Serospecific protection of mice against intranasal infection with Bordetella pertussis

Serospecific protection of mice against intranasal infection with Bordetella pertussis A. Robinson, A.R. Gorringe, S.G.P. Funnell and M. Fernandez The ability of purified serospecific agglutinogens from Bordetella pertussis to protect mice against intranasal infection has been examined. Immunization with agglutinogen 2 protected mice against infection with 1.2.0 or 1.2.3 serotypes of B. pertussis, whereas immunization with agglutinogen 3 protected mice against infection with all serotypes. More importantly immunization with serospecific agglutinogen resulted in immune selection so that organisms recovered following infection did not express the immunizing antigen. The results are consistent with the suggestions that protection of children with whole cell pertussis vaccine is to some extent serospecific and that agglutinogens should be considered as constituents of acellular pertussis vaccines.

Keywords:Bordetellapertussis;agglutinogens;flmbriae;serospecificprotection Introduction During the last decade a number of acellular pertussis vaccines have been proposed which usually contain purifed pertussis toxin (PT) alone or in combination with filamentous haemagglutinin (FHA) 1'2. We have developed a vaccine which contains, in addition to PT and FHA, purified agglutinogens3. It is now known that the main serotype specific agglutinogens of Bordetella pertussis correspond to fimbrial components. There are two types of fimbriae on B. pertussis: one with a subunit M r of 22 500 bears agglutinogen 2 and is found on 1.2.0 and 1.2.3 serotypes; the other has a subunit M r of 22 000, bears agglutinogen 3 and is found on 1.0.3 and 1.2.3 serotypes4 9. One group has considered the agglutinogen 3 subunit to be equivalent to agglutinogen 61°. There are three main reasons for including agglutinogens in the vaccine. Firstly, the agglutinogens of B. pertussis have been shown to correspond to the fimbriae of the organism and hence have a presumed role in pathogenesis of pertussis by mediating bacterial attachment to the respiratory tract of the child. At present, however, there is only indirect evidence that agglutinogens can act as adhesins 2,11. Secondly, isolated agglutinogens have been shown to protect mice against respiratory infection with B. pertussis7'12. Thirdly, epidemiological evidence suggested that the protection afforded to children by whole cell pertussis vaccines correlated with the agglutinin response in mice and children 13. Furthermore, evidence since the 1960s has suggested that the protection of children by whole cell vaccines is to some extent serospecific, e.g. in the UK, vaccines predominantly containing 1.2.0 serotype strains protected poorly against 1.0.3 serotypes of B. pertussis, and in Finland vaccines predominantly containing 1.0.3 serotype strains did not protect well against 1.2.0 serotypes 14'15. The WHO therefore recommended that whole cell pertussis vaccines should contain both 1.2 and 1.3 serotype strains. Division of Biologics, PHLS Centre for Applied Microbiology and Research, Porton, Salisbury, Wilts SP4 0JG, UK. (Received 6 December 1988; revised 3 February 1989) 0264-410X/89/040321-04$03.00 © 1989 Butterworth & Co. (Publishers) Ltd

The latter observations interested us in particular, since the recently acquired ability to purify agglutinogens 2 and 3 separately provided a means of testing this serotype specific protection in animal models. Previous experiments with whole cell vaccines are difficult to interpret because of the many protective antigens present on whole cells in addition to agglutinogens. Nevertheless, some evidence of serospecific protection and serotype variation has been found in the rabbit and marmoset 16,17. The animal model we chose was the mouse intranasal challenge test which measures protection against lung colonization 12. This test has several advantages over the mouse intracerebral challenge test 12 and is amenable to challenge with strains of B. pertussis of various serotypes.

Materials and methods Bacterial strains and growth o f bacteria B. pertussis strains W28, CN5476, CN4132, CN2992 were obtained from Dr P. Novotny, strain 10907 from the Natural Collection of Type Cultures, and strain Tohama from Dr C.R. Manclark. Strains were stored as freeze-dried suspensions and recovered by growing for 48 h at 35°C on charcoal agar containing 10% (v/v) defibrinated horse blood. The growth from plates was inoculated into 100 ml CL medium containing 1 g1-1 2-6-O-dimethyl fl-cyclodextrinis and incubated with shaking for 24 h at 35°C. From this primary culture 10 ml was used to inoculate 300 ml CL medium in 2.5 1 Thompson bottles and shaken incubation resumed for 48 h. The bacteria were harvested by centrifugation (5000g, 30 min) and briefly washed in water. Purification o f agglutinogens Agglutinogen 2. Bacterial paste (strain Tohama, serotype 1.2.4) was dispersed in ~10 vol. of 0.05 M phosphate/0.5 M NaC1 pH 7.2 and homogenized in a Silverson homogenizer for four periods of 5 min. The bacteria were removed by centrifugation (10 000g, 45 rain) and the fimbriae in the supernatant precipitated by the addition of ammonium sulphate to 30% saturation. After

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Serospecific protection against Bordetella pertussis: A. Robinson et al.

A

B C

D

E

F

G

H

I

J

K

were removed by centrifugation, washed in TSA buffer and finally dispersed in TSA containing 4 M urea. The fimbriae were examined by SDS-PAGE using either 15% or 20% (w/v) polyacrylamide gels (Figure 1, Lanes B and C). From 10 g wet weight cells, ~2 mg fimbriae were obtained.

Intranasal protection test. The test was performed essentially as described previously Iz. Male NIH mice (5-6 weeks) in groups of 5 or 10 were immunized once i.p. with 0.5 ml PBS/Alhydrogel (25%, v/v) containing agglutinogen 2 or 3 (20 #g ml-1). Control mice were immunized with either PBS or PBS-Alhydrogel. Three weeks after immunization groups of mice were infected intranasally with B. pertussis strains of defined serotype. To prepare the infecting inoculum, bacteria from 24 h growths on charcoal agar plates were dispersed in 1% (w/v) casamino acids and serially diluted to yield a suspension of ~ 2 x 107 organisms m1-1 (estimated by opacity and subsequently confirmed by viable counting). Mice were lightly anaesthetized with ether and 0.05 ml bacterial suspension (i.e. ,~ 106 bacteria) were pipetted on to the nostrils and allowed to be inhaled. After seven days groups of five mice were killed, the lungs excised and homogenized in 10 ml 1% (w/v) casamino acids. Viable organisms in the lung homogenates were counted by plating out serial dilutions on to charcoal agar plates. Rgure 1 SDS-PAGE of fimbriae isolated from B. pertussis. Lane A; standard agglutinogen 2 and 3 preparation. Lane B; agglutinogen 2 used as immunogen. Lane C; agglutinogen 3 used as immunogen. Lanes D-K are crude fimbrial preparations from B. pertussis cells recovered from the lungs of infected mice. Lanes D, E, F: mice infected with strain CN5476 (serotype 1,3) after immunization with PBS (lane D), agglutinogen 2 (lane E) or agglutinogen 3 (lane F). Lanes G, H, I; mice infected with strain CN4132 (serotype 1, 2) after immunization with PBS (lane G), agglutinogen 3 (lane H), or agglutinogen 2 (lane I). Lanes J and K; mice infected with strain 2992 (serotype 1.2.(3)) after immunization with PBS (lane J) or agglutinogen 2 (lane K)

standing at 4°C overnight the precipitated fimbriae were collected by centrifugation and the precipitate extracted 3-5 times with 0.014 M phosphate/0.14 M NaC1 pH 7.2. The suspended fimbriae were reprecipitated by the addition of ammonium sulphate to 15% saturation. The precipitated fimbriae were again collected by centrifugation and extracted as above and the 15% ammonium sulphate precipitation step repeated. The precipitated fimbriae were finally dissolved in 0.05 M phosphate/0.5 M NaC1, pH 7.2.

Agglutinogen 3. Yields of agglutinogen 3 using the above cell homogenization procedure were usually low and hence an alternative isolation procedure was used. Bacterial paste (strain NCTC 10907 or CN5476, serotype 1.3), was dispersed in 10 vol. phosphate buffered saline (PBS) containing 4 M urea and heated in a water bath to 60°C for 30 min. Bacterial debris was removed by centrifugation (30000g, 2 h) and the fimbriae in the supernatant precipitated by the addition of polyethylene glycol 600 to 4% (w/v). After standing at 4°C overnight the fimbriae were collected by centrifugation, dispersed in 0.01 M Tris/0.145 M NaC1 pH 7.4 (TSA) containing 4 M urea and dialysed against 0.01 M Tris/0.1 M MgC12 pH 7.0. Any precipitated fimbriae after 24-48 h dialysis

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Determination of serotype of B. organisms

pertussis

Two methods were used to determine the serotype of

B. pertussis colonies recovered from the lungs of infected mice. Only the presence of the major serotype antigens, i.e. agglutinogens 2 and 3, was determined. The preparation of monoclonal antibodies (mAb) has been described previously1 t.

Colony-blot serotyping. After growth on charcoal agar plates for 5 days, B. pertussis colonies (usually 36/group of mice) were picked off on to three identical charcoal agar plates in a 6 x 6 square grid. After 48 h at 35°C the colonies were blotted onto nitrocellulose filters which were then held at 4°C overnight in 30% (v/v) newborn calf serum (NCS) in PBS/0.1% (v/v) Tween 20. Excess bacteria were washed offthe filters with PBS/0.1% (v/v) Tween 20 and then one filter from each group incubated, with slight agitation for 2 h, in PBS/10% NCS/0.1% (v/v) Tween 20 alone (conjugate control) or containing 1% (v/v) AGG 3B (ascitic fluid of mAb to agglutinogen 3) or 10% (v/v) AGG 2A (hybridoma supernatant of mAb to agglutinogen 2). The filters were washed three times in PBS/0.1% (v/v) Tween 20, incubated in 0.001% (w/v) sheep anti-mouse-horseradish peroxidase conjugate in PBS/10% (v/v) NCS/0.1% (v/v) Tween 20, rewashed and then the enzyme substrate 0.02% (w/v)9-amino-3-ethylcarbazole/2.5% (v/v)DMSO/ 0.125% (v/v) hydrogen peroxide in 0.05 M acetate pH 5.0) added. The colour was allowed to develop and stopped by water washing when colour was observed on the conjugate control filters. By comparison with control filters each colony could thus be scored as either positive or negative for reaction with mAbs to agglutinogens 2 and 3.

Serospecific protection against B o r d e t e l l a pertussis: A. Robinson et al.

Slide agglutination. Alternatively many bacterial colonies, chosen at random, either directly from lung homogenate plates or from the grid plates used for transfer to nitrocellulose filters, were dispersed in PBS containing 0.03 % (v/v) formalin and 0.01% (w/v) thiomersal. The serotype of the suspension was determined by mixing one drop with one drop of mAb to agglutinogen 2 or 3 on a glass plate and macroscopicaUy determining agglutination over the next 30 rain. Isolation of fimbriae from B. pertussis recovered

from lungs

A random mixture of colonies from lung homogenate plates or from grid plates used to transfer colonies to nitrocellulose filters were inoculated into liquid medium and grown as described above. A small amount of harvested bacteria was dispersed in PBS and examined by SDS-PAGE. Crude fimbriae were prepared from 2 4 g wet weight of cells by heating the cells in PBS/4 M urea to 60°C for 30 min, centrifugation, precipitation with 4% (w/v) poly(ethylene glycol) and finally dispersing the precipitate in TSA/4 M urea.

ELISA for antibody to agglutinogens Antibodies to agglutinogens from sera of immunized mice prior to infection were determined using ELISA techniques 3 with microtitre plates coated with either agglutinogen 2 or 3 at 2 #g m1-1 and an anti-mouse Ig/horseradish peroxidase conjugate.

appeared to be the case in some (Table 2) but not all experiments. The anti-agglutinogen 2 activity of sera from agglutinogen 3 immunized mice could have arisen from contamination of agglutinogen 3 with agglutinogen 2. However, as shown in the Figure (lanes B and C), any contamination is at a very low level. This was confirmed by ELISA of the antigens with homologous and heterologous monoclonal antibody (data not shown). Alternatively the anti-agglutinogen 2 activity of agglutinogen 3 could be due to common epitopes which are perhaps exposed due to the use of urea in the purification of agglutinogen 3. There is considerable sequence homology between agglutinogen 2 and 3 8'1°'19'2°. However, the protection afforded against 1.2 serotypes by agglutinogen 3 differs from that afforded by agglutinogen 2, since organisms recovered from agglutinogen 3-immunized mice had not lost their agglutinogen 2 component, whereas those from agglutinogen 2-immunized mice had done so (see below). Hence agglutinogen 3 may not totally protect against 1.2 serotypes. In contrast, agglutinogen 2 totally protects against the 1.2 serotypes and allows a minor infection of 1.0.0 serotypes to develop. These points indicate that in addition to the numbers of organisms recovered their serotype should also be determined.

Table I

Serospecific protection of mice against infection with

Immunogen

Challenge strain: serotype

Number of organisms recovered from lungs (% control)

PBS Agglutinogen 2 Agglutinogen 3

CN5476:1.3 CN5476:1.3 CN5476:1.3

100 77 6

PBS Agglutinogen 2 Agglutinogen 3

W28:1.2.3 W28:1.2.3 W28:1.2.3

PBS Agglutinogen 2 Agglutinogen 3 PBS Agglutinogen 2 Aggiutinogen 3

Results and discussion The data expressed in Table 1 are typical of results where mice were challenged with 1.2.0, 1.0.3 or 1.2.3 serotype cells and are representative of three different experiments for each serotype challenge.

Orerall protection When the numbers of organisms recovered from agglutinogen-immunized mice were expressed as a percentage of organisms recovered from control (PBSimmunized) mice for any particular strain, it was apparent that agglutinogen 2 protected against challenge with 1.2.0 and 1.2.3 serotype strains but not against 1.0.3 serotype strains, whereas agglutinogen 3 protected against all serotypes (Table 1). There are a number of factors that could account for this apparent anomaly. Firstly, agglutinogen 3 may contain additional non-serospecific antigens found to a lesser extent in agglutinogen 2 preparations (e.g. outer membrane proteins). No obvious contaminants could be seen on S D S - P A G E with Coomassie blue staining. Lipooligosaccharide (LOS) would not be detected on the gels and could be present in agglutinogen 3 preparations due to the urea extraction procedure employed. In general agglutinogen 3 tended to contain more LOS than agglutinogen 2 preparations (perhaps up to 10% in some cases). Previous results have shown that purified LOS is non-protective in mice 12. However, low levels of LOS with agglutinogen 3 could possibly have a synergistic protective effect. Secondly, agglutinogen 3 may induce a higher level of cross-reacting antibodies than does agglutinogen 2. When this possibility was explored by ELISA ofsera from mice immunized with the homologous and heterologous agglutinogen, this

B. pertussis

Reaction of recovered organisms with mAb a AGG 2

AGG 3

0/36 0/35 0/35

32/36 20/35 4/35

100 2 0.4

35/36 12/34 33/36

+ + NT +

CN4132:1.2 CN4132:1.2 CN4132:1.2

100 0.6 3.0

+ + + + + +

+/+/+/-

CN2992:1.2(3) CN2992:1.2(3) CN2992:1.2(3)

100 0.7 0.03

+ + + -NT

+/+ + NT

aResults expressed numerically (e.g. 32136) were obtained by the colony blot technique using the appropriate monoclonal antibody. Results from slide agglutination tests are expressed as: + + +, (full agglutination) to -- (no agglutination). NT, not tested. Where both tests were performed on the same sample there was good agreement between the tests

Table 2

ELISA for antibody to agglutinogen in serum of immunized mice ELISA titre to agglutinogen

Immunogen

2

3

Agglutinogen 2 Agglutinogen 3

29300 3890

31 54700

ELISA titre is the reciprocal of the serum dilution equivalent to the midpoint of the dose-response curve for the standard monoclonal antibody preparation. Results are expressed as geometric means of 6 or 7 sera

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Serotype of recovered organisms As stated above, in addition to overall protection it is important to determine whether immunization with agglutinogen 2 or 3 has provided immunological selection for organisms not possessing those antigens (i.e. alternative serotypes). Using the colony-blot serotyping method it was apparent that immunization with agglutinogen 3, but not agglutinogen 2, had a marked effect in reducing the agglutinogen 3 content of recovered organisms compared with the infecting organisms, i.e. the serotype of recovered organisms had changed from predominantly 1.0.3 or 1.2.3 to predominantly 1.0.0 or 1.2.0 respectively (Table 1). Similarly, immunization with agglutinogen 2 changed the serotype of organisms from predominantly 1.2.3 to predominantly 1.0.3. Sometimes the results of the colony blot tests did not permit unequivocal assessment of the serotype of organisms and hence slide agglutination tests were performed. When mice were challenged with strain CN4132, there was a dramatic reduction in the ability of cells recovered from agglutinogen 2-immunized mice to agglutinate with mAb to agglutinogen 2. Similarly, for mice challenged with strain CN2992, which is a 1.2 serotype with a weak agglutinogen 3 response, the organisms recovered from agglutinogen 2, immunized mice did not agglutinate with mAb to agglutinogen 2 but had a qualitatively enhanced agglutination with mAb to agglutinogen 3 (Table 1).

Isolation of fimbriae from organisms recovered

from lungs

To confirm these observed changes in serotype, fimbriae were isolated from lung organisms following growth in liquid culture. In all cases the whole cell protein patterns, as determined by SDS-PAGE, were similar and typical of phase I B. pertussis cells (data not shown). Agglutinogen 3 could not be isolated from cells recovered from mice immunized with agglutinogen 3 and challenged with a 1.0.3 serotype (Figure 1, lane F) and similarly agglutinogen 2 could not be isolated from cells recovered from mice immunized with agglutinogen 2 and challenged with a 1.2.0 serotype (Figure 1, lane I). In the case where mice were challenged with the 1.2.(3) serotype strain CN2992, organisms from PBS-immunized mice showed agglutinogen 2 with a trace of agglutinogen 3, whereas organisms from agglutinogen 2-immunized mice possessed only the agglutinogen 3 fimbrial subunit (Figure 1, lanes J and K). These results confirm that immunization of mice with agglutinogen 2 causes selection of organisms deficient in this antigen, i.e. serotype 1.0.3 and 1.0.0 strains, whereas immunization of mice with agglutinogen 3 causes selection of organisms deficient in that antigen, i.e. serotype 1.2.0 or 1.0.0 strains. Whether this specific protection represents genuine serotype conversion, or selection of a small number of organisms of different serotype within the infecting inoculum, is not known at present. Nevertheless, these experiments demonstrate for the first time, that immunization with purified agglutinogens exerts a selective pressure for organisms not possessing the homologous agglutinogen. Although these experiments only demonstrate protection against colonization of the lungs of mice, they do support the suggestion that protection of the child with whole cell pertussis vaccine may to some extent be

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serotype specific. Furthermore, the results reaffirm the importance of agglutinogens as candidates for inclusion in acellular pertussis vaccines 1'2.

Acknowledgements The authors are grateful to Mr A.R. Blake for technical assistance and Dr I. Livey, Dr L.A.E. Ashworth, Dr L.I. Irons and Professor J. Melling for advice and constructive criticism.

References 1 Robinson, A., Irons, L.I. and Ashworth, L.A.E. Pertussis vaccine: present status and future prospects. Vaccine 1985, 3, 11 2 Robinson, A. and Ashworth, L.A.E. Acellular and defined component vaccines against pertussis. In: Pathogenesis and Immunity in Pertussis (Eds Wardlaw, A.C. and Parton, R.) John Wiley & Sons Ltd., Chichester, UK 1988, p. 399 3 Rutter, D.R., Ashworth, L.A.E., Day, A., Funnell, S., Lovell, F. and Robinson, A. Trial of a new acellular pertussis vaccine in healthy adult volunteers. Vaccine 1988, 6, 29 4 Irons, L.I., Ashworth, L.A.E. and Robinson, A. Release and purification of fimbriae from Bordetella pertussis. Dev. Biol. Stand. 1985, 61, 153 5 Ashworth, L.A.E., Irons, L.I. and Dowsett, A.B. The antigenic relationship between serotype specific agglutinogens and fimbriae of Bordetella pertussis. Infect. Immun. 1982, 37, 1278 6 Steven, A.C., Bisher, M.E., Trus, B.L., Thomas, D., Zhang, J.M. and Cowell, J.L. Helical structure of Bordetalla pertussis fimbriae. J. Bacteriol. 1986, 167, 968 7 Zhang, J.M., Cowell, J.L., Steven, A.C. and Manclark, C.R. Purification of serotype 2 fimbriae of Bordetella pertussis and their identification as a mouse protective antigen. Dev. Biol. Stand. 1985, 61, 173 8 Mooi, F.R., van der Herde, H.G.J., ter Avast, A.R., Welinder, K.G., Livey, I., van der Zeijst, B.A.M,J. and Gaastra, W. Characterisation of fimbrial subunits from Bordetella species. Microb. Pathogen. 1987, 2, 473 9 Fredriksen, J.H., Nanork, E. and Froholm, L.O. Immunoelectron microscopy of fimbriae-like structures on Bordetella pertussis serotype 1.3. J. Med. Microbiol. 1988, 25, 285 10 Cowell, J.L., Zhang, J.M., Urisu, A., Suzuki, A., Steven, A.C., Liu, T. et al. Purification and characterisation of serotype 6 fimbriae from Bordetella pertussis and comparison of their properties with serotype 2 fimbriae. Infect. Immun. 1987, 55, 916 11 Gorringe, A.R., Ashworth, L.A.E., Irons, L.I. and Robinson, A. Effect of monoclonal antibodies on the adherence of Bordetella pertussis to Vero cells. FEMS. Microbiol. Lett. 1985, 26, 5 12 Robinson, A., Ashworth, L.A.E., Baskerville, A. and Irons, L.I. Protection against intranasal infection of mice with Bordetella pertussis. Dev. Biol. Stand. 1985, 61, 165 13 MRC. Vaccination against whooping cough, final report. Br. Med. J. 1959, 1,994 14 Preston, N.W. Prevalent serotypes of Bordetella pertussis in nonvaccinated communities. J. Hyg. (Camb.) 1976, 77, 85 15 Kuronen, T. and Huovila, R. Seroresponse to pertussis vaccine. In: International Symposium on Pertussis, US Dept. Health Education and Welfare, Washington, 1979, p. 34 16 Preston, N.W., Timewell, R.M. and Carter, E.J. Experimental pertussis infection in the rabbit: similarities with infection in primates. J. Infect. 1980, 2, 227 17 Stanbridge, T.N. and Preston, N.W. Experimental pertussis infection in the marmoset: type specificity of active immunity. J. Hyg. (Camb.) 1974, 72, 213 18 Imaizumi, A., Suzuki, Y., Ono, S., Sato, H. and Sato, Y. Effect of Heptakis (2,6-O-dimethyl)/Y-cyclodextrinon the production of pertussis toxin by Bordetella pertussis. Infect. Immun. 1983, 31, 1138 19 Livey, I., Duggleby, C. and Robinson, A. Cloning and nucleotide sequence analysis of the serotype 2 fimbrial subunit gene from Bordetella pertussis. Mol. Microbiol. 1987, 1,203 20 Mooi, F.R. Bordetella pertussis fimbriae, gene structure and analysis of fimbrial mutants. Abstracts International Pertussis Workshop. Hamilton, USA Sept, 1988