Divisions of Bacteriology1 and Pathology2, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702-5011, USA
Author for correspondence: Susan Welkos. Tel: +1 301 619 4930. Fax: +1 301 619 2152. e-mail: welkos{at}ncisun1.ncifcrf.gov
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ABSTRACT |
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Keywords: antitoxin antibody, protective antigen, vaccines, phagocytosis
Abbreviations: AVA, Anthrax Vaccine Adsorbed; BHI, brain heart infusion; EF, (o)edema factor; H, heat-activated, ungerminated; HRP, horseradish peroxidase; LF, lethal factor; PA, protective antigen
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INTRODUCTION |
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METHODS |
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Sera and antibody preparations.
Immune sera from goats, guinea pigs, rabbits and monkeys were obtained from animals vaccinated with AVA, the Sterne strain, or purified PA. Antisera were also obtained from humans vaccinated with AVA. Rabbit antisera designated anti-St. PA and anti-rPA antisera were from animals hyperimmune to PA purified, respectively, from either the native Sterne strain or Sterne-1 CR4 (CR4), a plasmid pX01-cured derivative of Sterne that carries the recombinant plasmid pPA102, consisting of the PA gene from Sterne cloned into plasmid pUB110 (Ivins & Welkos, 1986
; Worsham & Sowers, 1999
). The former had a mean reciprocal anti-PA IgG titre of 1·2x106 and the latter had a titre of 1·35x106, as determined using an antigen-capture ELISA (Little & Knudson, 1986
). IgG was purified by Protein A column chromatography from rabbit anti-St. PA and anti-rPA sera and from normal rabbit serum (Pepper, 1990
). The anti-PA IgG preparations had anti-PA ELISA titres greater than 106. Sera collected from rabbits prior to vaccination (preimmune) and from non-immune animals, and the IgG preparation purified from the latter, had mean anti-PA titres of <50. Monkey antisera were from animals inoculated intramuscularly with two doses of AVA (diluted 1/12·5), given 28 d apart, and were obtained 2 weeks after the second dose. Affinity-purified rabbit anti-PA IgG was obtained by chromatography of anti-rPA antisera over a PA antigen column followed by a Protein A column. Monoclonal antibodies (mAbs) that are specific for two major epitopes on the PA molecule were prepared as described previously (Pepper, 1990
; Little & Lowe, 1991
; Little et al., 1988
, 1996
). The mAb PAI-2D5-1-1 recognizes an epitope between amino acids (aa) 581 and 601 and inhibits binding of LF to cell-bound PA; PA211-14B7-1-1 recognizes an epitope between aa 671 and 721 and blocks the C-terminal cell receptor site for PA.
Preparation of macrophages and in vitro phagocytosis of spores.
Peritoneal exudate macrophages from mice were cultured for 35 d at 37 °C in 5% CO2 on coverslips in 24-well plates, as described by Welkos et al. (1989) . The cells were washed and infected with opsonized spores of B. anthracis (mean of 1x107 c.f.u. ml-1, with a usual m.o.i. of 510 and a range of 320). The spores were opsonized by incubation on ice for 30 min with immune serum or IgG, preimmune serum or IgG, or medium alone. They were then added to macrophages and the culture was incubated for 45 min (at 37 °C in 5% CO2). The 45 min incubation period was the minimum time required to achieve maximal phagocytosis of spores by the C3H/HeN peritoneal macrophages, without observing significant spore clumping in the medium. The coverslips were washed ten times with HBSS (Hanks Balanced Salts Solution) containing 10 mM HEPES, pH 7·0 or 20 mM PBS without Ca2+ or Mg2+ to remove unphagocytosed spores. The coverslips were then removed from the wells and further washed by rinsing sequentially in three 150 mm3 disposable sterile beakers (Falcon) prior to fixation and staining. Phagocytosis was measured by direct microscopic counts of samples stained with WrightGiemsa and spore stains as described previously by Welkos et al. (1989)
. Specifically, the number of spores in
10 fields, and a total of at least 100 macrophages, were counted on each of the duplicate coverslips per sample. All the macrophages in a field, whether infected or uninfected, were included. The mean number of spores per infected macrophage and the percentage of macrophages harbouring one or more organisms were calculated, and the data were expressed as the phagocytic index (PI), where PI=(mean no. of spores per infected macrophage)x(% macrophages containing one or more spores).
Germination of serum-treated spores.
Heat-activated, refractile ungerminated spores of B. anthracis strain Ames (3x108 spores) were incubated on ice for 30 min with serum or IgG diluted in Dulbeccos Minimal Essential Medium (DMEM). The pre-treated spores were then added to germination medium, usually 1% (v/v) BHI in water, and incubated with shaking at 30 °C. At intervals, samples were removed to be read spectrophotometrically (at OD560), tested for heat-resistance, or fixed in 4% formaldehyde for microscopy. An unstained aliquot of the latter was examined with phase-contrast microscopy and a second aliquot was stained with Grams crystal violet and examined microscopically. The heat resistance of spores was determined by incubation of paired samples at 65 °C or on ice for 30 min; and plating dilutions on trypticase soy agar (TSA) plates for viable counts (Levinson & Hyatt, 1966 ). The extent of germination was assessed as described previously by (1) decline in OD560; (2) loss of refractility by phase-contrast microscopy; (3) increase in stainability; and (4) loss of heat resistance (Levinson & Hyatt, 1966
).
Immunoelectron microscopy.
Spores were fixed in 8% (v/v) formaldehyde (Tousimis Research Co.), incubated for 7 d at 4 °C, washed in PBS and processed as described previously (Fritz et al., 1995 ; Ezzell et al., 1990
). Sections were incubated with rabbit anti-rPA antiserum and then goat anti-rabbit IgG conjugated with colloidal gold. They were poststained and examined in a Philips CM100 transmission electron microscope. Negative controls included samples with no primary antibody or with rabbit preimmune serum.
Spore extraction.
Spores from which surface proteins were to be extracted were purified by density-gradient centrifugation and stored in sterile water for injection. Just before extraction, an aliquot containing approximately 4x1095x109 spores ml-1 (1x10103x1010 spores total) was heat-activated by incubation at 65 °C for 30 min and then chilled on ice. Samples of germinated spores were prepared by incubating the activated spores in BHI at 37 °C until germinated. The spore suspensions were centrifuged and the pellets were suspended in a volume of extraction buffer equal to that of the original volume. The extraction buffer consisted of 0·1 M DTT, 0·5% (w/v) SDS, 0·1 M NaCl, pH 10·0 and was prepared as described previously (Vary, 1973 ). Small volumes of spores were placed as aliquots in microtubes (0·6 ml per tube) and the tubes were incubated in a microtube incubator shaker (Eppendorf Thermomixer, Brinkman Instruments) at 1200 r.p.m. Larger volumes were aliquoted into 15 ml plastic tubes and incubated horizontally in a tabletop shaker (LabLine) at 120 r.p.m. The suspensions were incubated for 2·5 h at 37 °C. The spores were then centrifuged in a microfuge for 5 min and the supernatants collected and filtered through 0·2 µm filters. These samples were dialysed, washed in MilliQ water and concentrated three- to fourfold by centrifugation in Centricon-3 or -10 concentrators (Amicon). These preparations were stored at -70 °C until analysed by SDS-PAGE.
Toxin neutralization assay.
An in vitro colorimetric assay (Hansen et al., 1989 ) was used to determine the viability of J774A.1 cells exposed to lethal toxin in the presence of Ab. Antiserum was preincubated with lethal toxin (50 ng PA ml-1 with 40 ng LF ml-1) for 1 h at 37 °C before it was added to the cells. After 4 h, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added and 2 h later the cells were lysed. Absorbance values at 570690 nm were read, the titre was calculated by linear regression analysis (Instat, GraphPad Software), and expressed as the reciprocal of the dilution of antiserum that protected 50% of the J774A.1 cells.
SDS-PAGE and immunoblot analysis.
Samples were electrophoresed on 10% Tricine gels (Novex) by SDS-PAGE under non-reducing conditions. The separated proteins were either visualized by silver stain or electrophoretically transferred to 0·45 µm nitrocellulose membranes (Towbin et al., 1979 ). The membranes were blocked with 5% (w/v) non-fat dry milk in PBS before incubating with rabbit anti-rPA serum, affinity-purified IgG prepared from the latter, a mixture containing two mAb ascitic fluids to PA (described above), or preimmune rabbit serum. The membranes were washed in PBS containing 0·1% (v/v) Tween-20 before incubation in horseradish peroxidase (HRP) conjugated to goat antiserum against either rabbit or mouse IgG. Reactive components were visualized by enhanced chemiluminescence (ECL; Pierce).
Statistical analyses.
The data were analysed by standard statistical methods [means, standard errors of the means (SEM), analysis of variance and unpaired Students t-tests]. Macrophage phagocytosis values were expressed as the PI (described above). In germination inhibition experiments with the purified IgG from Ra anti-St. PA and monkey anti-AVA antisera, the data analysed were the mean values (±SEM), for seven and four experiments, respectively, for spores treated with the dilution of purified IgG from the antiserum that maximally inhibited germination (compared to spores treated with identically diluted preimmune IgG or serum). In comparing groups, a P value of 0·05 was considered to indicate a significant difference.
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RESULTS |
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Pretreating heat-activated, ungerminated spores (H) of Ames with rabbit antisera to PA purified from recombinant Sterne strain CR4 (Fig. 1a, sample 1) or from non-recombinant Sterne (Fig. 1a
, sample 2) significantly enhanced the uptake of spores by the peritoneal macrophages over uptake by preimmune serum (Fig. 1a
, sample 3). In contrast, the treatment of the pX01-
Ames strain (
Ames-H) with the antisera did not have a significant opsonizing effect on the spores (Fig. 1a
). The phagocytosis-stimulating activity of sera from rabbits vaccinated with a toxigenic live vaccine was previously reported to be determined largely by the IgM fraction of the immune sera (Stepanov et al., 1996
). However, we found that the IgG fraction purified from the rabbit anti-St. PA antiserum also had a phagocytosis-enhancing effect when compared to the uptake of spores pre-treated with comparable dilutions of preimmune IgG (data not shown). In addition to rabbit antisera, the sera from two of three monkeys vaccinated with the human AVA vaccine had phagocytosis-enhancing activities greater than those of preimmune or normal sera (Fig. 1b
).
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Although antitoxin Abs appear to play a major role in the enhancement of spore phagocytosis, other factors might be involved. The existence of the latter was suggested, for instance, by the enhanced uptake of pX01+ spores by antisera elicited in guinea pigs by the pX01- Sterne-1 strain (data not shown), and by the incomplete correlation between anti-PA Ab titre and the phagocytosis-enhancing activity of monkey antisera. Although the antiserum obtained from AVA-vaccinated monkeys 1 and 3 had anti-PA titres of 12800 and 800, respectively, the antiserum from monkey 3 had more phagocytosis-enhancing activity than did that from monkey 1 (Fig. 1b
). The toxin-neutralizing titres of the sera (1157 and 299, respectively, for monkeys 1 and 3) also did not correlate with the anti-spore activity of the sera.
Inhibition of spore germination by immune sera and anti-PA IgG
Incubation in BHI, or in BHI diluted in water to 2% or more, rapidly induced complete germination of both heat-activated and unheated spores of the Ames strain. There were no detectable differences in the rates of germination in this medium between spores preincubated with or without immune serum (data not shown). Heat-activated or unheated Ames spores pre-treated with water or preimmune sera and then incubated in 1% BHI exhibited up to 80% germination after 30 min at 30 °C. Most of the spores that germinated did so within 10 min. In contrast, pretreatment of spores with rabbit anti-rPA serum, purified rabbit anti-PA IgG and monkey anti-AVA sera was associated with the inhibition of spore germination (Fig. 2 and data not shown). Anti-PA-or anti-AVA-treated spores demonstrated significantly smaller changes (percentage decrease) in OD560 (Fig. 2a
) and fewer stained spores than did the samples pre-treated with preimmune serum or water alone (Fig. 2b
).
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Effect of anti-PA antiserum on loss of heat resistance during spore germination
When germination was assayed by the decrease in OD560 and increase in staining, the effect of pretreating spores with sera as measured by one assay was mirrored by a comparable change in the other assay. Different results were obtained when germination was measured by the loss of heat resistance. As expected, a higher percentage of spores treated with preimmune serum (>67%) than of anti-PA-treated spores (<30%) became stainable within 5 min of incubation in 1% BHI. However, no significant differences in resistance to killing of spores by heat were conferred by pretreatment with anti-rPA compared to preimmune serum (data not shown). Regardless of pretreatment, the loss of heat-resistance began within seconds, i.e. the viability after heating of samples collected immediately after transfer to BHI decreased by 1·5- to almost 10-fold compared to paired unheated samples. The basis of the apparent inability of anti-PA Abs to inhibit germination as assayed by changes in heat resistance is not known. Perhaps, as described for Bacillus megaterium (Levinson & Hyatt, 1966 ), Ames strain spores lose heat resistance first upon exposure to germinant, before the germination-inhibitory activity of anti-PA can have an effect.
Immunoelectron microscopy
The phagocytosis-enhancing and germination inhibition effects of sera containing anti-PA Abs suggested that PA or an antigenically similar protein might be present on the surface of the ungerminated spores. To detect anti-PA reactivity, activated spores of Ames strain were incubated with rabbit anti-rPA Ab or preimmune serum and PA was detected with goat anti-rabbit IgG conjugated to gold particles. As shown in Fig. 3, spores preincubated with the anti-PA Ab (Fig. 3a
) bound significantly more gold particles than those treated with the preimmune serum (Fig. 3b
). There were no significant differences in the extent of binding of the Abs by germinated and ungerminated spores, as determined by counting the gold particles on the spore surfaces (data not shown).
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Gel electrophoresis and immunoblot analysis
Proteins were extracted from intact spores with the DTT/SDS/NaCl extraction reagent and analysed by SDS-PAGE and immunoblotting. We observed a large number of proteins on silver-stained SDS-PAGE gels (data not shown). However, only extracts of the PA-producing strains yielded a band on the immunoblot recognized by the affinity-purified rabbit anti-rPA IgG and equal in size to the 83 kDa PA standard. The yield of the 83 kDa anti-PA-reactive material from the spore surface appeared to vary with the stage of germination of the spores at the time of extraction and with the strain. Extracts from germinated spores of Sterne, Ames, ANR-1 (the toxigenic unencapsulated derivative of Ames) and the PA+ LF- Sterne mutant RP42 reacted with anti-PA IgG on the blots (Fig. 4a, lanes 3, 5, 7 and 8, respectively). Extracts prepared from ungerminated spores from the same batches did not (data not shown). The extracts from the germinated spores of the PA+ and PA- strains also frequently yielded bands recognized by the rabbit anti-rPA IgG that did not co-migrate with 83 kDa PA (Fig. 4a
, lanes 39). Some may represent proteolytic fragments of PA (in PA+ strains); others may be the same as the entities recognized by anti-PA Ab in the extract from
Ames (Fig. 4a
, lane 6), a PA- pX01- derivative of Ames strain (Fig. 4a
, lane 6), and might represent cross-reacting proteins.
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When the immunoblot of extracts was developed using a mixture of PA mAbs, the results verified the presence of 83 kDa PA (Fig. 4b, lanes 3, 5, 7 and 8). It was present in low concentration in RP42 and appears as a light, 83 kDa band (Fig. 4a
, b
, lane 8).
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DISCUSSION |
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Protection can be induced by vaccination with live vaccine strains or component vaccines containing PA such as AVA (Ivins et al., 1996 ; Pitt et al., 1996
, 1999
). Recent studies suggest that the titre of anti-PA Ab in immune animals correlates with protection (Pitt et al., 1999
, unpublished data). Rabbits and monkeys vaccinated with AVA and challenged with virulent strains of B. anthracis only occasionally become detectably bacteraemic (Pitt et al., 1996
; B. Ivins & P. Fellows, unpublished data). The mechanism of protection against the early stages of infection afforded by AVA and other PA-based vaccines and the role of the antitoxin immune response are not fully elucidated.
Stepanov et al. (1996) reported that sera from animals vaccinated with an unencapsulated, toxin-producing, live vaccine strain had activities directed against the spores which may be important during the early stages of infection. Specifically, (1) germination was reported to be inhibited when the spores were incubated with antitoxin Abs (IgG fraction); and (2) the phagocytosis of spores by macrophages was stimulated by immune sera (IgM fraction) from animals vaccinated with the live spore vaccine. Our objectives were to develop in vitro systems for assaying the anti-spore activities of immune sera and to characterize these activities further using various antibody preparations. In agreement with the observations of Stepanov et al. (1996)
, made by using rabbit peritoneal macrophages and spores of B. anthracis, we found that sera from immune animals enhanced phagocytosis by murine peritoneal macrophages. The uptake of spores of both the fully virulent Ames strain and the pX02- Sterne vaccine strain was enhanced by preincubating spores with antisera to purified PA (Fig. 1
). Stepanov et al. (1996)
indicated that rabbit anti-anthrax sera promoted spore phagocytosis and we observed comparable activity in the antisera from this species (Fig. 1a
, c
) and from other species, such as monkeys vaccinated with AVA (Fig. 1b
). Although phagocytosis was attributed primarily to IgM Abs by Stepanov et al. (1996)
, we showed that anti-PA IgG Abs had significant activity.
The stage of germination when B. anthracis initiates synthesis of the toxin components, and when the spores become amenable to anti-PA stimulated phagocytosis, is not known. The spores do not germinate in the culture medium alone (DMEM) used in the macrophage phagocytosis experiments. However, the addition of animal sera from some species supports germination; e.g. the addition of fetal bovine serum, but not of horse or mouse serum, to DMEM stimulates germination of Ames spores (S. Welkos, unpublished data). Regardless of this, we observed that spores that are ungerminated (refractile) were phagocytosed at least as well as those that had completely germinated prior to phagocytosis (data not shown). Also, we demonstrated by immunoelectron microscopy the presence of an anti-PA reactive entity on the surface of ungerminated spores. We are currently pursuing studies to determine the kinetics of expression of the PA-reactive substrate during the process of germination and outgrowth.
Phagocytosis of the infecting spore appears to be a major step in the pathogenesis of inhalational anthrax, as reviewed recently (Dixon et al., 1999 ; Hanna & Ireland, 1999
). Ross (1957)
showed that inhaled spores were phagocytosed by alveolar cells and were either cleared locally or were transported within the macrophages to the regional lymphatics where they germinated and outgrew. Guidi-Rontani et al. (1999)
showed more recently that alveolar macrophages from infected mice can similarly phagocytose spores of B. anthracis and that the spores germinate within the phagocyte. Germination of the spores upon phagocytosis in vitro by peritoneal macrophages or macrophage-like tissue culture cells has also been demonstrated (Dixon et al., 1999
; Hanna & Ireland, 1999
; S. Welkos and others, unpublished data). The subsequent fate of the germinated spores is not well defined. Scenarios depicting both the outgrowth and release of the organisms (Hanna & Ireland, 1999
; Dixon et al., 2000
; J. Ireland, unpublished data) and the loss of viability of the phagocytosed spores (Guidi-Rontani et al., 1999
; Welkos et al., 1989
; S. Welkos and others, unpublished data) have been reported.
An antitoxin-mediated enhancement of phagocytosis could potentially contribute to the protection from infection induced by AVA vaccination. In recent studies on the effect of anti-PA Abs on the uptake and fate of phagocytosed spores, we observed that treating spores prior to phagocytosis was associated with an increased extent of intracellular germination and subsequent enhanced rate of killing (S. Welkos and others, unpublished data). The induction of such anti-spore activities in the vaccinated host might contribute to a protective immune response.
As reported by Stepanov et al. (1996) , another anti-spore activity associated with antitoxin antisera was the inhibitory effect on spore germination. Ames strain spores pre-treated with rabbit anti-rPA antiserum exhibited a significantly smaller decrease in OD560 and fewer stained spores after incubation than did the control samples (Fig. 2a
, b
and data not shown); similar differences were observed when germination was assessed by the loss of refractivity, but not by the loss of heat resistance (data not shown). The inhibition of germination could potentially be another Ab-mediated protective mechanism. If extracellular germinants are present at the inoculation site, the spores might be able to germinate and the bacilli multiply outside the macrophage. Abs directed to the spore might retard this process until activated macrophages arrive and phagocytose the spores. Further in vivo studies to define the sites (i.e. inside or outside of phagocytes) and stimulants of spore germination in the host are needed.
We are characterizing the entity on the surface of spores that is the target of the anti-spore immunity. It appears to be homologous antigenically to PA, but could be a variant of the toxin protein or a different cross-reactive surface protein. There also may be more than one target. We identified potential candidates by gel electrophoresis and immunoblotting of spore extracts. Extracts of germinated spores from all PA-producing strains except Vollum1B reacted with anti-PA Abs on immunoblots (Fig. 4); and they each produced an 83 kDa protein recognized by mAbs to PA (Fig. 4b
). In contrast to results using germinated spores, extracts from ungerminated spores from the same batches did not contain a detectable anti-PA reactive protein (data not shown). However, when binding of anti-PA Abs by spores was studied by electron microscopy (Fig. 3
), there were no detectable differences in the extent of binding of the Abs by germinated and ungerminated spores. Thus, although anti-PA-reactive material appears to be present in both germinated and ungerminated spores, it appears to be easier to extract or present in higher concentration in the former compared to the latter spore stage. The procedure used for spore extraction is optimal for extracting the spore coat proteins from several bacilli (Vary, 1973
; Aronson & Fitz-James, 1976
) and also solubilizes exosporium preparations (Beaman et al., 1971
; DesRosier & Lara, 1984
). In addition to the 83 kDa PA, other entities appeared to be recognized by the anti-rPA antiserum in immunoblots. Thus, further studies are being performed to elucidate several parameters, including: (1) the kinetics of PA expression in anthrax spores; (2) the localization of PA in the outer spore surfaces (i.e. the spore coats and the exosporium); and (3) the identities of all anti-PA-reactive antigens.
In summary, antisera to PA had an anti-spore effect(s) that was detectable by at least two activities: (1) the stimulation of phagocytosis by macrophages of spores of the Ames and Sterne strains; and (2) the inhibition of spore germination in 1% BHI. Activity was shown with rabbit antisera to recombinant and non-recombinant PA, IgG purified from rabbit antisera to PA, and monkey antisera to the human AVA vaccine. PA or PA-like antigen was detected by electron microscopy on immunogold-labelled spores, and proteins extracted from spores reacted with anti-PA IgG and mAbs on Western blots. It is conceivable that the anti-spore effect(s) of antitoxin antibodies in individuals vaccinated against anthrax might be protective early in infection before outgrowth and toxin secretion by bacilli. Our goals are to identify the genes encoding the spore proteins reacting with anti-PA Abs and to determine the role of the antitoxin-mediated anti-spore activity in early protection against infection.
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ACKNOWLEDGEMENTS |
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Received 24 August 2000;
revised 3 January 2001;
accepted 29 January 2001.