ( % of control)
Prior to challenge °
After challenge °
n-Pertactin r-Pertactin -
3.2 x 10a (0.32) 2.0 x 103 (0.2) 1.0 x 106
9000 9000
