Vol. 58, No. 8

INFECTION AND IMMUNITY, Aug. 1990, p. 2523-2528

0019-9567/90/082523-06$02.00/0 Copyright © 1990, American Society for Microbiology

Murine Antibody Response to Oral Infection with Live aroA Recombinant Salmonella dublin Vaccine Strains Expressing Filamentous Hemagglutinin Antigen from Bordetella pertussis N. CHRISTINE MOLINA AND CHARLOTTE D. PARKER* Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri-Columbia, Columbia, Missouri 65202 Received 27 November 1989/Accepted 9 April 1990

Two plasmids which express either nearly intact or truncated filamentous hemagglutinin (FHA) from Bordetella pertussis and which are marked with a tetracycline resistance (TcF) gene were transformed into Salmonela dublin SL1438, an aroA deletion mutant intended for use as an attenuated oral vaccine against salmonellosis. These S. dublin recombinants, when fed to mice, induced serum immunoglobulin, immunoglobulin M (IgM), and sometimes IgA antibody responses to FHA and S. dublin. In addition, IgA antibodies against FHA were found in gut wash fluids. S. dublin carrying pDB2300, a multicopy plasmid encoding truncated FHA protein, induced a better antibody response than did S. dublin carrying pDB2000, a low-copy-number plasmid encoding full-sized FHA. Administration of tetracycline to mice enhanced the stability of recombinant plasmids, and tetracycline-treated mice developed higher anti-FHA titers. Although neither strain examined is suitable for use in a human oral vaccine, these data demonstrated that an immune response against B. pertussis FHA could be induced by oral administration of live attenuated recombinant strains of S. dublin and suggested that development of a live oral attenuated vaccine against pertussis may be possible.

An improved and inexpensive pertussis vaccine would be highly desirable, especially for underdeveloped countries (27). Separation of protective but nontoxic Bordetella pertussis antigens, such as filamentous hemagglutinin (FHA) (32), from toxic or potentially toxic components, such as endotoxin, dermonecrotic toxin, pertussis toxin (for a review, see reference 39), adenylate cyclase (10, 36), and tracheal cytotoxin (19), has allowed development of new subunit vaccines containing largely detoxified pertussis toxin and FHA (20, 30, 33). FHA is a very large surface protein of B. pertussis (3) which confers protective immunity in the mouse intranasal challenge model (23). In a recent field trial, highly purified FHA with detoxified pertussis toxin and detoxified toxin alone were used as subunit vaccines, and they protected more against severe disease than against infection (1). An inexpensive vaccine which would deliver nontoxic B. pertussis antigens directly to mucosal surfaces and stimulate secretory immunity should be effective against this mucosal pathogen. Attenuated oral Salmonella vaccines have been used to deliver antigens and to elicit immune responses to both Salmonella and cloned antigens (6, 9, 11, 21, 31, 37). Attenuation of Salmonella spp. by deletion of the aroA gene is an established procedure which has recently been experimentally tested (15, 26). In addition, a Salmonella typhi Ty2l live attentuated vaccine has been tested extensively in humans (24). aroA deletion mutants have been found useful, although low-level dissemination to the reticuloendothelial system of the host may follow oral dosing (15). We used an aroA deletion mutant of Salmonella dublin as the carrier for the recombinant plasmids which encoded FHA production (7), and we challenged mice orally. At various times postchallenge, we examined mice for evidence of an antibody response to FHA. *

MATERIALS AND METHODS Bacterial strains and plasmids. The bacterial strains and plasmids are described in Table 1 and Fig. 1. Media. Bacteria were grown in either 2x TY medium (5) (in grams per liter of H20: tryptone, 16; yeast extract, 10; NaCl, 5) or LB broth (5) (in grams per liter of dH2O: tryptone, 10; yeast extract, 5; NaCl, 5). LB plates additionally contained 15 g of agar per liter, and they contained 15 ,ug of tetracycline or 100 ,ug of streptomycin per ml, as appropriate. MacConkey agar containing lactose was from Difco Laboratories, Detroit, Mich. Phosphate-buffered saline (PBS) contained the following in grams per liter of dH2O: KCI, 0.20; KH2PO4, 0.20; Na2HPO4, 1.15; MgSO4, 0.05; NaCl, 8.0 (pH 7.0). Genetic manipulations. Plasmids were transformed and isolated by standard methods (25). To minimize restriction problems, plasmids isolated from Escherichia coli LE392 were first transformed into restriction-minus modificationplus Salmonella typhimurium SL5651. Plasmid DNA from S. typhimurium SL5651 was then transformed into S. dublin SL1438. Experimental design. At intervals after oral inoculation, groups of three inbred BALB/c mice were simultaneously tested for the following: (i) excretion of S. dublin in feces; (ii) presence of S. dublin in spleens; (iii) retention of the recombinant plasmids in S. dublin colonies recovered from feces or spleens; (iv) serum antibody responses to FHA and sometimes to S. dublin; and, in some experiments, (v) presence of local (gut) antibody to FHA and S. dublin. We collected and pooled blood samples from three animals for each measurement to assure an adequate amount of serum for antibody determinations. Other samples collected from the same animals were also pooled. The necessity of pooling blood for adequate sample size, the time-consuming nature of the manipulations, and the use of an inbred strain of mice made it logical to pool samples of each type. Experiments were repeated once or twice for confirmation.

Corresponding author. 2523

2524

MOLINA AND PARKER

INFECT. IMMUN. TABLE 1. Bacterial strains and plasmidsa Relevant phenotype, genotype,

Strain or plasmid

and characteristicsb

(parent plasmid, length) S. typhimurium SL5651

Source

Reference

B. Stocker'

8

S. dublin SL1438 E. coli LE392

Restriction- modification' hsdLT6 hsdSA hsdSB strA120 strA, aroAS54::TnJO (Tcs, nonreverting) F- hsd R514 (r-K MiK) supE44 supF58 lacYl or A(lacIZY)6

B. Stocker Stock strain

37 25

pDB2000 (pCP13, 23 kb)

Tcr, low copy number

7, 14, 18 8.3-kb XhoI-ClaI fragment inserted into Kmr gene and polylinker 3-kb XhoI fragment inserted into Kmr gene 7, 22

pDB2300 (pMK16, 4.5 kb) Tcr, high copy number a kb, Kilobase pairs. b

c

Tc', Tetracycline resistance; Tcs, tetracycline sensitivity; Stanford University School of Medicine, Stanford, Calif.

Kmr, kanamycin resistance.

Preparation of the inoculum. Overnight cultures of bacteria were subcultured into 20 ml of broth containing 15 ,ug of tetracycline per ml. After 3 to 4 h of growth, the cultures were spun down and suspended in sterile PBS to a cell density of ca. 200 Klett units (green filter), as determined by using a colorimeter (Klett-Summerson, New York, N.Y.), which is equivalent to about 109 CFU/ml. Mouse inoculation. Four- to six-week-old BALB/c mice of either sex were obtained from a breeding colony at our institution and sorted into cages, separating males and females. Mice were given streptomycin (5 g/liter) in their drinking water for 48 h prior to inoculation, to suppress the normal colonic flora and to aid colonization by S. dublin (28). After removal of food and water for 4 h, mice were lightly anesthetized with ether on day 0 and fed 100 ,ul (about 108 CFU) of S. dublin. Mice were then maintained with free access to food and water; in some cases, water contained antibiotics, as noted above, in an attempt to prolong colonization (4). Mouse exsanguination and specimen collection. Fresh fecal pellets were collected from individual mice, two pellets per mouse, into sterile PBS and were held 1 to 2 h on ice to allow softening of the pellets. The mice were anesthetized with ether and exsanguinated by bleeding from the heart. The animals were immediately dissected, and the spleens were removed to sterile PBS. Gut washings were collected as follows. The gut was clamped offjust below the stomach and just above the anus and removed intact, and 3 to 4 ml of PBS containing protease inhibitors (EDTA, 5 mM; phenylmethane-sulfonyl fluoride, 2 mM; aprotinin, 0.05 U/ml) was gently washed through its length. Bacterial colonization and plasmid stability. Spleens for culture were pooled in sterile PBS, held on ice for up to 2 h

E

P

C PD B2000

pD82300 I

I

I

I

I

I

I

I

I

I

I

I

1 2 9 l0 l 12 3 4 5 6 7 8 FIG. 1. Map of complete FHA structural gene (29) and portions of it contained in plasmids used in this study (7). The open reading frame of secreted B. pertussis FHA is indicated by the thick line. Subclones are shown as thin lines below the entire gene. Scale at bottom is in kilobase pairs. E, EcoRI; C, ClaI; B, BamHI; X, XhoI; Pv, PvuII; P, PstI.

0

if necessary, homogenized, and plated on MacConkey agar. We found fewer than 100 S. dublin CFU per spleen, indicating systemic spread of the organism but demonstrating that the replication of S. dublin in vivo was limited. Fecal pellets were homogenized and plated in the same manner as the spleens. Plasmid maintenance was determined by scoring individual S. dublin colonies for plasmid-encoded tetracycline resistance. The in vitro control for plasmid maintenance was obtained by plating the inoculum on MacConkey agar, picking colonies, and scoring for tetracycline resistance. Immunoassays. Mouse blood samples were allowed to clot, and the serum was separated and maintained frozen until assay. Pooled gut wash samples were centrifuged, and the supernatants were stored frozen. Protein assays of gut wash fluid were performed by using a dye-binding microassay (Bio-Rad Laboratories, Richmond, Calif.) prior to immunologic assays, and gut wash fluids were adjusted by dilution to yield 5 jig of protein per well prior to assay. ELISA. Antibodies in serum and gut wash fluids were measured in duplicate wells by the enzyme-linked immunosorbent assay (ELISA), as described previously (17), with approximately 1.0 pLg of protein (FHA or S. dublin) to coat each well. Total immunoglobulin bound by the antigen was detected by using goat anti-mouse immunoglobulin G (IgG) (whole molecule), while IgM and IgA responses were detected by using specific goat anti-mouse IgM (,u-chain specific) or IgA (a-chain specific). The secondary antibodies, conjugated to peroxidase, were obtained from U.S. Biochemical Corp., Cleveland, Ohio. Anti-IgG and anti-IgM were used at 1: 2,000 dilutions, while anti-IgA was used at a 1:250 dilution. Protein immunoblot. Bacteria to be tested were grown overnight in 1.5 ml of LB broth plus tetracycline, the cultures were spun down, and the cell pellets were resuspended in approximately 150 ,u1 of the supernatant. An equal amount of sample buffer was added, and the samples were boiled for 10 min before 70 RI1 was loaded per well onto a 5 to 15% gradient sodium dodecyl sulfate-polyacrylamide gel containing 0.5 M urea (35). After electrophoresis, protein immunoblotting was performed by standard techniques (16, 38). Chemicals and reagents were purchased from Bio-Rad Laboratories, except prestained molecular weight markers, which were obtained from Bethesda Research Laboratories, Inc., (Gaithersburg, Md.). A monoclonal antibody specific for FHA, P12H3 (17), was used as the primary antibody to develop blots.

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ANTIBODY RESPONSE TO LIVE ORAL PERTUSSIS FHA VACCINE

VOL. 58, 1990

ABCDE FG

FIG. 2. Expression of cloned FHA by E. coli LE392 and S. dublin SL1438. The immunoblot was developed with anti-FHA monoclonal antibody 12H3. Lane A, Purified FHA (ca. 0.5 ,ug); lane B, S. dublin SL1438; lane C, E. coli LE392(pDB2000); lane D, S. dublin SL1438(pDB2000); lane E, S. dublin SL1438(pMK16); lane F, E. coli LE392(pDB2300); lane G, S. dublin SL1438(pDB2300). Bars at the right represent prestained molecular weight markers. Approximate molecular weights, from top: 219,600, 100,420, 68,030, 42,730, 27,420, 18,080, and 14,880.

RESULTS AND DISCUSSION Production of FHA antigen by S. dublin. S. dublin expressed cloned FHA fragments at least as well as E. coli did (Fig. 2). As was reported previously for E. coli (7), the product from S. dublin containing plasmid pDB2000 was slightly larger (Mw, -230,000) than FHA from B. pertussis (Mw, -215,000). The truncated FHA product from pDB2300 was consistently slightly larger in S. dublin than in E. coli. This finding suggested differences in mechanisms of protein processing between E. coli and S. dublin. Since S. dublin SL1438 was capable of producing cloned FHA antigen in quantity, it appeared feasible to proceed with in vivo tests.

S. dublin oral infection. Preliminary experiments (data not shown) demonstrated that within 5 days of oral immunization, S. dublin could only rarely be recovered from mouse feces or spleens; additionally, the majority of S. dublin colonies recovered from feces, even as early as day 1, were cured of the plasmid. Antibody to FHA appeared to be present in low titer, but the numbers were unimpressive. The rapid clearance of plasmid-bearing S. dublin may have prevented an optimal antigenic stimulus. Therefore, we treated orally inoculated mice so as to select for either plasmid or chromosomal drug resistances in an attempt to lengthen the duration of S. dublin colonization and to perhaps stabilize the plasmid. Either streptomycin (for chromosomal selection) or tetracycline (for plasmid selection) was administered in the drinking water in experiments with pDB2000 and pDB2300. S. dublin disappeared relatively quickly in such experiments (Table 2). However, use of tetracycline for 14 days following challenge had a slight effect in prolonging colonization with S. dublin SL1438 (pDB2000) and a stronger effect with S. dublin(pDB2300). Tetracycline treatment also improved plasmid stability in S. dublin(pDB2300). Since the small plasmid was more stable than the larger plasmid in vivo, especially with tetracycline selection, it was used for all further studies. We then tested the effects of various doses of tetracycline on length of colonization and plasmid stability of S. dublin(pDB2300) (Table 3). In this experiment, S. dublin colonized somewhat longer and pDB2300 was more stable than in typical experiments, for unknown reasons. Plasmid curing occurred at a detectable frequency when the inoculum was plated on MacConkey agar lacking tetracycline, as shown by the inoculum control (Table 3). The higher in vivo loss is striking, and it presumably reflects selection in the competitive setting of the gut. Also, intact FHA self-assembles into filaments (2, 34), and such assembly could be deleterious to S. dublin. The truncated mutant FHA encoded by pDB2300 may allow greater plasmid stability because it is less deleterious to host bacteria than pDB2000. Antibody response to FHA in infected mice. The data for mouse antibody responses to FHA in the experiment presented in Table 2 are shown in Table 4. No anti-FHA IgA

TABLE 2. Effects of oral antibiotics on stability of plasmids pDB2000 and pDB2300 in S. dublin SL1438 in mice' Group (dose [108 CFU])

Oral

treatmentb

Site

pDB2000 inoculum control pDB2300 inoculum control SL1438(pDB2000) (2.8)

SL1438(pDB2300) (1.8)

32/32 (100) 32/32 (100)

ND ND

ND ND

ND ND

ND ND

NR

Spleen

18/18 (100)

NR NR

NR NR

Tet

Feces Spleen

ND ND

11/12 (93) 16/16 (100)

NR NR

NR NR

Strep

Feces

Spleen

ND ND

NR 16/18 (89)

NR NR

NR NR

Feces

ND ND

7/12 (58) 16/16 (100)

9/28 (32) 16/18 (89)

NR 21/21 (100)

Strep

Tet

Feces

Spleen a

No. of Tcr isolates/no. scored (%) after no. of days postchallengec 2 7 9

Tcr, Tetracycline resistant.

bStrep, and Tet, Streptomycin (5 mg/ml) and tetracycline (25 ,ug/ml), respectively (in drinking water of mice).

c ND, Not done; NR, S. dublin not recovered.

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MOLINA AND PARKER

INFECT. IMMUN.

TABLE 3. Effect of oral tetracycline dose on stability of plasmid pDB2300 in S. dublin 1438 in mice Oral treatmenta

No. of Tcr isolates/no. scored (%) after no. of days postchallengeb

Site

0

1

5

7

29/30 (97)

ND

ND

ND

11/19 (58) 13/15 (87)

1/44 (2.3) ND

0/25 (0) 2/21 (9.5)

Feces

9/9 (100)

Spleen

21/24 (88)

36/40 (90) ND

23/25 (92) 21/25 (84)

Feces

17/17 (100) 21/22 (95)

44/44 (100) ND

25/25 (100) 23/25 (92)

Inoculum control No antibiotic

Feces

Spleen Tetracycline (25 ,ug/ml) Tetracycline (500 ,g/ml)

Spleen All doses were 4 x 107 CFU. b ND, Not done.

a

response was detected. As one might expect, the tetracycline-treated group, in which both S. dublin colonization and plasmid stability were best, showed the highest antibody

levels. Significantly, FHA appeared to be a successful antigen, and even a brief colonization induced an anti-FHA response. IgM results were comparable to the immunoglobulin response at the time points tested. The antibody response was not long lived with animals immunized once

(Table 4). More recent experiments (N. C. Molina and C. D. Parker, unpublished data) have shown that several oral immunization both heighten and prolong the anti-FHA response.

The data in Table 5 show the antibody responses for the experiment presented in Table 3. In this experiment, antibodies to both FHA and S. dublin were measured. IgA was found both in serum and in intestinal washings. The IgA values cannot be directly compared with total immunoglobulin values, as the sensitivities of the assays were not the same. While anti-FHA immunoglobulin was always detectable, anti-FHA IgA was detectable only in the most sensitive assays. We do not know why gut washings at day 7 from mice receiving the low dose of tetracycline (Table 5) showed no anti-FHA or anti-S. dublin antibody response but believe this result to be an artifact. Since the samples showed no antibody in repeat assays, sample thawing or failure of protease inhibitors may have occurred.

Mouse sera were tested at dilutions up to 1: 20 by immunoblotting against FHA but failed to show a positive reaction. This result was not surprising, since immunoblotting is less sensitive than ELISA and since we saw only modest anti-FHA antibody titers. The reasons for choosing S. dublin aroA as the carrier strain were that (i) aroA strains have been shown to effectively immunize against both salmonellosis and recombinant antigens; (ii) the aroA allele in S. dublin 1438 is a nonreverting deletion mutation; and (iii) S. dublin is not primarily a mouse pathogen, having no special predilection for systemic invasiveness, as might occur with S. typhimurium, the agent of mouse typhoid. Since S. dublin does not cause mouse typhoid, it may more successfully mimic a human S. typhimurium vaccine.

The studies presented here demonstrated that oral inoculation of mice with a live attentuated strain of S. dublin expressing a cloned portion of B. pertussis FHA induces an antibody response to FHA. Plasmid stability and the time course of S. dublin colonization affected the antibody response to FHA. While antibiotic administration to humans receiving a live attenuated bacterial vaccine is unacceptable, selective systems for stabilizing plasmids in avirulent Salmonella vectors based on nutritional requirements have been devised (11-13). Our results with FHA demonstrate

TABLE 4. Production of antibodies to FHA by mice infected orally with S. dublin SL1438 carrying either pDB2000 or pDB2300a Groups and treatment

SL1438(pDB2000) Streptomycin Tetracycline SL1438(pDB2300) Streptomycin Tetracycline

Immunoglobuln

2

ELISA (OD405) reading of 1:25 dilution of serum at dayb: 7 9 14 21

28

Total IgM

0.070

0.174 0.305 d

0.120

0.126

0.104

0.136

0.164

0.136

0.112

Total IgM

0.160

0.188

0.154

0.176

0.078

0.342d

0.212d

0.256d

0.178

0.132

Total IgM

0.159

0.195

0.192

0.330d

0.287d

0.153 0.170

0.124

0.222d

0.260d

0.162 0.178

Total IgM

0.090 0.196

0.422 d 0.526c,d

0.188

0.152

0.270d

0.260d

0.128 0.154

0.134 0.134

0.070 0.112

0.078 0.126

a Groups are described in Table 2. b Values for controls were as follows: no serum, 0.005; total immunoglobulin with normal mouse serum, 0.107; total immunoglobulin with immune FHA serum, 1.119; IgM with normal mouse serum, 0; IgM with immune FHA mouse serum, 0. OD405, Optical density at 405 nm. c Significant rise (defined as doubling of the optical density value in comparison with the day-2 value of this group). d Optical density was -0.2 units higher than that of normal mouse serum.

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ANTIBODY RESPONSE TO LIVE ORAL PERTUSSIS FHA VACCINE

VOL. 58, 1990

TABLE 5. Antibody responses to FHA and S. dublin(pDB2300) as assessed in sera or gut washings in mice treated with various doses of tetracyclinea Tetracycline dose

ELISA anti-FHA OD reading (anti-S. dublin reading) at indicated dayb 1:80 Dilutions of serum Gut washings'

Immunoglobulin

(p.g/ml)_

0 25 500

__

1

7

1

7

Total IgA

0.064 (0.148) 0.037 (NF)

0.240d (0.314") 0.386d (NF)

ND 0.036 (0.052)

0.190d (0.173)d

Total

IgA

0.075 (0.104) 0.010 (NF)

0.272d (0.216d) 0.322d (NF)

ND 0.020 (0.052)

ND 0.065" (0.050)

Total IgA

0.107 (0.110) -0.004 (NF)

0.302d (0.208) 0.206d (NF)

ND 0.060 (0.083)

ND 0.258" (0.135)

ND

a Groups are as described in Table 3. OD, Optical density. Values for controls were as follows: no serum with immunoglobulin, 0.008; no serum with IgA, 0.0; anti-B. pertussis serum with immunoglobulin, >2.0 (0.167); anti-B. pertussis serum with IgA, 0.098 (0.048). b NF, Not found. c 5 of gut wash fluid protein was added per well. ND, Not done. d Significant rise in titer of antibody, defined by doubling of optical density, compared with that of day 1, or by reading -0.2 optical density units over normal mouse controls.

p.g

that an oral Salmonella vaccine expressing cloned B. pertussis proteins may be feasible. ACKNOWLEDGMENTS This research was supported by Public Health Service grants A117682 and A125055 from the National Institutes of Health to C. Parker. Additionally, a portion of the research was supported by funds from the Research Council of the School of Medicine, University of Missouri, Columbia. The advice and assistance of Bruce A.D. Stocker, who kindly provided strains and expertise, are gratefully acknowledged. We also thank Karen Ehlert for excellent secretarial assistance. LITERATURE CITED 1. Ad Hoc Group for the Study of Pertussis Vaccines. 1988. Placebocontrolled trial of two acellular pertussis vaccines in Sweden: protective efficacy and adverse events. Lancet i:955-960. 2. Arai, H., and Y. Sato. 1976. Separation and characterization of two distinct hemagglutinins contained in purified leukocytosispromoting factor from Bordetella pertussis. Biochim. Biophys. Acta 444:765-782. 3. Armstrong, S. K., and C. D. Parker. 1986. Surface proteins of Bordetella pertussis: comparison of virulent and avirulent strains and effects of phenotypic modulation. Infect. Immun. 54:308-314. 4. Aserkoff, B., and J. V. Bennett. 1969. Effect of antibiotic therapy in acute salmonellosis on the fecal excretion of salmonellae. N. Engl. J. Med. 281:636-640. 5. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 1987. Current protocols in molecular biology. John Wiley & Sons, Inc., New York. 6. Brown, A., C. E. Hormaeche, R. Demarco de Hormaeche, M. Winther, G. Dougan, D. J. Maskell, and B. A. D. Stocker. 1987. An attenuated aro A Salmonella typhimurium vaccine elicits humoral and cellular immunity to cloned ,B-galactosidase in mice. J. Infect. Dis. 155:86-92. 7. Brown, D. R., and C. D. Parker. 1987. Cloning of the filamentous hemagglutinin of Bordetella pertussis and its expression in Escherichia coli. Infect. Immun. 55:154-161. 8. Bullas, L. R., and J.-I. Ryu. 1983. Salmonella typhimurium LT2 strains which are r- m+ for all three chromosomally located systems of DNA restriction and modification. J. Bacteriol. 156:471-474. 9. Clements, J. D., F. L. Lyon, K. L. Lowe, A. L. Farrand, and S. El-Morshidy. 1986. Oral immunization of mice with attentuated Salmonella enteritidis containing a recombinant plasmid which codes for production of the B subunit of heat-labile Escherichia coli enterotoxin. Infect. Immun. 53:685-692.

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