INFECTION AND IMMUNITY, Mar. 1990, p. 680-686

Vol. 58, No. 3

0019-9567/90/030680-07$02.00/0 Copyright X) 1990, American Society for Microbiology

Polyclonal Anti-Idiotypic Antibodies Exhibit Antigenic Mimicry of Limited Type 1 Fimbrial Proteins of Escherichia coli RONALD E. PAQUE,* RUTH MILLER, AND VIRGINIA THOMAS Department of Microbiology, The University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7758 Received 25 July 1989/Accepted 4 December 1989

Polyclonal anti-idiotypic antibodies (anti-Ids)(fim) developed against idiotypes on antibodies (Ab-1s) that specifically bind structural, organelle fimbrial proteins of Escherichia coli were able to modulate immune function in anti-Id(fim)-immunized mice. Proliferation or suppression of splenic lymphoid cell responses by polyclonal anti-Ids in tissue culture appeared to be dose dependent. Anti-Ids were able to induce a dose-dependent T-cell-mediated immunity specific for type 1 fimbrial antigen(s) in immunized animals when assessed in vitro, but they failed to elicit in vivo positive ear-swelling skin reactions. Anti-Ids were unable to induce protective immunity against an in vivo infectious challenge with E. coli in anti-Id-immunized adult animals, but they stimulated a specific, secondary antibody response in anti-Id-challenged mice. Anti-Ids stimulated the development of anti-anti-Ids (Ab-3s) specifically binding a fimbrial antigen(s) and revealed the presence of antibody idiotypes binding E. coli adhesin proteins in the 27- to 29-kilodalton range. Results suggest discrete, but subtle, immunomodulatory effects of the anti-Ids and potential vaccinoid properties capable of stimulating a specific humoral and cellular response in vivo.

Jerne's (12) original proposal of antibody idiotypic network regulation suggested that antigenic determinantbinding antibodies (Ab-1s) and antibodies against Ab-1 idiotopes (anti-idiotypes [anti-Ids] [Ab-2s]) belong to the same family of molecules that act in vivo in a regulatory fashion on the immune system (12, 13). Nisonoff and Lamoyi (19) proposed that such anti-Ids, or antibodies against internal image idiotypes, would represent molecular images of the original antigenic determinants and that such antibodies (Ab-2s) might act as immunogens in molecular mimicry of antigenic determinants associated with the original immunogen/pathogen (9, 13, 16, 19). Indeed, recent studies by several groups of investigators, including ourselves (15, 16, 21), have reported on the vaccinic/protective characteristics of polyclonal or monoclonal anti-Ids developed against idiotypes binding bacterial cell wall (18, 30, 31), protozoan membrane antigen (28, 29), or viral capsid and/or envelope (15-17, 20, 22) determinants. Type 1 fimbriae/pili are elongated, hairlike appendages that are produced by Escherichia coli as well as by other members of the family Enterobacteriaceae. Type 1 fimbriae mediate adherence to mannose-containing receptors on mammalian mucosal surfaces and, thus, have been suggested to be key factors mediating pathogenicity and virulence in these organisms (1-3, 8, 10, 19). The protein and structural nature of type 1 fimbriae and their principle role in bacterial adherence or virulence (2, 3, 8) suggested to us that such proteins might be reasonable and novel candidates for development of experimental, anti-idiotypic vaccines (16, 28, 30, 31) designed to specifically mimic adhesive properties of type 1 fimbriae, including the important mannose binding protein subunit, FimH (2). Mimicry of fimbrial adhesins by anti-Ids might permit vaccinic utilization of adhesin epitopes free of immunosuppressive, fever-producing endotoxins naturally associated with fimbrial antigen/adhesins. Moreover, specific anti-Ids representing nonendotoxic internal images (16, 25) of fimbriae and/or their subunits might offer specific *

immunological probes for understanding host idiotypic-antiidiotypic immune modulation operative during gram-negative bacterial infections (4-6, 11, 24, 25, 27). In this report, we describe some of the immunomodulatory and vaccinoid characteristics of polyclonal anti-Ids directed against idiotopes binding type 1 fimbrial antigens of E. coli. Evidence is presented that anti-Ids affect several parameters of the murine immune response to fimbrial proteins and that these anti-Ids demonstrate mimicry for at least one adhesin molecule. MATERIALS AND METHODS Mice and immunizations. BALB/c mice were purchased from Harlan Sprague-Dawley Laboratories, Indianapolis, Ind., and bred in University of Texas Health Science Center laboratory animal facilities. Donors of anti-fimbrial antibodies (anti-fim) were immunized subcutaneously with 20 to 50 jxg of type 1 fimbrial protein in Freund complete adjuvant, followed by weekly booster doses of antigen intraperitoneally (i.p.) for at least 6 to 8 weeks. BALB/c donors of anti-Ids were hyperimmunized subcutaneously with 50 ,ug of protein A affinity-purified immunoglobulin G (IgG) emulsified with Freund complete adjuvant. Groups of mice (three to five mice each) were boosted weekly with i.p. injections of 50 ,ug of IgG for 10 to 12 weeks and bled weekly after the sixth injection via the retro-orbital sinus, and antibody titers were assessed by the enzyme-linked immunosorbent assay (ELISA). Similar procedures were used to produce antianti-Ids (Ab-3), in this case immunizing mice with anti-Id IgG. Type 1 fimbrial antigen preparation. E. coli K-12 strain CSH50 was used as a source of type 1 fimbriae. An isolated colony of CSH50 from Trypticase soy agar was inoculated into a 1-liter tissue culture flask containing 50 ml of trypticase soy broth. For optimal piliation, the flask was placed flat on the incubator rack to allow maximal aeration of the broth culture with oxygen. The cells were incubated statically at 37°C overnight. Then, 1.0-ml amounts of the broth culture were transferred to each of eight 6-liter flasks con-

Corresponding author. 680

VOL. 58, 1990

taining 1 liter of Trypticase soy broth, which allowed for appropriate aeration of cultures. Bacteria were incubated statically overnight at 37°C. Pili were purified by the methods of Dodd and Eisenstein (7). Bacteria were centrifuged for 10 min at 10,000 x g at 4°C and washed with 200 ml of 0.5% NaCl. The pellet was then suspended in 100 ml of Tris buffer (pH 7.4). Fimbriae were removed by homogenizing for 5 min in a Virtis 45 homogenizer at 4°C, followed by two additional cycles of 30 s each. Bacteria were pelleted by centrifugation at 3,000 x g and washed twice with 60 ml of Tris buffer, combining supernatants from each pool. Supernatants were then centrifuged at 27,000 x g for 30 min, followed by ultracentrifugation at 227,000 x g for 2 h. The gelatinous fimbrial pellet was suspended in 8 ml of 5 M urea buffer and mixed for 2 h at 37°C. After the pellet and buffer were mixed, 32 ml of Tris buffer was added and 10-ml samples were layered in centrifuge tubes onto 10-ml Tris cushions consisting of 1 M urea, 1 M sucrose, and 5 mM Tris. Samples were centrifuged for 16 h at 200,000 x g, and the pellets were suspended in Tris buffer. DNP-ovalbumin preparation. 2,4-Dinitrophenol (DNP)ovalbumin was prepared by conjugation of 0.2 ml of dinitrofluorobenzene (DNFB) to 500 mg of ovalbumin (Sigma Chemical Co., St. Louis, Mo.) as described earlier (22, 23). Briefly, ovalbumin was dissolved in 100 ml of 0.85 M saline with DNFB by stirring at 25°C for 30 min. Sodium bicarbonate was slowly added to a pH of 8.0, and the solution was left to stand for 2.0 h at 25°C. The preparation was then refrigerated at 4°C overnight and dialyzed the next day in a 20:1 volume of 10 changes of 0.85 M saline. Preparations were dispensed in 0.5-ml increments and stored at -20°C until used. Preparation of spleen cell suspensions. BALB/c spleens were surgically extirpated from each experimental animal. The spleen tissue was finely minced with sterile, sharp scissors and expressed with a small syringe plunger through a 40-mesh stainless steel screen with RPMI 1640 medium. Expressed cells were gently aspirated with a Pasteur pipette, collected in 15-ml centrifuge tubes, and washed three times at 500 x g. After centrifugation, cells were suspended and then counted in a hemacytometer before being set up in a tritiated thymidine incorporation assay. Protein A separation of IgG fractions. Serum antibodies were purified by protein A-Sepharose (Sigma) separation. One gram of Sepharose was swollen in phosphate-buffered saline (PBS) to a volume of 3.5 to 4.0 ml. Sepharose gel was packed in a Bio-Rad Econo (Bio-Rad Laboratories, Richmond, Calif.) column (7 mm by 30 cm) and washed several times with PBS. Samples were diluted 1:5 in PBS, adjusted to pH 7.5, and layered on the gel surface. Samples were permitted to run into the column, and 2.5 to 5.0 ml of eluate was collected. After a washing, the IgG was eluted with 0.1 M acetic acid (pH 4.5) and immediately neutralized. Collected fractions were analyzed at 280 nm with a Beckman model DB-G spectrophotometer. The peak of absorbance was collected, and the amount of protein IgG was calculated from the optical density reading at 280 nm. Fab preparation. F(ab')2 fragments of Ab-1 or anti-Ids (Ab-2) were prepared by methods described earlier (22, 23). Briefly, serum IgG (3.0 mg) was dissolved in 0.14 M sodium acetate-sodium chloride buffer with 60 mg of pepsin at pH 4.0. Mixtures of antibody and enzyme were incubated for 18 h at 37°C and adjusted to pH 8.0 with 0.1 M NaOH. The preparation was then dialyzed against 0.15 M saline-borate



buffer at pH 7.5 with a molecular weight cutoff of 60,000, yielding 53% of F(ab')2 fragments. Affinity chromotography. Cyanogen bromide Sepharose 4B (Pharmacia, Inc. Piscataway, N.J.) was utilized for coupling anti-type-1(fim) IgG in purification and characterization of the anti-Ids. One gram of gel was swollen in 0.003 M HCl and washed for 15 min on a glass filter. Next, 500 ,ug of serum per ml was dissolved in 5 ml of coupling buffer consisting of 0.1 M NaHCO3 and 0.5 M NaCl and mixed with the swollen gel. The mixture was rocked slowly at 25°C for 60 min, and excess ligand was washed away with buffer. Remaining active groups were blocked with 0.1 M Tris hydrochloride buffer (pH 8.0). The gel was washed three times with cycles of 0.1 M acetate buffer (pH 4.0) and 0.1 M Tris buffer (pH 8.0). Coupled Sepharose (5 ml) was suspended in the column with twice the volume of PBS and allowed to incubate for 30 min at 37°C. Samples containing anti-Id were applied to the column and permitted to flow for 30 min. Eluate was washed over the column twice, and the column was washed with 200 ml of PBS. Anti-Ids were eluted by suspending the Sepharose and gently agitating it in 3 M MgCl2. This procedure was repeated twice, and the eluate was collected, dialyzed overnight against two changes of distilled water to remove excess salt, and suspended in PBS. Cell migration inhibition assay. Murine peritoneal exudate cells (PEC) were obtained from anti-Id-immunized mice 3 days after the injection of 2.5 ml of light peanut oil. Cells were collected by flushing the peritoneal cavity with Hanks balanced salt solution and were washed three times by centrifugation at 145 x g. After the final wash, cells were immediately suspended in a 1:4 ratio of cells (106) in a 2x concentration of tissue culture medium 199 containing 40% fetal calf serum mixed with an equal volume of 0.4% melted agarose (Marine Colloids, Rockland, Mass.) at 37°C. The cell suspension was dispensed in 2-,ul droplets on 96-well flat-bottom microtiter plates (Falcon Plastics, Cockeysville, Md.). Droplets were permitted to solidify at 4°C for 7 min in the cold, after which 0.2 ml of RPMI 1640 medium containing 20% fetal calf serum was added to each well. Antigen concentrations in the media included graded doses of antiIds, fimbrial antigen, or DNP-ovalbumin. Droplets were then covered and incubated at 37°C in 7% CO2 for 36 h. After 36 h, the droplets were examined for cell migration under an inverted microscope fitted with an ocular grid (100 x 100 mm). The number of squares of cellular migration from four equidistant points at the edge of each droplet were counted in double-blind fashion and recorded. Mean migration indices (MMI) were calculated according to a previously published statistical formula (21). Cell proliferation assay. Graded concentrations of antigens or anti-Ids were dispensed in RPMI tissue culture medium and added to spleen cells suspended at a concentration of 5 x 105 cells per ml in 95-microwell tissue culture plates. Cultures were incubated for 48 to 72 h at 37°C in 7% CO2. After 48 h, cultures were pulsed with diluted tritiated thymidine at 50 mCi/ml for 24 h. Cells were harvested with an Ilacon Microcell harvester on glass fiber filter paper (Whittaker M.A. Bioproducts, Walkersville, Md.). Filters containing harvested cells were deposited into numbered liquid scintillation vials and treated with tissue solubilizer to digest the cells at 56°C for 3 h. Scintillation fluid was added to the solubilized cells, and the vials were counted in a Tracor Analytic Model 6895 liquid scintillation counter. ELISA. Plastic 96-well microtiter plates were coated overnight with 2 ,ug of Fab fragments, fimbrial antigen, or



INFECT. IMMUN. present in our Ab-1 affinity-purified antibody populations and which were utilized to generate the anti-Ids(fim). Fimbrial proteins (25 to 75 jig) were layered on a sodium dodecyl sulfate gel and subjected to slab gel electrophoresis with appropriate molecular weight markers. Next, the separated gel proteins and marker bands were transferred to nitrocellulose, probed with affinity-purified Ab-1 antibodies, and finally developed by using a Western blot-grade goat anti-mouse IgG (Bio-Rad). Results are shown in Fig. 1. Ab-1 specifically bound six different protein bands, with the greatest intensity of staining in two bands in the 27-kilodalton (kDa) range and with one band, nearest the bottom of the gel, in the 17-kDa range. More faintly staining highmolecular-weight bands are shown at the top of the gel. Controls using Ab-ls developed against an unrelated immunogen, DNP-ovalbumin, failed to bind any of the type 1 fimbrial proteins (Fig. 1). Quantitation and specificity of affinity-purified polyclonal anti-Ids. Various groups of syngeneic BALB/c mice were hyperimmunized with affinity-purified antibodies (Ab-1s) specific for purified fimbrial proteins. Antibody titers of four separate pools of sera from each group ranged from 1:256 to 1:1,024 as assessed by ELISA. Seven days after the last boost with Ab-1, the anti-Id donors were bled. The resulting Ab-2 was purified by protein A affinity separation and titrated against F(ab')2 fragments of Ab-1, normal IgG, or F(ab')2 of idiotypes binding an unrelated immunogen DNPovalbumin. Results are shown in Table 1. Diluted preparations of each pool of protein A-separated anti-Id(fim) bound F(ab')2 fragments of Ab-1s; absorbance values ranged from 0.109 to 0.260, and titers ranged from 1:32 to 1:128 (Table 1). On the other hand, when these same preparations were tested against F(ab')2 fragments of Ab-1 binding an unrelated immunogen, DNP-ovalbumin, ELISA absorbance values with negligible titers were similar to control values (Table 1). Dose-response assessment of anti-idiotypes (fim) on stimulation and proliferation of anti-Id immunized murine spleen cells. To assess the immunomodulatory effects of polyclonal anti-Ids(fim) on lymphoid cell populations, spleen cells from anti-Id(fim)-immunized animals were set up in a tritiated thymidine cell proliferation-stimulation assay. Cells were incubated with anti-Ids(fim) or fimbrial antigen for 48 h, pulsed with radiolabel, and assessed for cell proliferation and stimulation. Results are shown in Fig. 2. The proliferation of lymphoid cells from anti-Id-immunized animals was markedly dependent on the amount of anti-Id(fim) incubated with spleen cell populations. For example, 500 ng of purified anti-Id(fim) per ml caused increased stimulation of lymphoid cells, with stimulation indices (SIs) ranging from 2.5 to 5.5 (Fig. 2). On the other hand, doses of anti-Ids(fim) with 10 ,ug of anti-Id(fim) per ml resulted in suppression of lymphoid cell stimulation and resulted in SIs comparable to those observed with nonspecific anti-Ids and/or controls (Fig. 2). were





A 13



FIG. 1. Immunoblot analysis of antibodies against type 1 fimbrial proteins. Lanes A, B, C, and D, Graded doses of 75, 50, 30, or 25 ,ug, respectively, of type 1 fimbrial proteins electrophoresed and probed with protein A-purified Ab-1. Binding of Ab-1 was assessed by using a peroxidase-conjugated goat anti-mouse IgG immunoblot grade antibody. The two intense bands of staining indicate migration corresponding to molecular weight marker controls at 27 kDa (not shown). The right side of the gel represents controls with Ab-ls developed against DNP-ovalbumin, an unrelated immunogen.

DNP-ovalbumin per well. The plates were coated with a 3% solution of bovine serum albumin as a filler and incubated overnight with the test antibodies. After incubation, the plates were washed 20 times in distilled water and incubated for 3 h with a horseradish peroxidase-conjugated goatanti-mouse Fc fragment. After incubation with the antimouse Fc serum, plates were washed 20 times and substrate was added to detect the presence of the enzyme and the binding of the anti-mouse Fc to the test antibodies. Intensity of color development was assessed with an MR580 MicroELISA Autoreader (Dynatech, Alexandria, Va.). Wells with color changes of 0.05 or above were considered positive on the basis of a statistical analysis of the assay with 100 negative wells containing all reagents in the assay except specific antiserum. Negative values ordinarily range from 0.000 to 0.050. RESULTS Fimbrial protein analysis. Western immunoblot analysis was carried out to assess the idiotypic specificity(ies) which

TABLE 1. ELISA assessment of anti-Ids against AB-ls specifically binding type 1 fimbria proteins of E. coli CHS50 Anti-Id prepn

Saline control

MicroELISA abosrbance values of anti-Id (Ab-2) tested against:

F(ab')2 Ab-1 fimbria

1 2 3 4

0.003 0.010 0.048 0.039

0.109 0.223 0.176 0.260

(1:128)a (1:32) (1:128) (1:64)



0.064 0.035 0.021 0.038

(1:2) (1:2) (1:4) (1:4)

Type 1 fimbrial antigen

0.029 0.067 0.049 0.051

(1:4) (1:8) (1:2) (1:4)


0.041 0.027 0.025 0.055

(1:5) (1:2) (1:4) (1:8)

Normal F(ab')2

0.060 0.069 0.071 0.063

(1:2) (1:4) (1:8)


a Represents the absorbance reading and the last dilution (endpoint) in the test with detectable, positive ELISA absorbance readings in a doubling dilution titration sequence.


VOL. 58, 1990

iments, the amounts of anti-Ids failed to stimulate isolated T cells and SIs were similar to control values, ranging from 1.3 to 1.5. Assessment of delayed hypersensitivity or cellular immunity in BALB/c mice hyperimmunized with anti-Ids(fim). Because preliminary experiments demonstrated the ability of polyclonal anti-Ids to stimulate a B-cell response of specific anti-fimbrial antibodies, (see Table 4), anti-Ids were assessed for their ability to stimulate T-cell immunity in vivo. BALB/c mice hyperimmunized with polyclonal anti-Ids were injected with light mineral oil i.p., and PEC were harvested three days later. PEC were set up in the agarose droplet cell migration inhibition assay/correlate of delayed sensitivity and tested for cellular inhibition in the presence of graded doses of anti-Id(fim), type 1 fimbrial antigen, and anti-Id (DNP-ovalbumin). Results are shown in Table 2. In experiments 1 to 4, doses of 10 or 20 ,ug of anti-Id(fim) per ml were able to specifically inhibit the migration of PECs, with MMIs ranging from 27 + 2 to 65 + 3 (Table 2). When identical concentrations of type 1 fimbrial antigen were used, PEC migration was clearly inhibited in the same four experiments; MMIs ranged from 21 + 3 to 65 + 3 (Table 2). It should be noted that 5 pug of fimbrial antigen was able to inhibit cell migration in these controls, whereas S p.g of anti-Id(fim) did not demonstrate significant PEC inhibition (Table 2). On the other hand, testing control anti-Ids made against idiotypes binding an unrelated immunogen, DNPovalbumin, failed to inhibit cell migration in the same four experiments; MMIs ranged from 95 + 3 to 105 + 4 (Table 2). PECs obtained from normal, unimmunized animals in two experiments (Table 2) were incubated with the same array of antigens and failed to demonstrate cellular inhibition for the highest concentrations; MMIs ranged from 88 1 to 110 4 (Table 2). Because we were able to demonstrate cellular immunity in vitro, in anti-Id(fim) immunized animals, parallel experiments using in vivo skin testing of fimbrial antigen-immunized animals was carried out. Individual animals were injected in one ear with 1 ,ug of anti-Id(fim), a specificity control anti-Id (DNP-ovalbumin), or fimbrial antigen to assess skin test/ear-swelling reactivity to these reagents. The other ear received a saline control injection. Results are shown in Table 3. Animals skin tested with anti-Id(fim) did not exhibit ear swelling when compared with control animals; ear swelling in skin-tested animals ranged from 27 to 29 ,um, while that in controls ranged from 25 to 34 ,um (Table 3). On the other hand, animals skin tested with 1 ,ug of fimbrial












E 0-











O 0





A AA*-




2 jg 5OOng 10MLg 5MLg 1'Mg FIG. 2. Proliferation-stimulation indices of spleen cells from individual BALB/c mice preimmunized with anti-Ids prepared against idiotypes of Ab-1 binding type 1 fimbrial protein incubated with graded doses of anti-Id (0), fimbrial antigen (A), DNPovalbumin (A), anti-Id (normal IgG) ([1), or phytohemagglutinin (0). SI represents mean counts per minute of 5 control culture wells divided by the mean counts per minute of 5 separate wells incubated with media alone.


Moderate stimulation of lymphoid cells from anti-Id(fim)immunized animals was observed in doses of anti-Ids(fim) ranging from 1 to 5 ,ug (Fig. 2). Controls with fimbrial antigen DNP-ovalbumin or anti-Id developed against normal mouse IgG failed to exhibit the same degree of cellular stimulation observed with the anti-Ids(fim); SIs ranged from 0.5 to 2.7 with such controls (Fig. 2). Positive control cultures having 10 ,ug of phytohemagglutinin per ml exhibited SIs that ranged from 5.6 to 9.8 (Fig. 2). Two experiments were performed to assess the effects of anti-Id on isolated T cells from antiId(fim)-immunizing animals. T cells were incubated with graded doses of anti-Id ranging from 500 ng to 10 jig/ml under the same conditions described above. In both exper-

TABLE 2. Assessment of cell-mediated immunity in anti-Id immunized, BALB/c mice

MMIb Expt

iuAnti-Id BALB/c PEG

Affinity-purified anti-ld(fim)




1 2 3 4 5 6

100 100 100 100 100 100


85 78 104

3 2 5

96 102

3 2

65 54 75 33 90 9

51 48 65 27 88





5 3 2 4 1 3

Anti-Id DNP-ovalbumin

Type 1 fimbrial antigen

(l.g) ± ± ± ± ±

3 5 3 2 4

43 39 55 85 91

±2 ± 1

±3 ±4 + 2


25 ± 3 44 ± 3 49 1 96 2 87 3 96 2


21 23 47 65 110

± 3 ± 2

±2 ± 3 ±4


ND 110 ± 4 ND ND 92 ± 3 112 ± 4


95 ± 99 ± 105 ± 101 ± ND 106 ±

3 3 4 3 2

a NPEC, Normal peritoneal exudate cell controls. MMIs of PECs of BALB/c mice immunized against affinity-purified anti-Ids (Ab-2) against Ab-ls binding type 1 fimbrial proteins; PECs were tested against graded doses of anti-Id, fimbrial antigen(s), or DNP-ovalbumin. c ND, Not determined. b




TABLE 4. Binding of protein A affinity-purified anti-anti-Ids (Ab-3)

TABLE 3. In vivo assessment of cellular immunity with skin testing of syngeneic, BALB/c mice Ear swelling (pum) at time (h) after injection witha:


1 2 3 4

Anti-Id (fim)

Saline controls


33 28 25


34 29 33 27


33 27 26


28 28 27 28


28 23 29 29

Anti-Id (DNP-

ovalbumin) 24


26 27

25 26



Fimbrial antigen


35 46 51 45

Antianti-Id (Ab-3)



37 51 56 47

1 2 3 4 5

MicroELISA abosrbance values and dilution titers of Ab-3 tested with': Normal




Type 1 fimbrial antigen(s)

Ab-2 F(ab')2





0.027 0.034 0.051 0.004 0.047

0.106 (1:100) 0.209 (1:32) 0.074 (1:256) 0.118 (1:320) 0.113 (1:256)

0.069 (1:1,000) 0.117 (1:128) 0.099 (1:256) 0.108 (1:256) 0.109 (1:32)

0.054 (1:10) 0.035 (1:4) 0.027 (1:2) 0.033 (1:10) 0.049 (1:4)

0.040 (1:10) 0.010 (1:2) 0.003 (1:4) 0.051 (1:2) 0.002 (1:4)



Ear swelling was measured with a Mitutoyo Series E3 adjustable micrometer (Mitutoyo Manufacturing Co., Japan) before injection of antigen and at 24 or 48 h after challenge. b Results are reported as the average number of micrometers of thickness of three individual animals' ears at site of injection of 20 p.1 of solution containing 1 ,ug of each antigen or reagent.

a Anti-Anti-Id (Ab-3) was diluted and tested on microtiter plates coated with the reagent designated at the top of each column. b Represents the absorbance and last dilution (endpoint) with a detectable, positive ELISA reading in doubling dilutions of Ab-3. Positive controls comparing Ab-1 from animals immunized with fimbrial antigen alone had titers that ranged from 1:256 and 1:512 in two experiments.

antigen exhibited positive ear swelling when compared to controls in experiments 2, 3, and 4; ear swelling ranged from 45 to 56 p.m (Table 3). Assessment of protective immunity. Each of the anti-Id(fim) preparations listed in Table 1 was used to immunize groups of syngeneic BALB/c mice to assess whether our anti-Ids could generate protective immunity in adult animals. In four experiments, mice in groups of three were challenged i.p. with doses of piliated E. coli ranging from 1 X 102 to 5 x 103. In each experiment, anti-Id-immunized mice were dead on day 1 or 2 after bacterial challenge, in addition to the unimmunized controls. Ab-1 titers in the challenged mice had endpoints that ranged from 1:2 to 1:16 before bacterial challenge; one experiment using pooled anti-Id(fim) preparations also failed to induce protective immunity. Characterization of polyclonal anti-Ids(fim). The specificity of the polyclonal anti-Ids used in these experiments was assessed by the development of anti-anti-Ids (Ab-3). Theoretically, Ab-3s generated against Ab-2s will presumably possess idiotypes similar to those of Ab-1 and should bind not only Ab-2s but also the original immunogen used to stimulate Ab-1 (12, 13, 16, 19, 24, 25, 27). Five groups of BALB/c mice were hyperimmunized with anti-Ids(fim), serum was collected, and the IgG was affinity separated by protein A column separation. Separate preparations of these Ab-3s were then assessed for their ability to bind F(ab')2 fragments of Ab-2 [anti-Id(fim)], the fimbrial antigen normal F(ab')2 fragments, or DNP-ovalbumin, an unrelated immunogen. Results are shown in Table 4. When five separate preparations of Ab-3 were assessed, type 1 fimbrial antigen was bound in each instance; ELISA absorbance values ranged from 0.074 to 0.209, while titers of the Ab-3 ranged from 1:32 to 1:320 (Table 4). These antibodies also bound F(ab')2 fragments of anti-Ids(fim) used to generate Ab-3; absorbance values ranged from 0.069 to 0.117, with dilutions that ranged from 1:32 to 1:1,000 (Table 4). Results from experiments using normal IgG F(ab')2 fragments or DNPovalbumin failed to result in significant binding of the Ab-3s to these reagents; negative absorbance values were comparable to negative saline controls in all instances at negligible titers that ranged from 1:2 to 1:10 (Table 4). Comparing these Ab-3 titers to titers of Ab-ls from animals hyperimmunized with fimbrial antigen alone resulted in fimbrial antigen titers with endpoints at dilutions of 1:256 and 1:512 in two experiments and comparable to the Ab-3 titers (Table 4).

DISCUSSION In this study, the vaccinic or immunomodulatory potential of the polyclonal anti-Ids against idiotypes binding fimbrial proteins was analyzed. The thrust of this approach was to attempt immunization with anti-idiotypes against antibodies binding structural fimbrial proteins present on a common urinary pathogen, E. coli (1-3), as a possible surrogate vaccine and also to demonstrate the mimicry (9) of these anti-Ids for E. coli adhesins. A second purpose of the study was to assess the immunomodulatory characteristics of these anti-Ids in vivo and in vitro. Utilizing a thymidine incorporation cell proliferation and stimulation assay, we demonstrated both proliferative and "suppressive" effects on splenic lymphoid cell populations that appeared to be anti-Id dose dependent. However, it must be pointed out that development of cellular suppression in this system may be due to excess anti-Id being unable to cross-link Ab-1 receptors, resulting in lack of cellular activation. Moreover, these same anti-Ids were able to induce T-cell immunity against fimbrial proteins when assessed in vitro by specifically inhibiting the migration of PEC from anti-Id-immunized animals, with the release of migration inhibition factor from anti-Id-sensitized T cells. Results measuring T-cell immunity in vitro also appeared to be dose dependent, and the anti-Ids did not evoke skin test reactivity in anti-Id(fim)-immunized animals as measured by the ear-swelling test in vivo. In vitro, our anti-Ids were not able to stimulate proliferation of isolated T-cell populations. Perhaps not surprisingly, we were unable to demonstrate protective immunity in antiId-sensitized animals. The lack of development of adequate delayed sensitivity might help to explain the inability of our polyclonal anti-Ids to protect animals upon challenge with E. coli. However, it should be pointed out that present animal models for approximating human urinary tract infections are less than satisfactory, and our challenge of anti-Id-immunized animals i.p. might not simulate classical urinary tract infection by E. coli. In terms of a B-cell response, these polyclonal anti-Ids were able to elicit a fimbria-specific, secondary antibody response in mice injected once i.p. with fimbrial protein. Regarding specificity, polyclonal anti-Ids were able to generate Ab-3 antibodies with mimicry of certain idiotopes of Ab-1 capable of binding fimbrial proteins in an ELISA as well as F(ab')2 fragments of the anti-Ids. Earlier studies in other systems have documented that anti-Id modulates the immune response, characterized by enhancement of idiotype in some instances (4, 5, 21, 27)


VOL. 58, 1990

and/or suppression in others (6, 22). The results we report for this system are consistent with earlier observations and, considering the dose dependency of results, confirm experimentally the subtle modulatory nature of the idiotypeanti-Id immune network regulation suggested by others (24, 25, 31). On the other hand, in their present form, the anti-Ids used in this study, while clearly inducing specific immune responses to fimbrial determinants, did not appear to have vaccinoid properties. There are several explanations for this, which focus generally on presently unanswered issues of differences in specificity, quantity, and/or idiotype expression in immunity developed by anti-Id or regular vaccines. For example, recent studies demonstrate that type 1 fimbrial protein is made up of eight subunits (A through H), which include one ancillary subunit protein, designated as a 29-kDa FimH (2). FimH is presumably responsible for the binding or adhesion of mannose receptors on mammalian mucosal surfaces and, thus, likely contains the key epitope(s) necessary for generating protective immunity (2, 3). Thus, anti-Ids developed against idiotopes binding the major structural protein component, FimA, might not generate protective immunity and lack sufficient numbers of anti-Ids against the FimH adhesin idiotypes. This might include fimbrial idiotypes unable to trigger the necessary B- and/or T-cell subpopulations or clones (11, 26-28, 31) specific for those fimbrial subunits (FimH?) responsible for protective immunity (14). Our in vitro and in vivo comparisons of T-cell immunity in this system would support this idea. On the other hand, Western blot analysis of our idiotypic antibodies showed substantial binding to two bands in the 27-kDa range; however, we cannot say at this time which functional subunit(s) this reactivity represents. The polyclonal nature of these anti-Ids suggests the majority of the anti-Ids are probably alpha in nature and reactive against multiple and/or major idiotypes recognizing the major structural subunits of fimbriae. Development of an ideal vaccine capable of stimulating anti-adhesin characteristics should generate not only specific antibody, but cellular immunity, in addition to clones of memory cells able to guard against subsequent exposure to infection. In this regard, Ward et al. (31) have reported differences in the quality of antibody formation and B-cell precursor populations against anti-Ids conjugated to carrier proteins. Since our anti-Ids were not tested in conjugation to carrier proteins, it is possible, again, that the correct idiotope-positive B-cell or T-cell precursors were not triggered, because of their apparent inability to recognize silent clones (11, 26, 27) or perhaps true protective beta internal images in the context of anti-Id and carrier determinants (31). These structural binding considerations have been reviewed by Roitt et al. (25). Future research in this system suggests exploring the vaccinic potential of our polyclonal anti-Ids conjugated to appropriate carrier proteins. The development of experimental polyclonal anti-Ids in this system may prove useful in the future for dissection of immunoregulatory events in response to gram-negative infection, but also in formulation of a vaccine in the absence of bacterial products useful in specialized disease situations. Moreover, development of monoclonal anti-Ids against diverse idiotopes binding not only FimH subunits, but possibly other fimbrial proteins, may prove useful in studying immunoregulation and relevant anti-Id mimicry in construction of an effective, protective anti-adhesin vaccine.



ACKNOWLEDGMENTS We thank Jacques Hiernaux and Ronald Kennedy for their advice concerning the manuscript, Farion Williams for excellent technical assistance, and Diana Hinojosa for typing of the paper. This work was supported in part by an Institutional Biomedical Research support grant from the University of Texas Health Science Center. LITERATURE CITED 1. Abraham, S. N., and E. H. Beachey. 1987. Assembly of a chemically synthesized peptide of type 1 fimbriae of Escherichia coli into fimbriae-like antigenic structures. J. Bacteriol. 169:

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Polyclonal anti-idiotypic antibodies exhibit antigenic mimicry of limited type 1 fimbrial proteins of Escherichia coli.

Polyclonal anti-idiotypic antibodies (anti-Ids)(fim) developed against idiotypes on antibodies (Ab-1s) that specifically bind structural, organelle fi...
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