Immunologic Characterization of a 35-Kilodalton RecombinantAntigen of Mycobacterium tuberculosis
HELLA S. RUMSCHLAG, MITCHELL A. YAKRUS,* MITCHELL L. COHEN, SUZANNE E. GLICKMAN, AND
ROBERT C. GOOD
Division ofBacterial Diseases, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 30333
Received 31 July 1989/Accepted 1 December 1989
A 35-kilodalton (kDa) recombinant antigen (35-kDa antigen) produced by Escherichia coli JM107 carryingDNA from Mycobacterium tuberculosis was purified and immunologically examined by in vivo and in vitromethods. A monoclonal antibody (2B2) was produced against the 35-kDa antigen. The protein was purifiedfrom the insoluble fraction of the recombinant E. coli strain by either affinity chromatography with the 2B2monoclonal antibody or preparative isoelectric focusing. In enzyme-linked immunosorbent assay and Westernblot (immunoblot) analyses, antibody to 2B2 reacted with whole-cell sonic extracts ofM. tuberculosis and otherslowly growing mycobacteria but not with two rapid growers, M. chelonae and M. fortuitum. An injection seriestotaling less than 1 mg of purified protein without adjuvant elicited a humoral response in guinea pigs. In one
guinea pig, 10 ,ig of purified protein injected intradermally elicited both a humoral and a cell-mediatedresponse. Results of these studies suggest that the 35-kDa antigen is a membrane-associated protein thatstimulates both humoral and cell-mediated immune responses and should be evaluated as a vaccine candidate.
Tuberculosis still presents a significant public health prob-lem, causing 2 million to 3 million deaths per year worldwide(10). In the United States alone, more than 20,000 new cases
are detected each year (2). A steady decline in the incidenceof tuberculosis in the United States was reversed in 1986 (5),presumably because of an increase in the susceptible popu-
lations, including persons with acquired immunodeficiencysyndrome, the homeless, and infected immigrants fromdeveloping countries. A variety of techniques in molecularbiology have been applied to the challenge of improvingcurrent methods for detecting, preventing, and controllingthe disease.Because of the unusual biochemical characteristics, long
generation time, and high degree of serologic cross-reac-tivity among members of the genus Mycobacterium, hybrid-oma and recombinant DNA technologies have becomewidely used as tools for dissecting individual cell constitu-ents. Mycobacterial antigens can be purified by affinitychromatography by using monoclonal antibodies or can bestudied as recombinant proteins in well-characterized Esch-erichia coli systems (13, 14). Individual components can beevaluated for potential use as reagents for delayed-typehypersensitivity (DTH) and serological tests and as vaccinecomponents.We described previously (4) the construction of a recom-
binant E. coli strain that produces a 35-kilodalton (kDa)Mycobacterium tuberculosis antigen (35-kDa antigen). In thepresent study, we further characterized the recombinantantigen and evaluated the potential of the purified protein as
a reagent for DTH testing and a stimulator of humoralimmunity.
MATERIALS AND METHODS
Bacterial strains. Construction of recombinant E. coli TB2[JM107(pLWM2020)] has been described previously (4). Thefollowing mycobacterial strains were used in this study: M.tuberculosis H37Ra (Takayama), M. tuberculosis DT 612,M. kansasii TMC 1203, M. gordonae TMC 1325, M. avium
* Corresponding author.
TMC 715, M. simiae TMC 1226, M. scrofulaceum TMC1309, M. intracellulare TMC 1405, M. fortuitum TMC 1530,and M. chelonei TMC 1524.
Preparation of sonic extract material. Mycobacterial cellswere grown in 500-ml Erlenmeyer flasks containing 200 ml ofMiddlebrook 7H-9 broth (Difco Laboratories, Detroit,Mich.). After 3 weeks, cells were killed by adding NaN3(0.2%) and Merthiolate (0.01%). The cells were incubated for24 h at 35°C and tested for viability by placing 0.1-ml sampleson Lowenstein-Jensen slants. The broth cultures were keptat 4°C, while the slants were incubated for 2 weeks at 35°Cand examined for any growth. Once the cells were deter-mined to be nonviable, they were transferred from flasks to250-ml centrifuge bottles, pelleted at 8,000 x g for 20 min at4°C, and suspended in 40 ml of 200 mM Tris hydrochloride(pH 8.0). Each cell suspension was transferred to a 50-mlround-bottom glass centrifuge tube and sonicated by using a
cell disrupter (model 375; Heat Systems-Ultrasonics, Farm-ingdale, N.Y.) equipped with a standard 0.5-in. (1.25-cm)tapped horn. The tube was immersed in an ice bath andsonicated at 80 W for 5 min by using 30-s bursts interruptedby 1-min cooling periods. The sonicated suspension was
centrifuged at 3,000 x g for 20 min at 4°C to remove largeparticles. The supernatant was then centrifuged at 175,000 xg for 2.5 h and the pellet was suspended in 1 ml of water.This pelleted material was used in enzyme-linked immuno-sorbent assay (ELISA) and Western blot (immunoblot)analyses.
Monoclonal antibody production. BALB/c mice were in-jected intraperitoneally with the 35-kDa recombinant proteineluted from nitrocellulose blots with 20% acetonitrile (11)and suspended in phosphate-buffered saline (PBS; pH 7.2).After intraperitoneal boosts at 4 and 6 weeks, blood was
drawn from the tail veins and tested for activity against therecombinant antigen by Western blot analysis. Reactivemice were boosted intravenously 3 days before they were
humanely sacrificed. Fusion of spleen cells with myelomaline P3-X63-Ag8.653 cells was performed as described pre-viously (3). Supernatants from clones were screened 7 to 14days after fusion against Western blots prepared from M.
tuberculosis sonic extracts with a Miniblotter (Immunetics,Cambridge, Mass.). The monoclonal antibody used in thisstudy (2B2) was determined to be immunoglobulin G2a withthe Clonotyping Kit III (Fisher Scientific Co., Orangeburg,N.J.).
Immunoblotting. Proteins separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (8) were trans-ferred to nitrocellulose (pore size, 0.2 ,um; BA85; Schleicher& Schuell, Inc., Keene, N.H.) with a semidry blottingapparatus (Polyblot transfer system; American Bionetics,Hayward, Calif.) as described by the manufacturer. Theseblots were then washed in PBS with 0.3% Tween 20 threetimes for 10 min each time. Tissue culture fluids diluted withan equal volume ofPBS were incubated with the blots for 1.5h at room temperature. Guinea pig sera diluted 1:100 in PBScontaining 0.05% Tween 20 were incubated in the same way.After one 10-min wash in PBS-0.3% Tween 20, blots werewashed in casein buffer (0.5% casein in 10 mM Tris hydro-chloride [pH 7.6], 0.15 M NaCl, 0.02% thimerosal [7]) for 10min. Bound antibody was detected with goat anti-mousehorseradish peroxidase conjugate at a dilution of 1:3,000 orprotein A-horseradish peroxidase conjugate (Bio-Rad Labo-ratories, Richmond, Calif.) at a dilution of 1:5,000 in caseinbuffer. Blots were developed with tetramethylbenzidine aspreviously described (12).ELISA. Titers of guinea pig antisera were determined
against a sonic extract of M. tuberculosis H37Ra by ELISA.Each of 96 wells in flat-bottom Immulon Il plates (Dynatech,Chantilly, Va.) was coated with 0.1 ml of sonic extractsolution containing 100 ,ug of protein per ml in 0.01 M PBS(pH 7.2) with 0.01% Merthiolate. The plates were incubatedin a 37°C water bath for 2 h and stored overnight at 4°C.Fluids were removed from the plates, and nonreactive siteswere blocked with 2% bovine serum albumin in PBS for 2 h.Plates were washed with PBS, and the wells were thenincubated with 0.1-ml amounts of antisera diluted twofold,from 1:100 to 1:51,200. Bound antibody was detected withprotein A-horseradish peroxidase conjugate (Bio-Rad) di-luted 1:5,000 in 2% bovine serum albumin-PBS. After 1 h theplates were washed and developed with o-phenylenediaminedihydrochloride (Sigma Chemical Co., St. Louis, Mo.) sub-strate. Reactions were stopped by the addition of 50 ,ul of 4.5M H2SO4 to each well, and the A490 was read. An opticaldensity above 0.200 was considered positive.
Protein purification. Membranes were extracted fromrecombinant E. coli cells grown at 37°C for 48 h in L brothcontaining isopropyl-,-D-thiogalactopyranoside (10 mg/ml)by previously described methods (1). Briefly, cells grownovernight in L broth were washed and broken by sonication.After the unbroken cells and large debris were spun out at10,000 x g, the supernatant was subjected to ultracentrifu-gation at 175,000 x g for 2 h. This pellet was designated thecrude membrane and was used for guinea pig injections afterpassage through a 0.2-,um-pore-size filter. The purified pro-tein used in tests for DTH was prepared from crude mem-brane (1 mg/ml) that was solubilized in 1% Empigen BB(EBB) (Calbiochem-Behring Diagnostics, La Jolla, Calif.) in20 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid; pH 7.4)-0.15 M NaCl at 4°C overnight. Aftercentrifugation at 175,000 x g for 2 h, the supernatant wasdiluted to a concentration of 0.1% EBB and applied to anaffinity column (Affigel 10; Bio-Rad) carrying monoclonalantibody 2B2. Bound antigen was eluted with 3 M KSCN-0.1% EBB, concentrated on a YM10 membrane (AmiconCorp., Danvers, Mass.), and precipitated by diluting theEBB concentration to 0.01%. Insoluble recombinant antigen
was pelleted by centrifugation at 175,000 x g for 2 h.Supernatants were combined, concentrated, and furtherdiluted to 0.001% EBB and centrifuged again to pelletadditional antigen.For other applications that required purified antigen, the
following procedure was used. Crude membranes, at aconcentration of 5 mg/ml, were solubilized in 4 M urea at65°C for 30 min. After centrifugation at 175,000 x g for 2 h,the supernatant was subjected to preparative isoelectricfocusing in a Rotofor cell by using 2% 3/10 ampholytes asdescribed by the manufacturer (Bio-Rad). Fractions wereseparated by sodium dodecyl sulfate-polyacyrlamide gelelectrophoresis and examined for recombinant antigen bysilver staining (9). Fractions containing a predominance ofrecombinant antigen with few contaminating proteins werepooled and refocused in the Rotofor cell again with 2% 5/7ampholytes. Fractions were again assessed by silver stainingsodium dodecyl sulfate-polyacrylamide gels, and those con-taining the recombinant antigen were pooled. The fractionpool was concentrated and washed on a YM30 membrane(Amicon) and dialyzed extensively against PBS (pH 7.2). Inorder to remove insoluble antigen, the fraction pool wascentrifuged at 175,000 x g for 2 h, and the supernatantcontaining antigen was passed over a column (Detoxigel;Pierce Chemical Co., Rockford, 111.) before injection intoguinea pigs. The purity of the antigen preparations wasassessed by silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.Guinea pig sensitization. Female Hartley strain guinea pigs
(weight, 300 to 400 g) were sensitized with killed cells of M.tuberculosis DT 612. A suspension of 100 mg of dried wholecells in 5 ml of sterile saline was mixed with 5 ml of sterileArlacel-mineral oil (1:9; vol/vol) in a sterile blender cup andemulsified at a high speed in an Omnimixer (Ivan Sorvall,Inc., Newtown, Conn.). Each animal was injected subcuta-neously in the nuchal area with 0.5 ml (5 mg) of emulsifiedproduct. Control animals were injected with Arlacel-mineraloil only.Guinea pigs were tested for DTH with 0.1-ml intradermal
injections of M. tuberculosis purified protein derivative(PPD-S) (0.1 ,ug), the purified 35-kDa protein (10 ,ug), andPBS. Zones of erythema were measured at 24 and 48 h.
Preparation of antisera. Antisera were prepared to thepurified 35-kDa protein, the M. bovis BCG vaccine (Univer-sity of Illinois, Chicago, 111.), and the E. coli crude mem-brane preparation from either parent or recombinant cells.Female Hartley strain guinea pigs (weight, 400 to 500 g) weregiven three intramuscular injections at 10-day intervals.Injections of the 35-kDa protein contained 10, 20, and 60 ,ugof protein, while guinea pigs inoculated with either BCGvaccine or E. coli membranes received 100, 200, and 600 ,ugof protein, as determined by the BCA protein assay (PierceChemical Co.). All inocula were prepared with sterile waterfor injection (American McGaw, Irvine, Calif.). No adju-vants were used. Animals were anesthetized and bled bycardiac puncture 16 days after the last injection.
RESULTS
Species specificity of monoclonal antibody 2B2. We previ-ously demonstrated the presence of the 35-kDa antigen innontuberculous mycobacteria with a polyclonal rabbit serum(4). Although the monoclonal antibody directed against the35-kDa antigen displayed reactivity with slowly growingmycobacteria, it did not react with the rapid growers M.fortuitum and M. chelonae (Fig. 1). These observations were
FIG. 1. Western blot (immunoblot) comparison of mycobacterialsonic extracts reacted with monoclonal antibody 2B2. Each lanerepresents 25 ,ug of protein from M. kansasii (lane 1), M. avium (lane2), M. intracellulare (lane 3), M. simiae (lane 4), M. scrofulaceum(lane 5), M. gordonae (lane 6), M. chelonei (lane 7), M. fortuitum(lane 8), and M. tuberculosis (lane 9). mw, Molecular weights (inthousands); 35k, 35-kDa antigen.
mwA B C D A* * ....** X__
M_.._
_ _l
I i_
1
B C D
53 kd
_m 35 kd
i2
confirmed by ELISA of sonic extracts (data not shown).Monoclonal antibody 2B2 also reacted with several high-molecular-weight (.53,000) and low-molecular-weight(<35,000) components, suggesting either that this epitope isassociated with other antigens or that the 35-kDa proteinexists in high-molecular-weight polymers.
Antigen purification. Monoclonal antibody 2B2 was able tobind cell sonic extracts but not to intact cells of recombinantE. coli or M. tuberculosis in the ELISA (data not shown),suggesting that the antigen is not surface exposed. However,differential centrifugation demonstrated the presence of the35-kDa protein in the crude membrane but not in the solublecytosol of both cells. Thus, purification of the antigennecessitated solubilization of the crude membrane fraction.Purification of the 35-kDa antigen from recombinant E. colistrains was initially attempted with the zwitterionic deter-gent EBB and a monoclonal antibody 2B2 affinity column.Antigen prepared in this way was used to test guinea pigs forDTH (see below). Although the protein resulting from thispurification exhibited only the recombinant 35- and 53-kDaprotein bands on silver-stained polyacrylamide gels (data notshown), injection into guinea pigs resulted in antisera withactivity to a wide variety of E. coli antigens. Since theantigen preparation was contaminated with low quantities ofE. coli antigens, possibly because of incomplete solubiliza-tion of the membrane complex by EBB, we developed a
protocol in which we used membranes treated with 4 M ureaat 650C.
Further purification of the 35-kDa antigen was accom-plished by preparative isoelectric focusing of urea-solubi-lized membrane. The isoelectric point of the antigen was
estimated to be 5.81, based on the DNA sequence (S.O'Connor, submitted for publication). Preparations thatcontained only the 2B2-reactive 35- and 53-kDa bands (Fig.2) were used for injections into guinea pigs for antibodyproduction. In contrast to the affinity-purified antigen, injec-tion of antigen resulting from preparative isoelectric focusingresulted in essentially monospecific antisera (Fig. 3).
Tests of DTH. Guinea pigs sensitized with either M.tuberculosis cells in adjuvant or adjuvant alone were exam-
ined 24 and 48 h after intradermal injection of PPD-S and the
FIG. 2. Preparations of recombinant E. coli whole cells (A),whole cells of parent E. coli (B), M. tuberculosis sonic extract (C),and purified recombinant protein (D) compared by silver staining(lanes 1) and Western blot (immunoblot) reacted with monoclonalantibody 2B2 (lanes 2). mw, Molecular weights (in thousands); 35kd,35-kDa antigen.
purified 35-kDa antigen. After 24 h, the control animalsdisplayed nearly as much erythema to the recombinant35-kDa antigen as did the sensitized guinea pigs, suggestinga nonspecific reaction to the antigen preparation. After 48 h,however, the reactions to the 35-kDa antigen in the controlanimals subsided, while the sensitized guinea pigs main-tained significant reactions (Table 1).One of the eight control animals that had not been sensi-
tized with M. tuberculosis was selected to receive an intra-dermal injection of 10 ,ug of the 35-kDa protein to test thequality of a new lot of purified antigen. No significantreaction was observed. Fourteen days later, when the sameanimal was again used as a negative control in the largerstudy summarized in Table 1, a strong (diameter, 16 mm)skin test reaction to the 35-kDa antigen was observed at 48 h.It was also the only control animal to demonstrate a reaction(diameter, 6 mm) to PPD-S 48 h after injection. Examinationto the serum of this animal on Western blot revealed a strongactivity to the recombinant antigen. Apparently, a singleintradermal injection of recombinant antigen was sufficientto evoke both a humoral and a cell-mediated response. Datafor this guinea pig were not included in the control groupdata presented in Table 1.
Serologic responses in guinea pigs. Serum samples fromguinea pigs injected intramuscularly with various antigenpreparations were tested against Western blots of recombi-nant E. coli cells and M. tuberculosis sonic extract to assessthe strength and specificity of the humoral response to the35-kDa antigen. The antibody response in animals injectedwith purified 35-kDa antigen was very specific; there wasvery little activity to E. coli proteins (Fig. 3B, lane 1). Theantibody response to the 35-kDa antigen elicited by therecombinant E. coli membrane (Fig. 3B, lane 2) was stron-ger, albeit much less specific. In guinea pigs injected with
FIG. 3. Western blot (immunoblot) strips of M. tuberculosissonic extract (A) and recombinant E. coli whole cells reacted withvarious guinea pig sera (B). The four guinea pigs in group 1 (lanes 1)were injected with purified recombinant antigen, group 2 (lanes 2)received recombinant E. coli membrane protein, group 3 (lanes 3)received parent E. coli membrane protein, group 4 (lanes 4) receivedthe BCG vaccine, and group 5 (lanes 5) were uninoculated controls.Lanes 6 were reacted with protein A-horseradish peroxidase conju-gate only, lanes 7 were reacted with monoclonal antibody 2B2 andgoat anti-mouse-horseradish peroxidase conjugate, and lanes 8 werereacted with goat anti-mouse-horseradish peroxidase conjugateonly. 35k, 35-kDa antigen.
recombinant or parent E. coli membranes (Fig. 3B, lanes 2and 3, respectively), a predominance of antibody was foundto a membrane protein that was just slightly larger than the35-kDa antigen. BCG vaccine evoked a strong response tomany components of the M. tuberculosis sonic extract (Fig.3A, lane 4), including a consistent response to the 35-kDaantigen (Fig. 3B, lane 4).
TABLE 1. Comparison of DTH in guinea pigs usingM. tuberculosis PPD-S and the recombinant protein
TABLE 2. Reactivity of sera from guinea pigs in groups 1 to 5against M. tuberculosis sonic extract as measured by ELISA
Group" Antigen prepn injected Titerb
1 Purified 35-kDa antigen 1,1312 Recombinant E. coli membranes 10,1593 Parent E. coli membranes 204 BCG 25,6005 Uninoculated controls 3
a Groups are defined in the legend to Fig. 3.b Expressed as a geometric mean of four individual titers.
The antibody titer of each guinea pig antiserum wasmeasured against M. tuberculosis sonic extract in an ELISA(Table 2). Serum titer and strength of the response seen onWestern blots (Fig. 3A) were closely correlated.
DISCUSSION
Recombinant DNA technology has become invaluable inthe study of individual mycobacterial antigens (13, 15). Inthis study, we used a monoclonal antibody and proteinpurification techniques to investigate the immunogenic po-tential of a previously described 35-kDa antigen of M.tuberculosis expressed by a recombinant E. coli strain (4).The results of this study demonstrate that epitopes of the
35-kDa antigen are not unique to M. tuberculosis; themonoclonal antibody bound to antigens with similar molec-ular weights in several other slowly growing mycobacterialspecies. The epitope recognized by the monoclonal antibodywas not present in the rapid growers M. chelonae and M.fortuitum; however, previous studies with polyclonal rabbitantiserum to the recombinant protein demonstrated thepresence of related antigens in these two species as well (4).Work is continuing in the effort to generate monoclonalantibodies against other, perhaps species-specific, epitopesof the 35-kDa antigen.The solubility characteristics of this antigen suggest that it
is associated with the membrane. In protein purificationprotocols, the 35-kDa antigen was always present in theinsoluble, i.e., membrane, fractions of both the recombinantE. coli strain and M. tuberculosis. Solubilization in detergent(EBB) or 4 M urea was necessary for affinity chromatogra-phy and isoelectric focusing, respectively. Even with thesereagents, purification methods did not consistently yield ahomogeneous preparation, apparently because of the asso-ciations of recombinant antigen with other insoluble cellconstituents. Since DNA sequencing indicated that the an-tigen has a molecular weight of 29,000 (O'Connor, submit-ted), the presence of higher-molecular-weight bands in puri-fied preparations of the recombinant E. coli and M.tuberculosis strains (Fig. 1 to 3) suggests that the antigenalso forms aggregates with itself and perhaps other proteins.Although insolubility and the tendency to aggregate
present difficulties in purification, such characteristics arebeneficial in evoking an immune response. It is generallyrecognized that insoluble antigens are better stimulators ofan immune response than are soluble antigens (6). We foundthat a very small amount (10 ,ug) of the 35-kDa proteininjected intradermally was sufficient to produce an antibodyresponse and DTH to both the 35-kDa antigen and PPD-S inat least one guinea pig. Because of this strong antigenicity,the 35-kDa antigen could not be used repeatedly and wouldthus be unsuitable for in vivo DTH testing.
This antigenicity might be very beneficial in the develop-ment of a vaccine. Good antibody responses (Fig. 3 and
Tale 2) were elicited in guinea pigs by injection with eitherpurified antigen or a mixture of insoluble antigens from therecombinant E. coli strain.
In conclusion, we characterized a 35-kDa antigen of M.tuberculosis that is readily available from a recombinant E.coli strain. Its immunogenic nature suggests that it may havepotential as a vaccine component, and further analysis willcontribute to the understanding of the immunology of tuber-culosis.
ACKNOWLEDGMENTS
We thank Ray Butler for providing the mycobacterial strains usedin this study. We also thank William F. Bibb, George M. Carlone,Leta O. Helsel, and Leonard W. Mayer for helpful advice.
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