Contribution of adjuvant to adaptive immune responses in mice against Actinobacillus pleuropneumoniae

Fernando San Gila,1, Bernadette Turner1, Mark J. Walker2, Steven P. Djordjevic1 and James C. Chin1

NSW Agriculture, Elizabeth Macarthur Agricultural Institute, PMB 8, Camden, NSW 2570, Australia1
Department of Biological Sciences, University of Wollongong, Wollongong, NSW 2522, Australia2

Author for correspondence: James C. Chin. Tel: +61 246 406359. Fax: +61 246 406384. e-mail: james.chin{at}agric.nsw.gov.au


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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The authors have previously demonstrated that adjuvant-mediated differences in early cellular responses to antigens significantly affect subsequent adaptive immune responses. To investigate further the contribution of adjuvant to adaptive immune responses, outer-membrane proteins (OMP) purified from the respiratory pathogen Actinobacillus pleuropneumoniae, given either alone (antigen group) or complexed with SAMA4 (vaccine group), were injected intradermally into groups of mice. Controls were given PBS. Inclusion of adjuvant did not significantly alter the kinetics of antibody responses against OMP in serum or respiratory tract washings (RTW) over 21 weeks. Re-exposure to OMP at 21 weeks also induced identical recall responses in both immunized groups. However, differences between the responses of the vaccine and antigen groups were apparent when sera and RTW were reacted against OMP and OMP-derived polysaccharide antigens (ODPA). Serum and RTW reactivity against protein antigens was stronger in the vaccine group than in the antigen group. Serum and RTW from the vaccine group also reacted against a greater number of proteins than did the antigen group. Although serum reactivity against ODPA was equivalent for both groups, RTW from the vaccine group reacted only faintly against ODPA compared with the antigen group. The results suggested that shifting of antibody reactivity away from polysaccharide antigens toward protein antigens was an adjuvant-mediated effect. The rapid death of controls following intranasal inoculation confirmed that protection was ultimately dependent on the presence of specific antibodies in the serum and respiratory tract. However, since both groups responded equally to intranasal infection with A. pleuropneumoniae, as seen by the rapid clearance of bacteria from the lungs, the biological significance of any differences between the groups was unclear. Knowledge of the effects of adjuvants may provide a rational basis for adjuvant selection and the ability to manipulate immunological outcomes more precisely.

Keywords: pleuropneumonia, outer-membrane protein, antigen, vaccine, respiratory pathogen

Abbreviations: ASC, antibody-secreting cells; CP, capsular polysaccharide; ELISPOT, enzyme-linked immunospot; FE, faecal pellet extracts; ODPA, polysaccharide antigen; OMP, outer-membrane protein(s); OVA, ovalbumin; RTW, respiratory tract washings; TSTw, Tris-saline-Tween; VW, vaginal washings

a Present address: Biochemistry Department, Illawarra Area Health Service Pathology, The Wollongong Hospital, Crown St, Wollongong, NSW 2500, Australia


   INTRODUCTION
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ABSTRACT
INTRODUCTION
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Intradermal immunization with a hybrid liposome-ISCOM adjuvant, SAMA4 (Australian Patent no. PCT/AU95/00206) promotes both mucosal and systemic antibody responses in different animal species without any adverse immunological or physiological side-effects (Chin et al., 1996 ). Further investigation in mice identified that intradermal immunization with SAMA4-adjuvanted vaccines triggers a series of co-ordinated responses characterized initially by large declines in T-cell precursors in the thymus, and haematopoietic progenitor cells and B-cell precursors in the bone marrow, followed by increased haematopoeisis and mobilization of granulocytes and monocytes to the spleen, lungs and other organs (Chin & San Gil, 1998 ; San Gil et al., 1998 ). These events were reproducible and independent of the type of antigen used, and were due to synergistic interaction between antigen and adjuvant (San Gil et al., 1998 ).

The loss of leukocyte precursors followed by increased haematopoeisis and leukocyte mobilization observed following intradermal immunization resembled the changes that occur in response to many diseases (Sebunya & Saunders, 1982a ; Bhatia et al., 1991 ). These events are important preludes to the subsequent development of adaptive immunity. Fearon & Locksley (1996 ) and others (George et al., 1989 ; Naito et al., 1989 ; Gajewski et al., 1991 ; DeKruyff et al., 1992 ; Levin et al., 1993 ; Gong et al., 1994 ; Scardino et al., 1994 ; Dempsey et al., 1996 ; Mikszta & Kim, 1996 ) have proposed that quantitative and qualitative differences in innate responses evoked against antigens, such as differences in the rate and/or efficiency of phagocytosis and antigen presentation, can direct and alter subsequent adaptive immune responses. If so, then the much greater cellular responses evoked against SAMA4-adjuvanted antigens was expected to promote different adaptive responses from those elicited following immunization with antigen alone, which elicited only minor cellular changes post-immunization (San Gil et al., 1998 ).

This expectation was confirmed in studies with ovalbumin (OVA), a weak and innocuous antigen, complexed with SAMA4 (SAMA4-OVA). In mice given SAMA4-OVA intradermally, the early strong cellular responses evoked by immunization were followed by strong anti-OVA antibody production and strong OVA-specific T-cell cytotoxicity against OVA-expressing cell lines (Chin et al., 1996 ; San Gil et al., 1998 ). These outcomes were not detected in mice given OVA alone and suggested that adjuvant-mediated changes to leukocyte homeostasis ultimately enhanced adaptive immune responses against thymus-dependent antigens.

The present study investigated further the contribution of adjuvant to the development of adaptive immune responses. A chemically heterogeneous and complex antigen, outer-membrane proteins (OMP) purified from the respiratory pathogen Actinobacillus pleuropneumoniae, was substituted for OVA. A. pleuropneumoniae, a Gram-negative coccobacillus, is the aetiological agent responsible for pleuropneumonia in pigs (Sebunya & Saunders, 1983 ). Mice were used because they are also susceptible to A. pleuropneumoniae infection (Sebunya & Saunders, 1982a ; Oettinger & Stenbaek, 1990 ). Differences in disease pathogenesis between mice and swine have been discussed by others (Sebunya & Saunders, 1982a , b ; Fenwick et al., 1986 ; Rosendal et al., 1986 ; Lenser et al., 1988 ; Bhatia et al., 1991 ; Stine et al., 1991 ) and such an investigation was not an aim of the present study. However, the susceptibility of mice meant that investigation of adjuvant-mediated differences in adaptive responses against OMP could be done at the serological level and extended to examine responses to live infection. Although numerous components of A. pleuropneumoniae are required for complete protection, in order to achieve the primary aim of the study, namely to investigate differences in adaptive responses induced by adjuvanted and non-adjuvanted vaccines, only OMP was used.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
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Mice.
Female BALB/c mice aged 8–12 weeks were purchased from the CSIRO Animal Production and Processing Facility, Delhi Road, North Ryde, NSW, Australia. Mice were maintained at 23 °C under a 12 h day/night cycle. Antibiotic-free commercial mouse pellets and water were supplied ad libitum.

OMP vaccine preparation and immunization schedule.
OMP were purified from A. pleuropneumoniae serovar 1, strain HS54, as described previously (Chin & Turner, 1990 ). SAMA4-adjuvanted OMP was prepared by incorporating OMP in unilamellar liposomes prior to complexing with various ingredients used in the formulation of ISCOMs (Chin et al., 1996 ).

Mice (78 per group) were vaccinated intradermally with 100 µl either phosphate-buffered saline (PBS), pH 7·2 (control group), OMP alone (antigen group) or SAMA4-adjuvanted OMP (vaccine group) on days 0 and 7. The amount of antigen given per mouse was equivalent to 30 µg protein, as determined by a commercial bicinchoninic-acid-based protein estimation kit (Pierce Chemical Co.), and 0·6 µg Quil A (Superfos Biosector).

Preparation of OMP-derived polysaccharides.
OMP purified from A. pleuropneumoniae contain polysaccharide components. To assess the antibody responses of immunized mice to both protein and polysaccharides, OMP-derived polysaccharide antigens (ODPA) were prepared by de-proteinization of OMP with proteinase K (Wood et al., 1989 ). After such treatment, OMP polypeptides were not detected by Coomassie brilliant blue staining of SDS-PAGE gels. A silver-staining method sensitive to polysaccharides (Tsai & Frasch, 1982 ) was used to visualize ODPA remaining after this procedure. An identical polysaccharide pattern was obtained when OMP preparations were extracted in several cycles of hot aqueous phenol (AMRESCO) (Sambrook et al., 1989 ) (data not shown). In this present study, polysaccharide components were not purified further, therefore the term ODPA was used. ODPA were stored at -20 °C until use.

Specimen collection and ELISA.
Mice from each treatment group were killed by intraperitoneal injection with 50 µl of 300 mg sodium pentobarbitone ml-1 (Jurox). Blood was collected after death and allowed to clot at room temperature for 1 h before centrifugation at 1500 g for 10 min. Respiratory tract washings (RTW) were obtained by repeatedly (five times) inflating and deflating the lungs with 1·0 ml PBS. Vaginal washings (VW) were obtained by repeatedly (20 times) flushing the vagina with 200 µl PBS. Fresh faecal pellets (10 per mouse) were collected from individual mice before killing at each time point. Faecal pellet extracts (FE) were prepared by homogenizing faecal pellets in PBS (200 µl per pellet) containing 0·05% (w/v) Thimerosal (BDH) and 1 mM phenylmethylsulfonyl fluoride (Sigma). Cells and other debris were removed from RTW, VW and FE by centrifugation (1500 g for 10 min). Serum, RTW, VW and FE were stored at -20 °C.

OMP (50 µl of 25 µg OMP ml-1 in 50 mM bicarbonate buffer, pH 9·6), or equivalently diluted ODPA, were dispensed into 96-well plates (Immulon) and incubated overnight at 4 °C. To determine the optimal concentration of sera and RTW for ELISA, serial twofold dilutions (from 1/100 to 1/800) in TSTw (Tris-saline-Tween 20: 25 mM Tris/HCl in 0·15 M NaCl and 0·05% Tween 20, pH 7·2) were assayed (data not shown). Serial twofold dilutions of RTW (from neat to 1/8 in TSTw) were also assayed (data not shown). From these data, a 1/200 dilution was used to determine the anti-OMP and anti-polysaccharide responses in individual sera and a 1/4 dilution was used for individual RTW. Individual VW and FE were assayed without further dilution. All samples were assayed in triplicate. Optimally titrated horseradish peroxidase (HRP)-conjugated anti-murine antibodies were added, as follows: anti-IgG (1/1000 dilution; Silenus), anti-IgA (1/400 dilution; Kirkegaard and Perry Laboratories), and anti-IgM (1/1000 dilution; Silenus). Plates were incubated for 1·5 h at 37 °C and antibody binding was visualized by adding freshly prepared 0·4% (w/v) o-phenylenediamine dihydrochloride (Sigma)/0·03% (v/v) H2O2 substrate to each well. Colour development was stopped after 10 min by adding 25 µl 2·5 M H2SO4 to each well. Absorbances were measured at 492 nm with a microplate reader.

SDS-PAGE and immunoblotting.
A. pleuropneumoniae cells (100 mg) were reduced in Laemmli buffer by boiling for 5 min. The suspension was clarified by centrifugation. Total subunit preparations of bacteria, OMP or ODPA were electrophoresed by discontinuous SDS-PAGE in a Mini-PROTEAN II assembly (Bio-Rad) under reducing conditions described by Laemmli (1970 ). Electrophoretically resolved OMP polypeptides or ODPA were transferred onto nitrocellulose membranes overnight in a Hoeffer TE series transphor blotting apparatus as described by Towbin et al. (1979 ). Blots were reacted for 1·5 h at 25 °C with either pooled sera (1/500 dilution) or pooled RTW (1/10 dilution) from control, antigen or vaccine groups. Blots were then incubated with a 1/2000 dilution of HRP-conjugated anti-murine immunoglobulin (Silenus) for 1·5 h at 25 °C. Antibody binding was visualized with 0·5 mg 3,3-diaminobenzidine substrate ml-1 (Sigma)/0·03% (v/v) H2O2.

Assessment of recall of immunity by enzyme-linked immunospot (ELISPOT) enumeration of OMP-specific antibody-secreting cells (ASC).
Recall of immunity against OMP was assessed 21 weeks after immunization by inoculating mice intranasally with a subimmunogenic dose (50 ng) of OMP. This dose of OMP was determined in a preliminary experiment by intranasally inoculating anaesthetized naive mice with either 5, 50, 500 or 5000 ng OMP (in 25 µl PBS, pH 7·2). Serological responses to OMP in serum and RTW of intradermally immunized mice were assessed over a 3 week period following intranasal inoculation. Anti-OMP IgG and IgA responses were detected in serum and RTW of mice given 500 or 5000 ng OMP, but were not detected in mice given 5 or 50 ng (data not shown).

The number of OMP-specific IgG- and IgA-ASC in the spleens and collagenase-digested lung tissue of mice were determined over a 3 week period following intranasal inoculation of mice with 50 ng OMP. Spleen cells were released by gently teasing apart the spleen in ice-cold FACS buffer [PBS containing 1% (v/v) foetal calf serum (Life Technologies) and 0·1% (w/v) NaN3, pH 7·2] and pelleted by centrifugation (200 g for 10 min at 4 °C). Erythrocytes were removed by hypotonic lysis in 4·5 ml distilled water. Isotonicity was restored by the addition of 0·5 ml 10xHanks’ balanced salt solution (Trace Scientific).

Single-cell suspensions from pools of four collagenase-digested lung tissue samples were prepared as described by Holt et al. (1985 ). Briefly, the pulmonary vasculature in killed mice was perfused through the pulmonary artery with PBS, pH 7·2. Lungs were then removed, finely minced with a scalpel blade and suspended in 10 ml RPMI-1640 (Life Technologies) supplemented with 10% (v/v) newborn calf serum (NBS; Life Technologies), 150 U collagenase type VIII ml-1, EC 3 . 4 . 24 . 3 (Sigma) and 50 U deoxyribonuclease I ml-1, EC 3 . 1 . 21 . 1 (Sigma). The tissue pieces were shaken at 250 r.p.m. for 90 min at 37 °C on a benchtop flask shaker and then passed through a stainless steel sieve to remove undigested debris.

Spleen and lung cells were washed twice with RPMI-1640/10% (v/v) NBS and resuspended in cell culture medium [RPM1–1640 containing 54 mg sodium benzylpenicillin ml-1 (CSL), 90 mg streptomycin sulfate ml-1 (CSL), 2 mM l-glutamine (Cytosystems), 2 mM sodium pyruvate (Cytosystems), 58 mM ß-melanocyte-stimulating hormone (Sigma) and 10% (v/v) foetal calf serum (Life Technologies)].

ASC were enumerated by the ELISPOT technique (Czerkinsky et al., 1983 ). 96-well nitrocellulose bottom plates (Multiscreen HA 96-well filtration plate, Millipore) were incubated with 100 µl of 50 µg OMP ml-1 in PBS, pH 7·2, at 4 °C overnight. Preliminary experiments were done using spleen and lung cells harvested from mice 28 d after intradermal immunization with SAMA4-OMP. The concentration of cells used for the ELISPOT assay described in the present study yielded detectable, reproducible and readily quantifiable reactivity (i.e. spots) to immobilized antigen. Routinely, 2x105 cells (in 100 µl volume) were added in triplicate to the wells and incubated for either 3 h (for splenocytes) or overnight (for lung cells) at 37 °C in a 5% CO2 humidified incubator. Following incubation, 100 µl of a 1/1000 dilution (in TSTw) of HRP-conjugated anti-murine IgG (Silenus) or 1/400 dilution of HRP-conjugated anti-murine IgA (Kirkegaard and Perry Laboratories) was added to each well.

Intranasal inoculation of mice with A. pleuropneumoniae.
A. pleuropneumoniae serovar 1, strain HS54, was grown on chocolate blood agar plates [4·5% (w/v) GC agar (Oxoid), 1% (w/v) soluble haemoglobin (Oxoid) and 40 µg NAD ml-1 (Calbiochem)] overnight at 37 °C in a 5% (v/v) CO2 incubator. Colonies were inoculated into 20 ml of brain-heart infusion broth (Oxoid) supplemented with 40 µg NAD ml-1 and grown overnight with shaking at 170 r.p.m. at 37 °C. A 5 ml aliquot of the overnight culture was transferred to 95 ml brain-heart infusion broth and incubated as before until an OD650 of 0·8 was reached (early exponential phase); this corresponded to about 8x108 c.f.u. ml-1. Bacteria were centrifuged at 30000 g for 10 min at 4 °C, washed twice with 10 ml sterile saline (0·85% NaCl) and resuspended in 2 ml sterile saline. Mice (24 per group) were anaesthetized and intranasally inoculated with about 1x108 c.f.u. A. pleuropneumoniae in a 20 µl dose.

The number of live bacteria in the inoculum was confirmed by incubating 10 µl aliquots of serial dilutions (in triplicate) of the bacterial suspension on chocolate blood agar plates overnight at 37 °C in a 5% (v/v) CO2 incubator. Bacteria were quantified according to the formula: c.f.u. ml-1=mean no. of coloniesxdilution factorx100

Assessment of bacterial clearance.
Mice were killed at 4, 12 and 24 h after inoculation, and blood, lung and spleen tissues collected. Blood was transferred to sterile 1 ml tubes and anticoagulated with 50 µl sodium heparin. The lungs and spleens were excised aseptically. Individual lungs and spleens were cut into pieces and homogenized for 7x1 min cycles in a Colworth Stomacher 80. Lung or spleen homogenates and blood were diluted in sterile saline and 10 µl aliquots were inoculated in triplicate onto chocolate blood agar plates. Plates were incubated overnight at 37 °C in a 5% (v/v) CO2 incubator.

Statistical analyses.
The means (±sem) of different groups at individual time points were compared by two-tailed t-test. Changes were considered significant at P<0·05.

RESULTS
Serum antibody responses
The serum antibody response against OMP over a 21-week period in mice vaccinated with PBS (control group), OMP (antigen group) or SAMA4-OMP (vaccine group) is depicted in Fig. 1(a–c). Compared to controls, mice in the antigen and vaccine groups elicited a rapid increase in IgG (P<0·005, Fig. 1a), IgM (P<0·02, Fig. 1b) and IgA (P<0·02, Fig. 1c). The rise in IgG was due to increases in IgG1, IgG2 and IgG3 (data not shown). The increases in each of the IgG subclasses were equivalent for both immunized groups and were significant (P<0·01) compared to levels in control mice.



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Fig. 1. Serum ELISA reactivity in control and immunized mice. Mean antibody isotype responses in mice from the control ({square}), antigen ({lozenge}) and vaccine groups ({diamondsuit}) against OMP (a–c) and ODPA (d). Each data point represents the mean (±SEM) ELISA reactivity from 4 mice. Arrows indicate the time of intradermal immunization.

 
Although the peak antibody response in both antigen and vaccine groups was similar, antibody levels declined at a faster rate in the former group. Serum IgG remained elevated for more than 21 weeks in mice from the vaccine group and at this time was significantly higher than in mice from the antigen group (P<0·05).

Intradermal immunization with OMP also stimulated a rapid increase in serum IgG directed against ODPA in both antigen and vaccine groups, but not in the control group (P<0·05; Fig. 1d). An IgA response against ODPA could not be detected over a 21-week period in the serum of mice from different treatment groups (data not shown).

Mucosal antibody responses
Intradermal immunization with OMP primarily induced an IgG response against OMP in RTW from antigen and vaccine groups (Fig. 2a). OMP-specific IgM or IgA was not detected (data not shown). RTW IgG rose significantly, reaching a peak at week 13 (P<0·05). The peak RTW IgG response in the antigen group was significantly higher (2·5-fold) than in the vaccine group (P<0·05) but also declined more rapidly.



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Fig. 2. IgG responses in (a) RTW and (b) FE against OMP, or (c) RTW responses against ODPA, in control ({square}) , antigen ({lozenge}) and vaccine ({diamondsuit}) groups. Each data point represents the mean (±SEM) ELISA reactivity from four mice. Arrows indicate the time of intradermal immunization.

 
A small but significant (P<0·05) IgG response against OMP was detected at week 13 in FE prepared from the vaccine, but not the antigen group (Fig. 2b). No significant rise in the level of OMP-specific FE IgA, or IgG and IgA in VW, was detected over the entire 21-week period (data not shown).

Intradermal immunization with OMP also induced an IgG response against ODPA in RTW of antigen and vaccine groups (Fig. 2c). RTW IgG rose significantly to a peak at week 13 (P<0·05) in the antigen group. A smaller but still detectable (2·5-fold) rise in RTW IgG was also evident at week 13 (P<0·05) in the vaccine group. This rise was significantly less (4·5-fold) than the peak response in the antigen group.

SDS-PAGE and immunoblotting
A typical Coomassie-blue-stained SDS-PAGE profile of A. pleuropneumoniae serovar 1 strain HS54 is shown in Fig. 3(a), lane 2. At least 36 polypeptide bands ranging in size from 14 to 94 kDa can be seen. Purified OMP consisted of five major bands with apparent molecular masses of 14, 22, 33, 37 and 39 kDa (Fig. 3a, lane 3). Outer-membrane polysaccharides derived by proteinase K digestion of OMP preparations (ODPA) were visualized by silver staining (Fig. 3a, lane 4). Two separate smears, one of molecular mass less than 22 kDa and the other of molecular mass greater than 45 kDa, were present. The types of polysaccharides present in OMP preparations were not identified in this study. However, the high molecular mass smear was consistent with capsular polysaccharides (CP) or aggregations of lipopolysaccharide (LPS) (Rapp & Ross 1986 ; Rapp et al., 1986 ; Byrd & Kadis, 1989 ). The low molecular mass smear was consistent with the core oligosaccharide and lipid A components of LPS (Rapp & Ross, 1986 ; Byrd & Kadis, 1989 ).



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Fig. 3. (a) Analysis of A. pleuropneumoniae antigens by SDS-PAGE. Pharmacia low-molecular-mass protein standards (lane 1), HS54 bacteria (lane 2) and OMP (lane 3) were stained with Coomassie blue; ODPA were silver stained (lane 4). The amount of antigen loaded per lane was equivalent to 10 µg protein except for the ODPA preparation, which contained an amount of non-protein antigen derived from 10 µg equivalent of OMP preparation prior to proteinase K treatment. (b) Immunoblot reactivity profiles of pooled serum against OMP (lanes 1, 3 and 5) and ODPA (lanes 2, 4 and 6) obtained from mice in control, antigen and vaccine groups at week 13 post-immunization. (c) Immunoblot reactivity profiles of pooled RTW against OMP (lanes 1, 3 and 5) and ODPA (lanes 2, 4 and 6) obtained from mice in control, antigen and vaccine groups at week 13 post-immunization.

 
The target reactivity of sera (Fig. 3b) and RTW (Fig. 3c) collected at week 13 (peak antibody responses) from mice in different treatment groups was assessed by immunoblotting against either OMP or ODPA. Serum or RTW antibodies from the control group did not react against either OMP or ODPA (Fig. 3b, lanes 1 and 2; Fig. 3c, lanes 1 and 2). When reacted against OMP, pooled serum and RTW from the antigen group showed weaker reactivity against the 22, 26, 28, 33, 36 and 37 kDa OMP polypeptides (Fig. 3b, lane 3; Fig. 3c, lane 3) than the vaccine group (Fig. 3b, lane 5; Fig. 3c, lane 5). Both groups showed strong reactivity against a 14 kDa band.

A 36 kDa polypeptide band present on Western blots was absent from Coomassie-blue-stained gels. The apparent absence of the band on Coomassie-stained gels may be explained in several ways. The intensity of the 37 kDa band on the gel may have masked the presence of a smaller 36 kDa band. Alternatively, the 36 and 37 kDa bands detected by Western blotting may represent antibody reactivity against different exposed immunodominant epitopes of the same protein.

The apparent lack of high-molecular-mass polypeptides in the Western blots was also interesting. Rapp & Ross (1986 ) and others have reported weak or absent antibody reactivities against proteins present on Coomassie-stained gels. Immune competition between antigens, lower immunogenicity of high-molecular-mass proteins, the lower concentration of the higher-molecular-mass proteins in the antigenic mixture compared to the lower-molecular-mass proteins, or variability in the efficiency of transfer and binding to nitrocellulose of high-molecular-mass proteins (Loeb, 1984 ) are possible explanations for the apparent absence of antibodies against the higher-molecular-mass proteins.

When reacted against ODPA, both groups showed strong serum reactivity, but only against the high-molecular-mass polysaccharide antigens (Fig. 3b, lanes 4 and 6). Only the antigen group had strong reactivity against polysaccharide antigens in RTW (Fig. 3c, lane 4). By comparison, the vaccine group had weak reactivity against both high- and low-molecular mass ODPA antigens (Fig. 3c, lane 6).

Enumeration of ASC during the recall response
To determine if intranasal inoculation with a subimmunogenic dose of antigen (50 ng) stimulated an increase in antigen-specific B-cell activity, the number of ASC was determined by ELISPOT. A parallel analysis with mouse spleen cells was also carried out. Before intranasal inoculation, very few antigen-specific IgA-ASC (Fig. 4a, c) or IgG-ASC (Fig. 4b, d) were present in either lung or spleen tissue. However, at 1 week post-intranasal inoculation, the numbers of IgA-ASC or IgG-ASC in the lungs and spleens from both antigen and vaccine groups had risen significantly (P<0·01). Thereafter, the numbers of ASC declined in the antigen and vaccine groups in both lungs and spleens. Although IgA-ASC declined at a slower rate than for other ASC, there was little difference between the two immunized groups at each time point. No increases in OMP-specific ASC were detected in controls.



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Fig. 4. Enumeration of IgA (a, c) and IgG (b, d) OMP-specific ASC in collagenase-digested lung tissues or spleens from mice in control ({square}), antigen ({circ}) or vaccine ({bullet}) groups after intranasal inoculation with 50 ng OMP. Results represent responses in pools of four lungs or spleens. Group means are designated by +.

 
Bacterial clearance in mice
To assess the protective efficacy of antibody responses, mice were intranasally challenged with 1x108 c.f.u. A. pleuropneumoniae, at 5 weeks post-immunization. Bacterial clearance from the lungs of mice was determined at 4, 12 and 24 h after intranasal inoculation with bacteria (Fig. 5). At 4 h the mean number of viable bacteria in the lungs of mice from each group was similar and ranged from 2·4x106 to 6x106 c.f.u. per lung. The number of bacteria in the lungs of control mice increased significantly (eightfold) at 12 h (P<0·005) before declining slightly at 24 h. Control mice rapidly displayed clinical signs of illness within the first 12 h.



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Fig. 5. Pulmonary clearance of A. pleuropneumoniae following intranasal inoculation in control (a) , antigen (b) and vaccine groups (c). Results represent c.f.u. estimates from four individual lungs per experimental group sampled at 4, 12 and 24 h post-inoculation. NG, no growth.

 
Bacteria were recovered from three of four mice in the antigen group at 12 h, with the mean bacterial count being 7x104 c.f.u. per lung. At 24 h, bacteria were recovered from only one of four mice in the antigen group. This mouse was extremely ill and had 7·2x106 c.f.u. per lung. Bacteria were also recovered from three of four mice in the vaccine group at 12 h, with the mean bacterial count being 2·7x105 c.f.u. per lung. At 24 h bacteria were recovered in two of four mice in the vaccine group. The mean number of bacteria in these mice was 9·5x103 c.f.u. per lung.

A. pleuropneumoniae was cultured, but not enumerated, in spleen and blood samples taken from all mice at 4 h. No bacteria were detected in spleen or blood samples taken at 12 or 24 h from vaccinated mice.

DISCUSSION
Immunization with adjuvanted vaccines triggers a range of leukocyte responses different from those seen with non-adjuvanted vaccines (Chin & San Gil, 1998 ; San Gil et al., 1998). The different responses are due to synergistic interaction between antigen and adjuvant and are independent of the antigen used (San Gil et al., 1998 ). Early leukocyte responses, such as those that occur in disease and described for vaccination, are believed to be important modulators of subsequent adaptive immunity (George et al., 1989 ; Naito et al., 1989 ; Gajewski et al., 1991 ; DeKruyff et al., 1992 ; Levin et al., 1993 ; Gong et al., 1994 ; Scardino et al., 1994 ; Dempsey et al., 1996 ; Fearon & Locksley, 1996 ; Mikszta & Kim, 1996 ; Chin & San Gil, 1998 ).

The present study investigated the effects of adjuvant-mediated differences in cellular responses on the development of adaptive immune responses. A chemically heterogeneous and complex antigen, OMP purified from the respiratory pathogen A. pleuropneumoniae, was used. Surprisingly, the inclusion or absence of adjuvant from the vaccine had no effect on the kinetics of IgG, IgA and IgM antibody production and antibody isotype patterns in serum, with both groups eliciting almost identical, strong responses. Both groups also developed IgG responses in the lungs, and re-exposure to a subimmunogenic dose of OMP caused identical increases in IgA- and IgG-ASC in the lungs and spleens of both groups.

These findings contrasted prominently with the responses previously reported for OVA following immunization with or without adjuvant (Chin et al., 1996 ; San Gil et al., 1998 ). Adjuvanted OVA evoked rapid cellular responses in mice that were followed by strong anti-OVA antibody production and strong in vitro OVA-specific cytotoxic T-cell (CTL) responses against OVA-expressing cell lines. OVA alone elicited negligible cellular responses and subsequently evoked no detectable OVA-specific antibody or CTL responses (Chin et al., 1996 ; San Gil et al., 1998 ). The results suggested that adjuvant-mediated changes to leukocyte homeostasis ultimately enhanced adaptive immune responses against thymus-dependent antigens. Unlike OVA, OMP was sufficiently immunogenic to elicit strong antibody responses in the absence of adjuvant and indicated that antigen type also played an important role in determining adaptive immune responses.

At first glance, the results also indicated that the strong cellular responses induced by adjuvant were not necessary to induce strong adaptive immune responses if the antigen was strongly immunogenic. However, several important differences in responses were detected which may be attributed to the adjuvant-mediated effects during vaccination. Compared with the antigen group, the duration of serum and RTW antibody responses in the vaccine group were significantly extended beyond 21 weeks. Examination of FE also revealed an increase in OMP-specific IgG in the vaccine group but not the antigen group. Together, these findings suggested that the cellular response evoked by adjuvant induced proliferation of greater numbers of antigen-specific ASC than with OMP alone. Immunoblot analyses also highlighted a number of important qualitative differences between the antigen and vaccine groups. Serum and RTW from the antigen group reacted more weakly, and to fewer, OMP polypeptides than did the vaccine group. Conversely, although serum from both groups reacted equally strongly against ODPA, RTW reactivity against ODPA was much weaker in the vaccine group than in the antigen group.

The reasons for the difference in reactivity to ODPA in RTW between antigen and vaccine groups are not fully understood. However, the immunoblot results were consistent with those obtained by ELISA. Higher dilutions of both serum and RTW were used for immunoblotting to avoid the blots being ‘swamped’ by excess antibody. The most likely explanation was that the lower concentrations of antibodies in RTW compared with serum allowed better resolution of differences between the groups. The differences in immunoreactivity reinforced the notion that inclusion of adjuvant enhanced antibody responses against protein (thymus-dependent) antigens and shifted the immune responses away from polysaccharide (thymus-independent) antigens.

Since the primary focus of the paper was identifying differences in adaptive responses due to the contribution of adjuvant, the biological significance of differences in antibody reactivity was not investigated. However, the rapid bacterial clearance in immunized mice compared with control mice following intranasal infection indicated that antibodies against OMP were important. Despite differences in antibody specificities, however, no differences in the rate of bacterial clearance or morbidity were apparent between the two immunized groups. Studies with infected swine and mice have shown that protection against A. pleuropneumoniae is principally due to immunity against surface-exposed antigens, such as OMP, LPS and CP (Udeze et al., 1987 ; Fedorka-Cray et al., 1990 ; Byrd & Kadis, 1992 ; Negrete-Abascal et al., 1994 ). These antibodies mediate opsonophagocytosis of bacteria in the lungs (Inzana et al., 1988 ;Thwaits & Kadis, 1991 , 1993 ; Byrd & Kadis, 1992 ; Cruijsen et al., 1992 ; Stine et al., 1994 ). It was likely that enhanced bacterial clearance in immunized mice was also due to the same mechanism. Future studies utilizing vaccines containing purified OMP-derived proteins or polysaccharides, or passive immunization of animals with antisera against each component may determine the relative contributions to protection of each component.

The results of this and previous studies (Chin & San Gil, 1998 ; San Gil et al., 1998 ) indicate that adjuvant-mediated differences in early cellular responses to vaccination are responsible for differences in the subsequent adaptive immune response. However, the different findings with OVA (San Gil et al., 1998 ) and OMP also emphasize that antigen type also directs the development of adaptive immune responses. These findings are especially important because of the widespread use of adjuvants to ensure strong immunological responses to vaccines (Powell & Newman, 1995 ). Knowledge of the effects of adjuvants may provide a rational basis for adjuvant selection and the ability to manipulate immunological outcomes more precisely. Such investigations may minimize the use of adjuvants and lower the incidence of adverse reactions to required adjuvants (Straw et al., 1985 ; Lascelles et al., 1989 ; Powell & Newman, 1995 ).


   ACKNOWLEDGEMENTS
 
This work was supported by funds provided by Johnson and Johnson research.


   REFERENCES
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METHODS
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Received 22 December 1999; revised 26 April 1999; accepted 18 May 1999.



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