Polymer of Haemophilus (Actinobacillus) pleuropneumoniae. Serotype 5

Transcription

1 INFECTION AND IMMUNITY, Aug. 1988, p /88/ $02.00/0 Copyright 1988, American Society for Microbiology Vol. 56, No. 8 Virulence Properties and Protective Efficacy of the Capsular Polymer of Haemophilus (Actinobacillus) pleuropneumoniae Serotype 5 THOMAS J. INZANA,lt* JIANNENG MA,1t TERESA WORKMAN,1 RONALD P. GOGOLEWSKI,1 AND PALMER ANDERSON2 Departments of Veterinary Microbiology-Pathology' and Clinical Medicine and Surgery,2 Washington State University, Pullman, Washington Received 14 January 1988/Accepted 25 April 1988 The role of the capsule of Haemophilus (Actinobacilus) pleuropneumoniae serotype 5 in bacterial virulence, and the protective efficacy of antibody to serotype 5 capsule was investigated. Encapsulated H. pleuropneumoniae serotype 5 were resistant to killing by complement and antibody to capsule or somatic antigens, whereas a noncapsulated mutant was sensitive to killing by the alternative complement pathway alone. Antiserum to whole H. pleuropneumoniae serotype 5 bacteria or monospecific antiserum to capsule was capable of opsonizing bacteria of the homologous serotype for phagocytosis by swine polymorphonuclear leukocytes but was not opsonic for a heterologous serotype. An immunoglobulin M monoclonal antibody to the serotype 5 capsule was not opsonic for any serotype. Mice were protected against lethal, intranasal challenge with the homologous or heterologous serotype after immunization with live encapsulated or noncapsulated bacteria, but not after immunization with killed bacteria, lipopolysaccharide, or a capsule-protein conjugate vaccine. The protection induced by immunization with live bacteria was transferred to nonimmune, syngeneic mice by serum but not by spleen cells. Nonimmune pigs passively immunized with monospecific swine serum to capsule were protected from lethal infection but not from development of hemorrhagic lung lesions, whereas pigs passively immunized with swine antiserum to live bacteria did not develop severe respiratory lesions. Thus, the capsule of H. pleuropneumoniae serotype 5 was inhibitory to the bactericidal activity of serum and was antiphagocytic. Antibody to the capsule was opsonic but was not fully protective. Capsular polysaccharides are recognized as important bacterial virulence factors. Capsules enhance bacterial invasion by protecting the bacteria from host defenses (32, 33), in part by inhibiting activation of the complement cascade due to somatic antigens (e.g., lipopolysaccharide [LPS]) (17). Therefore, capsules enhance bactericidal serum resistance and prevent phagocytosis in the absence of specific antibody (1, 3, 11, 24). Formation of specific capsular antibody by the host results in opsonization and, for most gram-negative bacteria, in vitro bacteriolysis (32, 33). The presence of antibody specific for capsular polysaccharide is sufficient to protect the host against disease by a variety of bacterial pathogens, including Haemophilus influenzae type b, Neisseria meningitidis, Streptococcus pneumoniae, Escherichia coli, and others (32). Haemophilus (Actinobacillus) pleuropneumoniae is the etiologic agent of swine pleuropneumonia, which is an acute or chronic disease characterized by hemorrhagic, fibrinous lesions of the respiratory tract (38). The bacteria are encapsulated by a negatively charged, carbohydrate polymer (13) that is serotype specific (15). The role of the capsule of H. pleuropneumoniae in virulence or in immunoprotection, however, has not been adequately studied. Current vaccines for swine pleuropneumonia consist of killed bacteria. Vaccines have some efficacy in preventing mortality, but chronic infections and potential spread of H. pleuropneumoniae to nonimmune animals are still major problems in the swine * Corresponding author. t Present address: Department of Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, VA industry (9, 27; S. C. Henry and T. A. Marsteller, Abstr. Int. Pig Vet. Soc. Congr. 1982, p. 72). To improve vaccine efficacy, antigens that are capable of inducing protective antibodies to H. pleuropneumoniae need to be identified. In this report we describe the use of a noncapsulated mutant of H. pleuropneumoniae serotype 5 to evaluate the role of the H. pleuropneumoniae serotype 5 capsule in virulence. Because the purified capsule is nonimmunogenic (15), monoclonal antibodies to the capsule and polyclonal whole cell antiserum adsorbed with the noncapsulated mutant were used to determine the protective capacity of antibody to the capsule. Noncapsular antigens that may also play a role in virulence and protection are discussed. MATERIALS AND METHODS Bacterial strains and growth conditions. The source and culture conditions of H. pleuropneumoniae serotype 5 strains K17, 178, J45, a noncapsulated mutant of strain K17 (K17-C), and serotype 1 strain 4045 have been previously described (13, 15). Bacteria were grown in brain heart infusion broth supplemented with 5,ug of NAD per ml (BHI-N) to 109 CFU/ml with shaking. The bacteria were washed two times with phosphate-buffered saline, ph 7.4 (PBS), and suspended in PBS to 109 CFU/ml. Serum reagents. Rabbit antiserum to strain K17 was produced as previously described (15). Swine antiserum to strain K17 was raised in pigs 6 to 10 weeks old by immunization with 5 x 108 CFU of live bacteria intramuscularly in Freund incomplete adjuvant twice at 2-week intervals. Blood samples were obtained weekly throughout the immunization schedule and tested for antibody to capsule, LPS, or whole K17-C cells by an enzyme-linked immunosorbent 1880

2 VOL. 56, 1988 assay (ELISA) (15). Immunizations were continued until the ELISA serum titer to purified capsule was 1:6,400. Monospecific antiserum to the serotype 5 capsule was prepared by adsorption of antiserum to K17 with K17-C (15). Inhibition of antibody to the serotype 5 capsule was prepared by absorption of antiserum to K17 with purified capsule (15). Serum from neonatal, colostrum-deprived pigs and normal 3-month-old pigs (obtained from the Washington State University Swine Center) was used as a source of complement. The blood was allowed to clot at 4 C and centrifuged to separate the serum, and the serum was stored at -70 C in small working samples. Once thawed, serum was used immediately and any remaining serum was discarded. All serum from noninfected pigs was negative for antibody to H. pleuropneumoniae serotype 5 by ELISA or radioimmunoassay (15). Normal and C4-deficient guinea pig sera were obtained from Miles Scientific, Naperville, Ill., and Diamedix (Cordis Laboratories) Miami, Fla., respectively. Fresh, neonatal calf serum was kindly provided by Lynette Corbeil, University of California at San Diego. Monoclonal antibodies to the capsule. Murine monoclonal antibodies to the H. pleuropneumoniae serotype 5 capsule were prepared by a modification of previously described methods (18, 20). Briefly, BALB/c mice were immunized intraperitoneally (i.p.) with 25,ug of purified capsule in Freund complete adjuvant. Mice were boosted after 2 weeks with 25 p.g of capsule in Freund incomplete adjuvant and weekly thereafter with 5 x 108 to 1 x 109 CFU of Formalinkilled strain K17 intravenously. Mice were immunized with whole cells until an antibody response to the capsule was 1: 2,048 by ELISA. Cell fusions were performed as described by Kennett et al. (18) with P3U1 cells, kindly provided by Gerald Schwaber, Boston Children's Hospital, Boston, Mass. Hybridoma cell lines producing monoclonal antibodies to purified capsule, LPS, or K17-C were identified by ELISA as previously described (15) and cloned by limiting dilution. Immunoglobulin classes of the monoclonal antibodies were determined by radial immunodiffusion with reagents purchased from Tago Immunologicals (Tago, Inc., Burlingame, Calif.). Ascites fluid containing an immunoglobulin M (IgM) monoclonal antibody to the H. pleuropneumoniae serotype 5 capsule was produced in BALB/c mice primed with pristane. Virulence of H. pkuropneumoniae serotype 5. Various concentrations of strains K17, K17-C, and 178 in 50,ul of PBS were given by intranasal inoculation to 6- to 7-week-old Swiss Webster mice under deep narcosis with halothane (Fort Dodge Laboratories, Inc., Fort Dodge, Iowa) (37). The 50% lethal dose (LD50) of each strain was determined by the method of Reed and Muench (30). Quantitative bacteremia in mice was determined after intranasal inoculation of 3 x 107 CFU of strain K17 or K17-C. At regular time intervals 20,u1 of blood was obtained from the tail vein and cultured on BHI-N agar at 37 C overnight for determination of viable plate counts. Serum bactericidal assay. The bactericidal assay used has been previously described (14). Briefly, various concentrations of heat-inactivated (56 C for 30 min) normal swine serum or antiserum were mixed with 20% fresh normal swine serum as a complement source, and 104 CFU of bacteria were added per ml. Samples (15 p.1) of the mixture were plated in duplicate before and after 60 min of incubation at 37 C. After overnight incubation at 37 C, the percent viabil- CAPSULE OF H. PLEUROPNEUMONIAE 1881 ity was determined by the following equation: (number of colonies present after 60 min of incubation/number of colonies present before incubation) x 100. For some experiments, all complement activity was inactivated by heating the serum at 56 C for 30 min, or the classical complement pathway was selectively inhibited by the addition of 10 mm ethylene glycol-bis(p-aminoethyl ether)-n,n'-tetraacetic acid (Sigma Chemical Co., St. Louis, Mo.) and 7 mm MgCl2.6H20 (EGTA-Mg). All sera treated at 56 C for 30 min were confirmed to lack complement activity (at 1:2 dilution) by failure to effect lysis of opsonized sheep erythrocytes. The classical complement pathway was confirmed to be inactivated in swine serum treated with EGTA-Mg by lack of antibody-mediated lysis of H. influenzae type b (14). Forty percent rabbit serum to H. influenzae type b supplemented with 40% swine serum killed 100% of the cells in 30 min, whereas the same antiserum lacking swine serum or supplemented with swine serum containing EGTA-Mg completely lacked bactericidal activity. Serum supplemented with EGTA-Mg and C4-deficient guinea pig serum contained normal alternative complement pathway activity, as determined by direct lysis of rabbit erythrocytes, which lack sialic acid and activate the alternative pathway directly (31). Phagocytosis assays. Porcine polymorphonuclear leukocytes (PMNs) and 3H-labeled H. pleuropneumoniae serotype 5 were prepared by modification of previously described procedures (16). Swine PMNs from normal animals were isolated from heparinized whole blood by sedimentation of erythrocytes in 6% dextran. The cells from the resulting leukocyte-rich plasma were centrifuged over Histopaque-1077 solution (Sigma), and the sedimented cells were collected (40). The remaining erythrocytes were lysed with distilled water, followed by the addition of an equal volume of 1.8% NaCl to restore isotonic conditions. The PMNs were washed in Dulbecco PBS with 0.2% D-glucose and suspended to 107 cells per ml in Hanks balanced salt solution (GIBCO Laboratories, Grand Island, N.Y.) containing 1% gelatin. Leukocyte preparations contained greater than 95% PMNs that were greater than 98% viable, as determined by Wright stain and trypan blue exclusion, respectively. Bacteria were radiolabeled (16) and incubated with a 1:10 dilution of fresh serum for 30 min at 37 C with shaking. The treated bacteria were washed three times in PBS, suspended in the original volume with Hanks balanced salt solution-1% gelatin, vortexed, and centrifuged again at 800 x g for 5 min to remove aggregated bacteria. Phagocytosis of opsonized, radiolabeled bacteria by swine PMNs was determined exactly as described (16). Similar mixtures of PMNs and opsonized, unlabeled strain K17 organisms were harvested and fixed with 5% glutaraldehyde and 3% formaldehyde in 0.05 M sodium cacodylate buffer. The mixtures were postfixed with osmium tetroxide, centrifuged in agar, sectioned, stained with 2% uranyl acetate and lead citrate, and examined with a JEOL 100 CX combination scanning-transmission electron microscope (JEOL, Peabody, Mass.). Preparation of capsule and LPS. The capsular polymer and LPS of H. pleuropneumoniae serotype 5 were purified as previously described (13, 15). The purified capsule was conjugated to bovine serum albumin (BSA) through an adipic acid dihydrazide spacer as described by Schneerson et al. (35). Briefly, BSA was activated with 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide (Polysciences Inc., Warrington, Pa.) and conjugated to adipic acid dihydrazide at ph 4.7. Free amino groups were quantitatively assayed by trinitrobenzene sulfonic acid (10.6 mol of adipic acid hydrazide per mol of protein) (12). The capsule was activated with cyanogen bromide at ph 10.5 and conjugated to BSA through the hydrazide spacer at ph 8.5. Only a relatively

3 1882 INZANA ET AL. E E~ c O o vt57 conjugated to BSA from Sepharose CL-4B. Cyanogen bromideactivated capsule was conjugated to BSA through an adipic acid dihydrazide spacer. Protein (i) was monitored by absorption at 280 nm, and carbohydrate(0) was monitored by absorption at 488 nm, after treatment of fractions with phenol and sulfuric acid (5). The elution profile for unconjugated capsule was identical to that previously reported (13). small amount of total capsule was conjugated to BSA, as determined by column chromatography on Sepharose CL-4B (Fig. 1). Fractions at or near the void volume containing protein (monitored by absorbance at 280 nm) and carbohydrate (5) were pooled, dialyzed against distilled water, and lyophilized. Determination of protection by immunization. Swiss Webster mice were immunized i.p. with 1 x 107 CFU of live strain K17 or K17-C (determined to be a sublethal dose i.p.), 5 X 107 rdg CFU of Formalin-killed strain K17, 100 of capsule conjugated to BSA (11% protein, determined by Bio-Rad protein assay) (Bio-Rad Laboratories, Richmond, Calif.), or 50cig of purified LPS in Freund incomplete adjuvant twice at 2-week intervals. For the second immunization the concentration of bacteria was doubled, but the dose of purified antigen remained the same. Control mice were immunized with Freund incomplete adjuvant alone or with PBS. All mice were bled from the tail vein before immunization and after the last immunization and inoculated intranasally with 3 times the LD50 of the challenge strain 2 weeks after the last immunization. After death or, for survivors, 7 days after challenge, the lungs, spleen, and livewemnre examined grossly and cultured for H. pleuropneumoniae serotype 5. Passive immunization studies. BALB/c mice were immunized i.p. with 1 x 107 to 2 x 107 CFU of live strain K17 organisms twice at 2-week intervals. Mice were challenged intranasally with 5 x 107 CFU of the homologous strain 2 weeks after the final immunization to confirm protection. Antiserum was collected by cardiac puncture. The spleen was removed aseptically and placed in a small petri dish containing 5 ml of Dulbecco modified Eagle medium. The spleen was teased apart with a needle and syringe, and the spleen cells were collected and washed by centrifugation at 1,000 rpm for 10 min at 10HC. The remaining erythrocytes were lysed by suspending the cell pellet in 10 ml of cold 17 mm Trizma base (Sigma) containing 150 mmnh4co. The spleen cells were collected by centrifugation and suspended in 10 ml of Dulbecco modified Eagle medium. Cell viability was greater than 95%, as determined by trypan blue exclusion. BALB/c mice were passively immunized i.p. with 6 x 107 to 1.6 x10c spleen cells or with 1 ml of antiserum (or both) and challenged intranasally 24 h after injection with 3 times c INFECT. IMMUN. the LD50 of strain K17. The antibody titer for capsule, LPS, or whole cells of the donor and recipient serum was determined by ELISA. Mice were also passively immunized with 1 ml of ascites fluid containing IgM monoclonal antibody specific for the capsule of H. pleuropneumoniae serotype 5. Mice were challenged as described above with the homologous serotype 20 h after passive immunization. Swine antiserum to live strain K17 bacteria was adsorbed with live K17-C (15) to obtain monospecific antiserum to capsule. Outbred pigs 6 weeks of age were passively immunized with 20 ml of normal swine serum, antiserum to whole cells, or monospecific antiserum to capsule 20 h before intratracheal challenge with 5 x 107 CFU of strain J45. Blood was collected from all pigs before immunization, immediately before challenge, and assayed for antibody to serotype 5 capsule, LPS, or whole cells of K17-C by ELISA. Three pigs were challenged with bacteria that were preincubated with 1 ml of 10% monospecific rabbit serum to the capsule for 30 min at 37 C, followed by three washes in PBS. All animals were necropsied as soon after death as possible; survivors were necropsied 7 days postchallenge. Lungs were examined grossly for pathological changes, and samples were taken for culture and histological examination. Statistics. Statistical analysis of the opsonizing effect of serum on H. pleuropneumoniae serotype 5 was calculated by the Student t test. The effect of active or passive immunization of mice was calculated by the chi-square test for 2 x 2 contingency tables. Standard deviations were calculated from at least three experiments done in duplicate. Geometric mean antibody titers were calculated by the equation: 'Vx1.x2.. xn. RESULTS Bactericidal antibody and complement. The bactericidal activity of normal swine serum, swine antiserum to K17, monospecific swine antiserum to the serotype 5 capsule, and an IgM monoclonal antibody to Hp5 capsule was tested against serotype 5 strains K17, 178, and J45 and serotype 1 strain Although strain 178 is encapsulated, it contains about 10 times less capsule than strain K17 or J45, determined by radioimmunoassay (unpublished data). All encapsulated strains were 100% resistant to the bactericidal activity of each serum reagent, tested fresh at 90% concentration. An IgM monoclonal antibody to the serotype 5 capsule, supplemented with fresh swine serum, fetal calf serum, or guinea pig serum as a complement source, was also not capable of killing any of the strains tested (data not shown). Thus, encapsulated H. pleuropneumoniae was resistant to the bactericidal activity of complement, even in the presence of antibody to capsule or somatic antigens. To confirm that the complement sources tested contained bactericidal activity, H. influenzae type b strain Eag (kindly provided by Porter Anderson, University of Rochester Medical Center, Rochester, N.Y.) was mixed with 50% heat-inactivated antiserum to purified strain Eag LPS (14) and 20% of each complement source. Each complement source supplemented with antiserum killed greater than 90% of H. influenzae type b organisms within 30 min (data not shown). Fresh 90% rabbit antiserum to K17, or 45% rabbit antiserum and 45% fresh neonatal calf serum, was also not bactericidal for strains K17 or 178. This same lot of neonatal calf serum was bactericidal for "H. somnus" in the presence of antiserum to "H. somnus" (T. J. Inzana et al., submitted for publication). In contrast to encapsulated strains, strain K17-C was very sensitive to killing by various concentrations of fresh normal

4 VOL. 56, 1988 CAPSULE OF H. PLEUROPNEUMONIAE oor ) 0 I- C.) 0 4c I.- z A. 20- z % Ul (a 40 F \ 1.. I I \ _ I * I; * * * *, PERCENT SERUM FIG. 2. Dose-response bactericidal activity of various sera for H. pleuropneumoniae K17-C (noncapsulated mutant). Swine serum containing active complement was mixed with 104 CFU of strain K17-C per ml, and 20,ul was spread on BHI-N agar before and after 1 h of incubation at 37 C. Percent survival, determined from the equation (number of CFU after 1 h incubation/number of CFU before incubation) x 100, is shown for normal swine serum (-), normal swine serum adsorbed with K17-C (...), and precolostral swine serum (--- -). Each point represents the mean of three experiments done in duplicate. The standard deviation for each point was always less than 10%. swine serum, normal swine serum adsorbed with K17-C, or precolostral swine serum in a dose-dependent manner (Fig. 2). As little as 11% pormal serum adsorbed with K17-C was bactericidal for strain K17-C. The bactericidal activity of 90% normal serum for K17-C was completely eliminated by heating at 56 C for 30 min. Normal swine serum containing EGTA-Mg to block activation of the classical pathway killed % of strain K17-C (data not shown). Therefore, noncapsulated H. pleuropneumoniae could activate, and be killed by, the alternative complement pathway in the absence of specific antibody. Incubation of C4-deficient guinea pig serum with 5 x 108 CFU of K17-C at 37 C did not result in bacterial killing but did deplete the serum of 75% of its cornplement activity, determined by lysis of rabbit erythrocytes. Incubation of C4-deficient serum with 5 x 108 CFU of strain K17 or with no cells at 37 C for 30 min resulted in 25% loss of alternative pathway activity, indicating that K17-C was activating the alternative complement pathway (data not shown). Phagocytosis of H. pleuropneumoniae. Phagocytosis of 3Hlabeled H. pleuropneumoniae serotype 5 after incubation in various sera is shown in Fig. 3. Incubation of strain K17 with homologous fresh or heat-inactivated antiserum effectively opsonized the bacteria, resulting in 36.3 to 37.3% phagocytosis. Adsorption of antiserum with K17-C reduced phagocytosis to 29.4%, but the difference was not significant (P = 0.24). Incubation of bacteria with antiserum adsorbed with purified capsule reduced phagocytosis to 19.4%, which was significantly less than phagocytosis after opsonization with L Li d 0 o T. I 3 K17 K1 7-C J FIG. 3. Phagocytosis of H. pleuropneumoniae by swine PMNs after incubation in various sera or monoclonal antibody. 3H-labeled bacteria were incubated for 30 min at 37 C in 10% fresh, normal swine serum, in 10%o antiserum, or in 10% fresh, normal swine serum and 25% ascites fluid (containing IgM monoclonal antibody to capsule). The bacteria were washed and mixed with swine PMNs at a ratio of 80:1 for 30 min at 37 C; controls to differentiate adherence from uptake were incubated at 0 C. The PMNs were thoroughly washed and suspended in a small volume of PBS and scintillation cocktail, and the counts per minute were determined in a liquid scintillation counter. Percent phagocytosis was determined by [(counts per minute of cells incubated with bacteria at 37 C - counts per minute of cells incubated with bacteria at 0 C)/counts per minute of the total bacteria available] x 100. Symbols: 0, fresh swine antiserum to strain K17; U, heat-inactivated swine antiserum to strain K17; E, monospecific swine antiserum to serotype 5 capsule; U, swine antiserum to strain K17 adsorbed with serotype 5 capsule; M, IgM monoclonal antibody to serotype 5 capsule; US, normal swine serum; U, swine antiserum to strain K17 adsorbed with K17-C; O, precolostral swine serum. Standard deviations were calculated from assays done three or more times in duplicate. whole cell antiserum (P = 0.01). The opsonic activity of monospecific antiserum to the serotype 5 capsule for strain J45 was similar to that of K17, but neither strain was opsonized by normal serum. Neither whole cell or monospecific capsular antiserum was opsonic for strain 4045 (serotype 1). Of interest was that strain K17-C was efficiently opsonized with fresh or heat-inactivated antiserum to strain K17, but not by fresh, normal serum adsorbed with K17-C or by precolostral swine serum. Thus, complement was not opsonic in the absence of specific antibody for H. pleuropneumoniae. These results indicated that antibodies to the capsule were primarily responsible for opsonization, but that antibodies to somatic antigens were also opsonic. However, murine IgM monoclonal antibody to the capsule did not opsonize the homologous serotype for phagocytosis by porcine PMNs in the presence of swine complement. Phagocytosis of opsonized strain K17 was confirmed by identification of a bacterium within a phagosome by transmission electron microscopy (Fig. 4). A double membrane and hairlike projections surrounding the bacterium were clearly visible. Virulence studies. The LD50 of strain K17 in Swiss Webster mice was 1.5 x 107 CFU, only slightly lower than that of its noncapsulated mutant, K17-C (2.0 x 107 CFU). Although strain 178 contained 10 times less capsule than K17, the LD50 of strain 178 was 8.6 x 106 CFU. Therefore, the amount of capsule present on the H. pleuropneumoniae strains tested had little influence on lethality for mice after intranasal challenge. The capacity of the capsule to impart invasive potential on H. pleuropneumoniae serotype 5 was evaluated by blood culture of Swiss Webster mice after challenge of five mice each with 3 x 107 CFU of strain K17 or K17-C. Death

5 1884 INZANA ET AL. INFECT. IMMUN. 4(4~~~~~~4 s ^ :l.; K sv.,,,;syl,,,+.e az ti S' ' 4 P 'tffi ss SS.$ ip: Downloaded from FIG. 4. Transmission electron micrograph of opsonized H. pleuropneumoniae serotype 5 within the phagosome of a swine PMN. The bacterium is approximately 1,um in diameter (magnification, x42,250). The double membrane of the bacterium is clearly visible, as are hairlike projections surrounding the bacterium. occurred in all animals challenged with either strain, but varied between 2 and 44 h postchallenge. Bacteria were detected in the blood of all mice challenged with strain K17 by at least 1.5 h postchallenge. Variation of the level of bacteremia was greatest between the mice 1.5 h after challenge (0.1 x 103 to 1.5 x 103 CFU/ml). Peak numbers of bacteria were present in the blood of mice challenged with strain K17 between 2 and 4 h after challenge and were similar for each mouse (2.3 x 103 to 4.7 x 103 CFU/ml). In the one animal that survived longer than 8 h postchallenge, bacteremia declined from 2.3 x 103 CFU/ml at 4 h postchallenge to 0.2 x 103 CFU/ml by 8 h postchallenge and was not detected at 28 h after challenge. Although bacteria had been eliminated from the blood, the animal still died within 44 h after challenge. Intranasal challenge of mice with strain K17-C, however, did not result in detectable bacteria in the blood of any of the animals, although each of the animals died between 3 and 44 h after challenge. The lungs of all animals challenged with either strain were grossly hemorrhagic and edematous and were culture positive for H. pleuropneumoniae. Reversion of K17-C to the encapsulated form after successive, lethal in vivo passages in mice did not occur, as determined by inhibition radioimmunoassay (15). Protection after active or passive immunization. Active immunization of mice with live or Formalin-killed H. pleuropneumoniae serotype 5 or with purified antigens is shown in Table 1. Mice immunized i.p. with sublethal doses of live TABLE 1. Protection of mice against H. pleuropneumoniae K17 after active immunization Geometric mean antibody Immunogena Survival ratiob titerc Capsule LPS Whole cell Live strain K17 8/ ,500 Live strain K17d 5/ ,500 Formalin-killed K17 1/ ,363 Live K17-C 7/8 <10 ND 2,560 Purified K17 LPS 0/5 ND 320 ND Serotype 5 capsule-bsae 0/9 95 ND ND Nonimmunized controls 0/11 <10 <10 <10 a Swiss Webster mice (6 weeks old) were immunized i.p. with 1 x 107 to 2 x 107 whole bacteria without adjuvant, 50 p.g of capsule-bsa, or LPS in Freund incomplete adjuvant at 2-week intervals. b Mice were challenged 2 weeks after the last immunization with 5 x 107 washed, log phase K17 bacteria in 50,ul of PBS intranasally. The total numbers of mice reported are the sum of animals tested on two or more separate occasions. creciprocal antibody titers were determined by ELISA with purified serotype 5 capsule or LPS, or Formalin-killed, whole K17-C as antigens. ND, Not determined. d Mice were challenged with 3 x 107 CFU of serotype 1 strain H. pleuropneumoniae serotype 5 capsule was covalently conjugated to BSA through an adipic acid dihydrazide spacer to enhance immunogenicity. on November 17, 2018 by guest

6 VOL. 56, 1988 CAPSULE OF H. PLEUROPNEUMONIAE 1885 TABLE 2. Protection of BALB/c mice against H. pleuropneumoniae K17 after passive immunization with spleen cells or antiserum to live strain K17a Expt Immune-factors Geometric mean antibody titer of donor' No. of spleen Challenge Survival passively transferred Capsule LPS Whole cells cells transferred dose (CFU) ratioc 1 Spleen cells 6.0 x x 107 0/6 Serum ,800 6/6 Spleen cells and serum , x 5/6 Controls <10d <10 <10 0/3 2 Spleen cells 1.6 x x 107 0/7 Serum ,263 4/5 Spleen cells and serum , x 108 3/3 Controls <10 <10 <10 0/4 3 Spleen cells 1.0 x x 107 1/ Serum ,800 3/5 Spleen cells and serum , x 108 3/3 Controls <10 <10 <10 0/5 4 Monoclonal antibody 128 ND ND 4/11 to serotype 5 capsule a BALB/c mice were immunized i.p. with live strain K17 twice at 2-week intervals and challenged intranasally with 3 times the LD" to confirm protection; spleen cells, serum, or both were transferred to nonimmune, syngeneic recipients 3 days after challenge. b Reciprocal antibody titers were determined by ELISA. ND, Not determined. The total number of mice used was the sum of two or more separate experiments. dreciprocal titers <10 by ELISA were considered negative. or Formalin-killed strain K17 raised antibody titers to capsule, LPS, and whole cells. Immunization with live K17 protected all eight mice against lethal challenge with strain K17 or five of six mice against challenge with strain 4045 (serotype 1). Seven of eight mice immunized with live K17-C were also protected against lethal challenge with strain K17. Mice immunized with Formalin-killed or heat-killed (100 C for 1 h; data not shown) strain K17 were not protected against lethal challenge. Mice immunized with purified capsule conjugated to BSA or with purified K17 LPS made an antibody response to the immunizing antigen, but none of the mice was protected against challenge with strain K17. Therefore, protection of mice against lethal challenge after active immunization was not dependent on antibody to capsule or LPS, was not serotype specific, and was not induced by killed cells. To determine whether protection of mice after active immunization was humoral or cell mediated, spleen cells, serum, or spleen cells and serum were transferred from BALB/c mice immunized i.p. with a sublethal dose of live strain K17 to syngeneic, nonimmune recipients (Table 2). All actively immunized mice responded with positive antibody titers to capsule, LPS, or K17-C whole cells. Actively immunized mice were challenged with 2 to >3 times the LD50 of strain K17 to confirm the mice were protected against lethal infection. Three days after challenge, serum or spleen cells (or both) from actively immunized mice were passively transferred to syngeneic, nonimmune mice. Most nonimmune mice receiving only spleen cells were not protected against any challenge dose tested. Most of the nonimmune mice receiving serum alone or spleen cells and serum from immune mice were protected against challenge with 3 X 107 to 5 x 107 CFU of K17. Due to the small numbers of mice used in each group, however, we could not determine whether the combination of spleen cells and serum provided significantly greater protection than serum alone. Nonetheless, humoral immunity was of primary importance in the protection of mice against virulent H. pleuropneumoniae serotype 5. Passive transfer of an IgM monoclonal antibody to capsule protected 4 of 11 mice against lethal challenge but was significantly less efficacious than antiserum to live H. pleuropneumoniae serotype 5 (P = 0.02). Surviving mice demonstrated substantial morbidity for several days after challenge. Therefore, monoclonal antibody to capsule provided partial but inadequate protection against mortality and morbidity in the mouse model. Passive protection in young pigs was examined by i.p. transfer of swine antiserum to live, strain K17, antiserum to K17 adsorbed with strain K17-C, or normal swine serum. Twenty hours later the animals were challenged intratracheally with 5 x 107 CFU of strain J45 (Table 3). Strain J45 was used for challenge studies because this strain is a relatively fresh porcine isolate that has been used in other pathogenesis studies (6). Antiserum to strain K17 was used for passive TABLE 3. Protection of swine against H. pleuropneumoniae J45 after passive immunization' Antibody titer' Survival Bacteria Serum rateb isolated from Capsule K17-C lung (no./total) Normal <10 <10 0/3 3/3 Monospecific to capsule <10 3/3 3/3 Swine antiserum to live c 40c 2/3 1/3 K17 a Pigs were challenged intratracheally with 5 x 10' CFU of serotype 5 strain J45 20 h after passive transfer of 20 ml of immune or normal swine serum. bserum antibody titers to capsule or K17-C were determined by ELISA. Blood was collected before and 20 h after passive immunization (immediately before challenge). Reciprocal ELISA titers to capsule and K17-C were <10 for all pigs before passive immunization. c The reciprocal antibody titer to capsule or K17-C of the one pig that died was <10.

7 1886 INZANA ET AL. immunization because the noncapsulated mutant was derived from this strain. Pigs passively immunized with normal serum had no detectable antibody titer to capsule or whole cells immediately before challenge. Each of these pigs died within 2 days after challenge. Grossly, all control pigs had focally extensive, severe, necrotizing pleuropneumonia. Histologically, affected lungs were characterized by vasculitis and marked accumulation of neutrophils, mononuclear cells, fibrinous fluid, and necrotic cellular debris. Vasculitis and edema were consistent findings, and H. pleuropneumoniae serotype 5 was isolated from all lesions cultured. Pigs passively immunized with monospecific serum to the capsule survived challenge; although lesions were qualitatively similar to those of control pigs, lesions were less severe and less extensive. Cultures for H. pleuropneumoniae serotype 5 were positive from all lesions. Before challenge, antibody to capsule was present in pigs receiving monospecific serum, but antibodies to somatic antigens were not detectable. One pig passively immunized with antiserum to whole bacteria died shortly after challenge due to severe, hemorrhagic pleuropneumonia and had no detectable antibody titers to H. pleuropneumoniae serotype 5 before challenge; this pig was considered nonimmune. The other pigs given antiserum to live cells survived challenge and had antibodies to capsule and K17-C; pleuropneumonialike lesions were not seen at necropsy, and no bacteria were isolated from the lungs. Pigs challenged with bacteria that were preincubated with monospecific rabbit serum to the capsule did not die, lesions were not seen at necropsy, and no bacteria were cultured from the lungs (data not shown). DISCUSSION Molecular genetic studies done with E. coli, S. pneumoniae, and H. influenzae have clearly shown that capsules are capable of enhancing bacterial virulence (1, 2, 23). In addition, antibody to the capsule is sufficient for protection against many bacterial pathogens (32). We were interested in determining whether the capsule of H. pleuropneumoniae serotype 5 enhanced virulence and whether antibody to the capsule was protective. Since studies concerning the genetics of H. pleuropneumoniae have not been reported, our basic approach was to use a noncapsulated mutant for comparative in vitro and in vivo virulence studies. The mutant we used was hemolytic, and the LPS and major outer membrane protein electrophoretic profiles were identical to those of the parent. The presence, absence, or amount of capsule did not significantly alter the LD50 of each strain of H. pleuropneumoniae serotype 5 tested in mice. Strain 178, which contained 10 times less capsule than strain K17, had the lowest LD50, and strain K17-C was almost as lethal for mice as the parent strain. These findings were in marked contrast to reports that noncapsulated mutants of H. influenzae type b (24, 44) and E. coli (1, 19) are significantly less virulent than the parent strains. However, strain K17 was capable of causing bacteremia in mice, whereas strain K17-C could not, even though both strains caused death within 2 days. None of the organs other than lungs had any gross lesions or contained viable H. pleuropneumoniae serotype 5, indicating the bacteria probably did not localize in organs after bacteremia. These results indicated that, in the mouse model, mortality due to H. pleuropneumoniae serotype 5 was not dependent on bacteremia, but that death was probably due to localized infection of the lung. Our results were consistent with those of Fenwick et al. (8), who INFECT. IMMUN. reported that although the mouse is a good model for acute disease due to H. pleuropneumoniae, it is not a good model for chronic infection. Therefore, studies of H. pleuropneumoniae serotype 5 in mice should be interpreted with caution when considering natural, chronic disease in swine. Capsule-mediated resistance of gram-negative bacteria to the bactericidal activity of normal serum may occur through inhibition of complement activation and is influenced by the composition and molecular weight of the capsule (3, 19, 32, 39). Capsule clearly enhanced the resistance of H. pleuropneumoniae serotype 5 to the bactericidal activity of normal serum. In addition, strains K17 and 178 were completely resistant to killing by complement in the presence of hyperimmune antiserum to whole cells, to monospecific antiserum, and to IgM monoclonal antibody to capsule. Such results were unexpected because specific antibody to capsule (particularly IgM) or somatic antigens is bactericidal for many encapsulated gram-negative bacteria (32, 33, 36). The resistance of H. pleuropneumoniae serotype 5 to somatic antibodies may be explained by inaccessibility of somatic antigens due to steric interference by capsule. Resistance of the bacteria to antibody to capsule could be explained if the capsule prevented fixation of complement on the membrane of the bacterium. Confirmation of the mechanism of resistance of H. pleuropneumoniae serotype 5 to antibody and complement requires additional investigation. In contrast to the parent strain, K17-C was susceptible to killing by normal serum adsorbed with K17-C, to precolostral swine serum, and to normal swine or guinea pig serum containing EGTA- Mg in a dose-dependent manner. Killing was abrogated by heating serum at 56 C for 30 min. K17-C was not killed, however, by C4-deficient guinea pig serum. Since this serum is genetically deficient in C4, it is possible that other factors that are required for bactericidal activity may also be deficient in this serum. Therefore, in the absence of capsule the bacteria were apparently activating and being killed by the alternative complement pathway. Encapsulated bacteria are generally resistant to opsonization and phagocytosis in the absence of specific antibody, whereas noncapsulated mutants are efficiently opsonized in the presence of complement alone (10, 17, 32, 42). Furthermore, infections due to some gram-negative bacteria may be controlled primarily through opsonization and phagocytosis, rather than by the bactericidal activity of serum (10, 26, 42). Our results concerning opsonization of H. pleuropneumoniae serotype 5 were consistent with similar studies done with other encapsulated bacteria. Swine antiserum to whole cells or to capsule alone efficiently opsonized H. pleuropneumoniae serotype 5 for phagocytosis by swine PMNs. Heat inactivation of the serum did not significantly affect opsonization, indicating that complement did not enhance the phagocytic activity of antiserum. Of particular interest was that antiserum was also required for opsonizing K17-C. Serum containing active complement, which can be activated by K17-C (determined by bactericidal assays and consumption of complement from C4-deficient serum), but lacking specific antibody was not opsonic. Complement receptors may not have been accessible, or complement alone may not have been sufficient, for phagocytosis by swine PMNs. In addition, our murine IgM monoclonal antibody was not opsonic, which suggests that swine PMNs do not have Fc receptors for murine IgM. The lack of opsonization of H. pleuropneumoniae serotype 5 in the presence of murine IgM and swine complement suggests that either swine PMNs may not have efficient receptors for activated complement components, murine IgM cannot effi-

8 VOL. 56, 1988 ciently activate swine complement, or complement receptors for PMNs are not efficiently expressed after fixation of complement on H. pleuropneumoniae serotype 5. Schreiber et al. (36) reported that purified human IgG to capsule was highly opsonic for H. influenzae type b, but purified anticapsular IgM was only poorly opsonic in the presence of complement. Optimal opsonization of bacteria by human PMNs may require simultaneous binding of C3b and the IgG Fc receptor (25). A similar and even more substantial simultaneous binding may be required for phagocytosis of bacteria by swine PMNs. Phagocytosis of encapsulated, opsonized H. pleuropneumoniae serotype 5 was confirmed by electron microscopy. A bacterium was clearly visible within a PMN phagosome. In addition to a double membrane on the bacterium, fingerlike projections covered the surface. It was not determined if these projections were pili or antibody-coated capsular material. The presence or absence of pili in H. pleuropneumoniae has not been reported, and additional investigation of the ultrastructure of this bacterium is warranted. Whether H. pleuropneumoniae serotype 5 can survive within swine PMNs was not adequately examined in this study. It is possible that the leukotoxin produced by H. pleuropneumoniae (38) interferes with the capability of PMNs to kill these bacteria. Therefore, a detailed investigation of the interaction and survival of H. pleuropneumoniae serotype 5 in PMNs is needed. Although antiserum absorbed with purified capsule still opsonized H. pleuropneumoniae serotype 5 for phagocytosis by swine PMNs, activity was significantly reduced. This reduction in opsonic activity may have resulted from steric interference of antibodies to somatic antigens by capsule. Adsorption of serum with K17-C, however, did not significantly reduce activity, indicating that most of the opsonic antibody was directed to the capsule. Furthermore, the opsonic activity of antiserum to strain K17 was clearly type specific, because another serotype 5 strain was efficiently opsonized, but a serotype 1 strain was not. Therefore, these results support earlier data that the serotype 5 capsule is the serotype-specific antigen (15). Rosendal et al. (34) reported that immunization of pigs and mice with crude capsular extracts of H. pleuropneumoniae serotype 1 provided only partial protective immunity. Our results with purified capsule and monospecific or monoclonal antibody to capsule supported the evidence that antibody to capsule provides incomplete protection to H. pleuropneumoniae. Immunization of mice with Formalin-killed cells or purified capsule conjugated to BSA did not provide protective immunity, even though antibody to capsule was raised. Passive transfer of an IgM monoclonal antibody protected 4 of 11 mice from lethal challenge and delayed death in others. Such partial protection may have resulted from the mice receiving more antibody to capsule by passive transfer than was raised by active immunization. Whether the class of antibody that was transferred (IgM) was critical for protection was not determined. The quantitative importance of capsular antibody was supported by studies showing that when bacteria were preopsonized with rabbit antibody to capsule before challenge in pigs, apparent infection did not occur. Antibodies specific for LPS were also not capable of providing protective immunity in mice. These results in mice were in contrast to those reported by Fenwick et al. (6), that immunization of pigs with E. coli J5 LPS provided partial immunity to infection by H. pleuropneumoniae serotype 5 strain J45. These discrepancies may be due to differences in the immunizing antigens used and because immunization CAPSULE OF H. PLEUROPNEUMONIAE 1887 with E. coli J5 may induce a nonspecific, protective immune factor (41). We found that maximum protection (which was cross-reactive with a heterologous serotype) in mice was induced only when animals were immunized with live encapsulated or noncapsulated bacteria. Maximum protection against lethal infection did not require antibody to capsule and was not serotype specific. These results are compatible with those of Neilsen (28, 29), who reported that immunization with live H. pleuropneumoniae provided protection against heterologous serotypes but that immunization with a killed, whole cell vaccine induced only serotype-specific immunity. Immunization with live bacteria, is known to induce greater cell-mediated immunity than did immunization with killed bacteria due to prolonged stimulation of local lymphoid tissues (43). Components of live bacteria are also presented to the host in their natural antigenic state, which may be altered by heat, Formalin, or other agents that kill the bacteria. To evaluate the role of humoral and cellmediated immunity in protection provided by live H. pleuropneumoniae serotype 5, syngeneianonimmune mice were passively immunized with serum, spleen cells, or serum and spleen cells from immune donor mic'e. Transfer of immune serum or spleen cells and serum, but not spleen cells alone, protected nonimmune syngeneic recipients, indicating that protective factors were present in serum. Furthermore, the protection transferred by serum to live H. pleuropneumoniae serotype 5 was cross-reactive against serotype 1. These results suggest that certain protective antibodies may be made in response to immunization with live bacteria, but not in response to Formalin-killed or heat-killed bacteria, and that protective antibodies were cross-reactive with heterologous serotypes. Further evidence that antiserum to live bacteria provides maximum protection was obtained by passive transfer of immune swine serum to nonimmune pigs. Pigs receiving monospecific serum to capsule survived lethal challenge, but their lungs contained lesions qualitatively similar to controls challenged with viable H. pleuropneumoniae serotype 5. In contrast, two of three animals that received serum to live bacteria did not develop lesions, and bacteria were not recovered from the lungs. One animal that received antiserum to live cells developed acute pleuropneumonia but did not have detectable specific antibody in its blood at the time of challenge. Due to the limited amount of immune swine serum available for passive transfer and the space available for pigs in isolation, the number of pigs in each group was small. Nonetheless, results of passive transfer of immune swine serum in pigs was consistent with those obtained in mice. Protection provided by antiserum to capsule was similar to the level of protection provided by active immunization with killed bacteria (27). Our results were consistent with the views of Fenwick and Osburn (7), that the serotypespecific protection from vaccination is primarily due to antibody to capsule. In contrast to work done with encapsulated bacteria that do not produce an exotoxin, antibody to H. pleuropneumoniae serotype 5 capsule was not fully protective (32). The identity of the protective antigen(s) was not determined, but it may be a labile protein exotoxin such as hemolysin (22). Protection due to antitoxin would explain why only live, toxic cultures were protective and why the protection was cross-reactive with a heterologous serotype. Our protection results were similar to those reported for Pasteurella haemolytica Al, which is encapsulated and produces a labile leukotoxin. As for H. pleuropneumoniae serotype 5, immu-

9 1888 INZANA ET AL. nization with Formalin-treated P. haemolytica is not adequately protective, whereas immunization with live bacteria provides more complete immunity (4, 21). The role of the hemolysin in promoting the virulence of H. pleuropneumoniae serotype 5 and the protection provided by antibody to hemolysin require investigation. ACKNOWLEDGMENTS We thank Gerhardt Schurig and Craig Hammerberg for critical review of the manuscript and for helpful suggestions, Steve Kania for technical advice regarding monoclonal antibody production, and Thomas Caceci for assistance analyzing samples by electron microscopy. This work was supported, in part, by grants from the Washington State University Grant-in-Aid Program and by Praxis Biologics. LITERATURE CITED 1. Allen, P. M., I. Roberts, G. J. Boulnois, J. R. Saunders, and C. A. Hart Contribution of capsular polysaccharide and surface properties to virulence of Escherichia coli Kl. Infect. Immun. 55: Avery, 0. T., C. M. MacLeod, and M. 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