Vol. 59, No. 8

INFECTION AND IMMUNITY, Aug. 1991, p. 2595-2600

0019-9567/91/082595-06$02.00/0 Copyright © 1991, American Society for Microbiology

Immunological Characterization of Recombinant Antigens Isolated from a Mycobacterium avium Xgtll Expression Library by Using Monoclonal Antibody Probes DAVID A. ROUSE,* SHELDON L. MORRIS, ARTHUR B. KARPAS, JULIA C. MACKALL, PETER G. PROBST, AND SOTIROS D. CHAPARAS Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892 Received 18 March 1991/Accepted 13 May 1991

Nontuberculous mycobacteria, particularly Mycobacterium avium, have been isolated from a significant percentage of patients with AIDS. Early detection of M. avium infection is difficult, and treatment regimens are often ineffective. Much needs to be learned about antigens and factors responsible for immunity to and pathogenesis of the disease. Specific antigens and diagnostic procedures for infection need to be developed. To address some of these problems, we have generated 25 different monoclonal antibodies against a serovar 4 strain of M. avium isolated from a patient with AIDS. Protease sensitivity studies have demonstrated that each of these antibodies recognizes a protein-associated epitope. Immunoblot analyses suggest that seven of these monoclonal antibodies react specifically with M. avium and M. intracellulare epitopes. Immunoreactive bacteriophages were identified from an M. avium Agtll expression library with two of these monoclonal antibodies (3808 C3 and 3954 B12). Lambda lysogens, generated from the immunoreactive bacteriophages, overproduced ,-galactosidase fusion proteins which were reactive with the two monoclonal antibodies in immunoblot assays. The purified fusion proteins were shown to elicit skin test reactions in sensitized guinea pigs.

The Mycobacterium avium-Mycobacterium intracellulare complex (MAC) rarely caused serious infections in humans prior to 1981 (6, 7). However, during the past decade, the incidence of MAC disease has increased dramatically, primarily in patients with AIDS (15, 21, 22, 26). Approximately 50% of AIDS patients in the United States have been reported to be infected with MAC (6). Disseminated MAC infections found in a significant proportion of these individuals contribute to the high mortality seen in AIDS patients (15, 21, 22). Determination of infection with MAC and isolation of the organisms at present are difficult and time-consuming. As a result, many of the initial MAC infections were not detected in AIDS patients until postmortem examination (6). The incidence of tuberculosis in the AIDS population of the United States has also increased significantly. Recent studies have indicated that as many as 10% of AIDS patients have active tuberculosis (20). In fact, tuberculin testing is now suggested for all human immunodeficiency virus-seropositive individuals. However, because of the wide antigenic cross-reactivity among mycobacteria, the tuberculin test lacks specificity and does not necessarily indicate a tuberculosis infection. Of great value would be monospecific skin test antigens capable of differentiating between MAC and M. tuberculosis infections. In this report, we describe the production and characterization of 25 monoclonal antibodies (MAbs) directed against M. avium protein antigens. Western blot (immunoblot) analyses suggested that four of the MAbs were specific for M. avium epitopes and three were specific for MAC determinants. In addition, four of the MAbs failed to bind to tuberculous sonic extracts. Immunoreactive bacteriophages were identified from an M. avium Agtll gene expression *

library which expressed recombinant proteins recognized by MAbs 3808 C3 and 3954 B12. The immunoreactivity of 3-galactosidase fusion proteins recognized by these MAbs was evaluated in vivo by skin testing. Finally, the potential applicability of these MAbs and recombinant proteins in the rapid diagnosis of MAC disease is discussed. MATERIALS AND METHODS

Preparation of mycobacterial antigens. Most of the mycobacterial strains were obtained from the Trudeau Mycobacterial Culture Collection, Saranac Lake, N.Y., currently located at the American Type Culture Collection, Rockville, Md. Ten M. avium strains were isolated from AIDS patients and were kindly provided by Frank G. Witebsky of the National Institutes of Health Clinical Center. The serovars of these strains were determined at the Centers for Disease Control, Atlanta, Ga. Mycobacterial sonic extracts used in immunoblot analyses and immunizations were prepared as described earlier (19). Production of MAbs. BALB/c mice were injected intraperitoneally three times in 1 week with 8 ,ug of an ammonium sulfate precipitate from an M. avium (serovar 4) strain per injection. Intermittent bleedings of these animals indicated that M. avium antibody levels in serum remained high for several months. After 5 months, when specific antibody levels had fallen, the immunized mice were given an intravenous booster of the same M. avium precipitate. Cell fusions were performed 3 days later by a modification of the Kohler and Milstein method (14, 19). The fusions yielded 892 hybridomas, 96 of which were shown to produce antibodies directed against M. avium antigens. Screening of hybridomas. Preparative Western blots containing 150 to 200 ,ug of M. avium (serovar 4) ammonium sulfate-precipitated proteins were prepared, using the MiniProtean II gel electrophoresis system (Bio-Rad Laborato-

Corresponding author. 2595

2596

ROUSE ET AL.

ries, Richmond, Calif.). The blots were blocked at room temperature with 5% nonfat dry milk in Tris-buffered saline (TBS; 10 mM Tris [pH 8.0], 150 mM NaCI). After 1 to 2 h of blocking, these Western blots were washed with TBS and placed in a Miniblotter 28 apparatus (Immunetics, Cambridge, Mass.). The blots were then incubated with approximately 75 ,ul of hybridoma supernatant per channel for 1 to 2 h at room temperature. After the blots were washed with 0.05% Tween 20 in TBS (TTBS), they were incubated with goat anti-mouse immunoglobulin (immunoglobulin G [IgG] whole molecule) alkaline phosphatase conjugate (Sigma Chemical Co., St. Louis, Mo.). Antibody binding was detected with the Bio-Rad alkaline phosphatase detection system.

Specificity and isotype of M. avium MAbs. The specificity and isotype of M. avium MAbs were evaluated as described earlier (19). Briefly, the immunoglobulin class and subclass were evaluated by using an enzyme-linked immunosorbent assay screening/isotype kit (Boehringer Mannheim Biochemicals, Indianapolis, Ind.) as described by the manufacturer. The specificity of the 25 MAbs was determined by immunoblot analyses. Equal amounts of each mycobacterial sonic extract (15 p.g/lane) were applied to 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels, and gels were electrophoresed by using the Mini-Protean II gel system (Bio-Rad). Separated proteins were then transferred to nitrocellulose, incubated with the appropriate antibody, and developed with the alkaline phosphatase blot detection system as described above. Preparation of M. avium expression library. Production of the M. avium library closely followed the protocols established by Young and coworkers (25). M. avium (serovar 4) organisms were cultured in 150 ml of Long's synthetic medium (with 1% glucose) in 800-ml tissue culture flasks (Nunc, Roskilde, Denmark). Cells were incubated at 37°C until growth had reached late log phase. Cycloserine and ampicillin were both added to a concentration of 100 ,ug/ml, and DNA was extracted 24 h later (8). The DNA recovered by this protocol was then mechanically sheared to a molecular size range of 2 to 7 kb by repeated passage through a 30-gauge needle. Shearing of the DNA was monitored by agarose gel electrophoresis. Endogenous EcoRI sites were protected from EcoRI digestion by using EcoRI methylase, and the ends were made flush with T4 DNA polymerase. EcoRI linkers were removed by digestion with EcoRI and passage through a 1% low-melting-point gel (Bethesda Research Laboratories, Gaithersburg, Md.). The treated DNA was then ligated into Agtll arms and packaged by using the Packagene system (Promega Corp., Madison, Wis.). The packaged recombinant bacteriophage were used to infect Escherichia coli Y1090. This protocol was repeated until approximately 8 x 105 recombinant bacteriophage had been generated. The pooled recombinant bacteriophage were amplified to a final titer of 1011 PFU/ml. Random plaques were isolated from a plating of the library, and the bacteriophage DNA was isolated by standard techniques (16). Restriction analyses with EcoRI indicated that approximately 80% of the library contained DNA inserts. Screening of M. avium Agtll library. Cultured supernatants of two MAbs (3808 C3 and 3954 B12) were used to screen approximately 2 x 105 bacteriophage per MAb. E. coli Y1090 overnight cultures were pelleted, resuspended in an equal volume of TM10 (10 mM Tris [pH 8.0] 10 mM MgSO4), and then infected with 2 x 104 recombinant bacteriophage at 37°C for 20 min. The infected cells were then plated onto LBM (Luria-Bertani medium supplemented with

INFECT. IMMUN.

10 mM MgSO4) plates containing 100 plg of ampicillin per ml (16). These plates were incubated at 42°C for 2.5 h and then overlaid with nitrocellulose filters presoaked in 10 mM isopropylthiogalactoside. After incubation at 37°C for 3.5 h, the filters were washed with TBS and blocked with 5% nonfat milk in TBS at 4°C for 16 to 18 h. Several washes with TBS were required to remove residual milk. The anti-M. avium antibodies were then incubated with the filters for 1 to 2 h at room temperature. After several washes with TTBS, the filters were incubated with conjugate for 1 to 2 h, washed with TTBS, and developed with alkaline phosphatase substrate as described above. Immunoreactive plaques isolated from the primary screening were allowed to elute in 1 ml of

TM10 for 2 to 6 h. Repeated infections (usually two additional screenings) with increasing dilution factors allowed for purification of immunoreactive bacteriophage. Isolation and characterization of recombinant bacteriophage DNA. Recombinant bacteriophage DNA was isolated by the plate lysate method (16). The recombinant Agtll DNA of both bacteriophages was subjected to restriction digestion, using several enzymes as described by the manufacturer (Bethesda Research Laboratories), and analyzed by agarose gel electrophoresis. Production of MAb 3808 C3- and 3954 B12-reactive X lysogens. The protocol for generating A lysogens in E. coli Y1089 has been described by Huynh and colleagues (10). Briefly, approximately 103 E. coli cells were infected with 107 phage from each purified recombinant bacteriophage stock. After 20 min of infection at 30°C, the cells were plated onto LBM plates containing ampicillin at 30°C overnight. Single colonies were selected and streaked onto two LBM plates containing ampicillin and incubated at two temperatures, 30 and 42°C. Cells which proliferated at 30°C but not at 42°C were then grown in liquid culture at 30°C with ampicillin until the A600 reached an optical density of approximately 0.5. After incubation at 42°C for 20 min, the cultures were placed at 38°C for an additional hour in the presence of 10 mM isopropylthiogalactoside. The cells were harvested by centrifugation at 1,000 x g for 10 min and resuspended in 1/25 volume of a solution composed of 10 mM Tris (pH 8.0), 1 mM EDTA, and 2 mM phenylmethylsulfonyl fluoride. After one cycle of freezing and thawing, the cultures were sonicated for 45 s at 50% output. Sonic extracts were then precipitated with an equal volume of saturated ammonium sulfate and incubated at 4°C for 16 h. The precipitate was recovered by centrifugation at 10,000 x g at 4°C for 5 min and resuspended in a minimum volume of phosphate-buffered saline. Protein concentrations were determined by using the Bio-Rad protein assay system. Fifteen micrograms of ammonium sulfate precipitate of the K lysogens was electrophoresed through 10% SDSpolyacrylamide gels with the Mini-Protean II system (BioRad Laboratories). Lanes containing sonic extracts of M. avium and E. coli Y1089 served as controls. Proteins were transferred to nitrocellulose filters, and immunoblots were developed as described in Materials and Methods. Purification of M. avium recombinant antigens. The n-galactosidase fusion proteins were purified to a single spectro-

photometric peak by high-performance liquid chromatography (HPLC). The chromatographic system consisted of a Hewlett-Packard 1090 series M liquid chromatograph equipped with a photodiode detector. A Toso Haas TSK Gel 3000 SW column (7.5 by 300 mm) was developed with 0.2 M sodium phosphate (pH 7.2) containing 0.1% SDS. The flow rate was set at 0.5 ml/min. Bacterial sonic extracts were fractionated in ammonium sulfate (30 to 40%), taken up in

M. AVIUM MAbs AND RECOMBINANT ANTIGENS

VOL. 59, 1991

RESULTS

TABLE 1. Characterization of anti-M. av'ium MAbs MAb

Isotype

Reactive

Specificity'

3805 F9 3808 C3 3809 A12 3913 C12 3913 G10 3917 Fl 3918 D12 3920 B10 3921 E4 3936 E2 3954 B12 3958 B4 3968 E10 3982 A3 3989 Cl 3994 B8 4004 F3 4028 G9 4034 Eli 4045 H12 4046 Hi 4055 E6 4057 D2 4061 B10 4064 E6

IgG3 IgG2a IgGi IgGl IgM IgG2b IgG3 IgM IgG2a IgG2b IgG3 IgG3 IgM IgM IgGl IgG2b

44 26 66 27 66 34 27 30 22 70 33 25 64 12 30 71 20 45 30 25 120 35 23, 17 64 36

CR NTB NTB None NTB MAC MAIS None None None MA None CR CR MA None MAC NTB MAC None None MA None CR MA

IgGi IgM IgG2b

IgGi IgG3 IgGl IgG3 IgGl IgGl

aMA, M. avium; NTB, nontuberculous; CR, common reactivity.

0.2 M sodium phosphate (pH 7.2) at a concentration of 2 mg/ml, and treated with equal volumes of 0.2 M sodium phosphate (pH 7.2) containing 5% SDS and 5% mercaptoethanol for 40 min at 60°C. Samples were filtered through 0.45-pum filters, and 100-p.l aliquots (100 pLg) were injected onto the column. Protein concentrations were monitored by measuring the A230 and A280Skin testing of purified recombinant antigens. Hartley strain guinea pigs were sensitized with sonic extracts of M. avium (serovar 4), M. intracellulare (serovar 14), or M. tuberculosis (H37 Rv) as described earlier (4). After a minimum of 6 weeks, sensitized and nonsensitized guinea pigs were skin tested with intradermal injections of 3 ,ug of purified recombinant antigen and 0.1 p.g of homologous sonic extract each in 100 p.l of 0.85% sodium chloride-0.001% Tween 80. Reactions were measured approximately 18 h after injection.

2597

Production and characterization of MAbs. Immunological characterization of 96 antibody-producing clones demonstrated that at least 25 different MAbs had been generated. The antibody isotype and sizes are listed in Table 1. A composite of antibody reactivity in Western blots containing M. avium sonic extracts is shown in Fig. 1. Twenty of the MAbs to M. avium were of the IgG subclass and five were IgM. Although many of the antibodies recognize 20- to 45-kDa antigens, four reacted with larger proteins of approximately 65 kDa and two bound to approximately 70-kDa antigens. None of the MAbs bound proteinase K-pretreated M. avium extracts in immunoblot assays. Consequently, all of these MAbs recognize protein-associated epitopes. The specificity of the anti-M. avium MAbs was evaluated by immunoblot analyses. Western blots containing 16 different mycobacterial sonic extracts were incubated, using the hybridoma supernatants. Antibody binding was detected with the alkaline phosphatase blot detection system. The specificity results are shown in Table 2 and summarized in Table 1. Table 2 shows that eight of the antibodies reacted with only M. avium, MAC, or M. avium-M. intracellulare-M. scrofulaceum (MAIS) determinants. Four antibodies, 3954 B12, 3989 Cl, 4055 E5, and 4064 E6, recognized M. avium-specific epitopes. Three of the MAbs, 3917 Fl, 4004 F3, and 4034 Eli, reacted only with MAC antigens. Antibody 3918 D12 bound only to MAIS epitopes. Expanded studies with more mycobacterial strains will be needed to confirm the specificities of these antibodies. To determine the spectrum of reactivity against several different M. avium serovars, the MAIS-specific antibodies were incubated with Western blots containing sonic extracts of 10 different M. avium strains. Nine of the M. avium isolates were derived from patients with AIDS. These isolates include serovars 3b, 4, 4b, 8, and 20a and an untypeable strain. The remaining M. avium isolate was the Weybridge strain. Each of the eight antibodies that reacted with M. avium, MAC, or MAIS groups (Table 1) reacted with the 10 different M. avium strains tested (data not shown). Two other groups of antibodies listed in Table 1 may have clinical or laboratory significance. Four antibodies, 3808 C3, 3809 A12, 3913 G10, and 4028 G9, failed to react with tuberculous mycobacterial preparations. These MAbs may be useful in differentiating between infections with M. tuberculosis and infections with nontuberculous mycobacteria. Also, MAb 4028 G9 reacted with the Glaxo strain of M. bovis

1 2 3 4 5 6 7 8 9 101112 131415161718 1920 2122 2324 25

97-

66-

_

-

424

._

2717FIG. 1. Composite photograph of immunoblots illustrating the reactivities of the 25 anti-M. avium MAbs against M. avium protein antigens. The order of the MAbs is analogous to those listed in Table 1. Molecular weights (103) of protein standards are given on the left.

2598

INFECT. IMMUN.

ROUSE ET AL. TABLE 2. Species specificities of anti-M. avium MAbs"

MAb

AV1

AV2

BCG1

BCG2

Chel

Fort

Intr

Kan

Mar

Phl

Scr

Sme

TB1

TB2

Vac

Xen

3805 F9 3808 C3 3809 A12 3913 C12 3913 G10 3917 F1 3918 D12 3920 B10 3921 E4 3954B12 3958B4 3968E10 3982 A3 3989 C1 3994 B8 4004 F3 4028 G9 4034E11 4045 H12 4046 H1 4055 E6 4057 D2 4061 B10 4064 E6

++ ++

++ ++

++

++ -

++ -

++ -

++ +

++ +

++ +

++

++ ++ ++ ++ ++ ++ ++

++ ++ ++ ++ ++ ++ ++

++ + ++

++ + ++

-

+ ++ ++

++ ++ + + + ++ ++

++ + +

+ ++ +

++ +

++ + + ++ ++ ++ +

++ + ++ +

++ + ++ +

++ + ++ +

++

-

+ +

++ ++ ++ ++

++ ++

++ ++

-

-

-

-

-

-

-

-

-

-

-

-

+

+

+

++

++ ++

++ ++

++ ++

+ ++

++

++ ++

++ ++

++ ++

++ ++

++ ++

+ + ++

+ ++ ++

-

-

-

-

-

-

-

-

-

-

++

++

-

-

++ ++ +

++ ++ +

++ +

++

++ ++ ++

_

- +++ + + _ _ _ + ++ ++ + -

++ ++

+ +

-

++ ++

-

-

-

++

++

++ ++

++ ++

++ ++

++ ++

-

-

-

-

++

++

+

+

+

-

+

_

_

_

_

_

_

_

_

_

++ -

++ +

+ -

+ +

+ -

++ +

++ +

+ -

+ -

+

+

++

++

-

-

++ ++

++ ++

++ +

++ +

+ ++

++ ++

+ ++

+ ++

++

++

++

+

++

++

++

++

++

++

++ ++

++

++

-

-

-

-

-

-

-

-

-

-

-

-

-

-

+

a AV1, M. avium (Weybridge strain); AV2, M. avium (serovar 4); BCG1, M. bois (Glaxo BCG); BCG2, M. bovis (Tice BCG); Chel, M. chelonei; Fort, M. fortuitum; Intr, M. intracellulare; Kan, M. kansasii; Mar, M. marinum; Phl, M. phlei; Scr, M. scrofulaceum; Sme, M. smegmatis; TB1, M. tuberculosis (Erdman strain); TB2, M. tuberculosis (H37 Rv); Vac, M. maccae; Xen, M. xentopi. + +, strong reaction; +, moderate reaction; -, no reaction observed.

BCG but not with the Tice strain. Four other antibodies, 3805 F9, 3968 E10, 3982 A3, and 4061 B10, bound all of the mycobacterial sonic extracts tested and recognize common mycobacterial determinants. Isolation and characterization of recombinant bacteriophages. The M. avium Agtll library was screened with MAbs 3808 C3 and 3954 B12 as described in Materials and Methods. After purification of immunopositive plaques, bacteriophage DNA was isolated by the plate lysis method (16). EcoRI restriction analysis of DNA isolated from the recombinant bacteriophage recognized by MAb 3808 C3 revealed an M. avium DNA insert of 0.9 kb. This DNA fragment was found to have a HincIl site approximately 250 bp distal to lacZ (Fig. 2). EcoRI restriction of DNA extracted from MAb 3954 B12-reactive bacteriophage yielded no insert fragment. However, further restriction analyses clearly demonstrated that this phage contained a 2.8-kb DNA insert possessing 3808 C3

E H

E

0.9 kb 3954 B12

K

s

2.8 kb

FIG. 2. Restriction maps of the immunoreactive Agtll bacteriophages producing recombinant antigens which react with MAbs 3808 C3 and 3954 B12. The M. avium inserts for each bacteriophage are 0.9 and 2.8 kb, respectively. Restriction enzyme abbreviations: E, EcoRI; H, HincII; K, KpnI; S, SstI. Symbols: M, lacZ; C, left arm.

KpnI and SstI restriction sites (Fig. 2). Successful subcloning of predicted KpnI and SstI subfragments into plasmid pUC18 supported the predicted map of this M. avium DNA fragment. Production and characterization of A lysogens. Lambda lysogens were produced following established protocols (10). As seen in Fig. 3A, MAb 3808 C3 reacts in immunoblots with a 26-kDa protein in the M. avium sonic extract and with 140- and 26-kDa proteins in the X lysogen sonic extracts. The immunoreactive band observed at 140 kDa is the P-galactosidase hybrid protein. The 26-kDa band may be the result of proteolytic cleavage of the P-galactosidase fusion protein. Reactivity to this antibody was not seen in the E. coli Y1089 control. Figure 3B shows that antibody 3954 B12 reacts with a 33-kDa protein in the M. avium sonic extracts and with a 147-kDa fusion protein in the 3954 B12 X lysogen sonic extracts. The control Y1089 lysogen sonic extracts showed no reactivity to this antibody. Both of the large immunoreactive proteins were shown to react with an anti-,B-galactosidase MAb (data not shown). These results strongly suggest that these proteins contain a f-galactosidase component. Skin testing of purified recombinant antigens. Purified recombinant ,B-galactosidase fusion proteins were evaluated for their ability to induce delayed-type hypersensitivity (DTH) in Hartley strain guinea pigs sensitized with M. avium, M. intracellulare, and M. tuberculosis and in nonsensitized controls. As seen in Table 3, the 140-kDa 3-galactosidase fusion protein recognized by MAb 3808 C3 elicited significant skin test reactions. Intradermal challenge with this protein evoked a 7-mm reaction in animals sensitized with M. avium and a 12-mm reaction in M. intracellulare-immunized guinea pigs. This antigen also evoked a 9-mm reaction in animals sensitized with M. tuberculosis. Although the 147-kDa fusion protein recognized by MAb 3954 B12 induced only a weak skin test reaction in the M.

VOL. 59,

1991

M. AVIUM MAbs AND RECOMBINANT ANTIGENS

A

1

2

DISCUSSION

3

200 9768

42-

28 18B

1

2

3

2009768 42 -

28 FIG. 3. Western blots of 15 ,ug each of sonic extracts of (1) M. avium, (2) E. coli Y1089, and (3) 3808 C3 X lysogen. Each immunoblot was incubated with (A) MAb 3808 C3 or (B) MAb 3954 B12 and developed with the alkaline phosphatase blot detection system.

avium-sensitized animals (4 mm), moderate cell-mediated responses were detected for the M. intracellulare (7 mm)and M. tuberculosis (6 mm)-sensitized animals. This larger DTH response to specific purified antigens in heterologously sensitized animals has been observed previously (23). Neither of these fusion proteins evoked skin test responses in control nonsensitized animals. In addition, native ,B-galactosidase did not induce DTH reactions in control or sensitized animals. Therefore, the observed skin test responses appear to be due predominately to sensitivity to the M. avium portion of these hybrid proteins. TABLE 3. Skin test reactivities of M. avium fusion proteins in guinea pigs (n = 3)

Sensitization

Controls M. avium M. intracellulare M. tuberculosis

Avg diam (mean ± SD) of skin test reaction (mm) MAb MAb 3808 C3 3954 B12 Homologous (147 kDa) (140 kDa) soiexrc

NRa 7 2b 12 ± 2C 9 2C

NR 4 1 7 ± 2b 6 ± 1

2599

NR 12 1 12 ± 2 11 ± 1

a NR, nonreactive. b Significant (P < 0.1). c Significant (P < 0.05; Student's t test) increase compared with control.

In the past decade, the prevalence of MAC disease has increased dramatically in both the immunocompromised and the immunocompetent patient populations (7, 18). Disseminated MAC disease has become a frequent complication of AIDS patients (5, 9, 15, 21, 22). Because of the wide antigenic sharing among mycobacterial species, skin test and serologic tests are often not definitive in attributing an immune response to an infecting species. Individuals infected with MAC or M. tuberculosis, who may be immunocompromised and candidates for preventative therapy, need to be accurately diagnosed in order to select the appropriate treatment regimen. Cultured specimens are usually not available early in the infectious process. Even when a culture is available, cultivation and identification at the present time may require weeks. Our primary goal is to develop reagents which may be used as aids in the rapid and accurate diagnosis of mycobacterial infections. We have generated 25 MAbs against M. avium antigens. Four of the antibodies, 3954 B12, 3989 Cl, 4055 E6, and 4064 E6, are apparently specific for M. avium epitopes. Three additional antibodies, 3917 Fl, 4004 F3, and 4034 Eli, recognized only MAC determinants. MAbs 3808 C3, 3809 A12, 3913 G10, and 4028 G9 could be of clinical use in differentiating between M. avium and M. tuberculosis since they recognized antigenic determinants of M. avium and not M. tuberculosis. Ivanyi and coworkers (11) have successfully used two MAbs (TB 68 and TB 72) in the serodiagnosis of infections with the tubercle bacillus. Several of the antibodies described above may be similarly useful in direct serodiagnosis of MAC infections. There is considerable need for monospecific skin test reagents for differentiating sensitivities due to MAC infections (1-3). Our present focus is on the identification of immunologically active M. avium protein antigens which elicit significant skin test responses. The methodology we have used to achieve this goal is to generate MAbs directed against MAC protein antigens which can then be used to screen mycobacterial gene libraries. MAbs 3808 C3 and 3954 B12 were used to screen an M. avium gene library and to isolate recombinant bacteriophages which express M. avium fusion proteins. HPLC-purified recombinant mycobacterial proteins elicited significant DTH reactions in guinea pigs sensitized with homologous or heterologous mycobacterial preparations. Unfortunately, in contrast to the serologic specificity, the skin test responses induced by these fusion proteins were not specific. Previous assays with other purified and native recombinant antigens have also failed to demonstrate monospecific skin test reactivity (13, 17, 23, 24). Even the 38-kDa antigen of M. tuberculosis, which has been shown to be serologically monospecific, cross-reacted in delayed-type skin tests (12). The nonspecific DTH responses elicited by purified but multideterminant mycobacterial antigens, including the two M. avium fusion proteins, emphasize the need for the identification of MAC species-specific T-cell epitopes. Peptides inferred from the DNA sequence analyses of MAC antigen genes are being evaluated in our laboratory for their reactivity and specificity. We have generated peptides representing potential M. intracellulare T-cell epitopes and demonstrated that they elicit significant skin test responses. These data suggest that the production of monospecific skin test or serodiagnostic reagents developed from speciesspecific peptides may be feasible in the near future.

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REFERENCES 1. Chaparas, S. D. 1979. Why monospecific tuberculin skin test antigens have not been isolated. Bull. Int. Union Against Tuberc. 54:156-162. 2. Chaparas, S. D. 1984. Immune response, vaccination and antigen specificity in tuberculosis, p. 337-343. In D. Schlessinger (ed.), Microbiology-1984. American Society for Microbiology, Washington, D.C. 3. Chaparas, S. D. 1985. Tuberculin test-variability with the Mantoux procedure. Am. Rev. Respir. Dis. 132:175-177. 4. Chaparas, S. D., C. J. Maloney, and S. R. Hedrick. 1970. Specificity of tuberculins and antigens from various species of mycobacteria. Am. Rev. Respir. Dis. 101:74-83. 5. Collins, F. M. 1986. Mycobacterium avitum complex infections and development of the acquired immunodeficiency syndrome: casual opportunist or casual cofactor? Int. J. Lepr. 54:458-474. 6. Collins, F. M. 1988. AIDS-related mycobacterial disease. Springer Semin. Immunopathol. 10:375-391. 7. Collins, F. M. 1989. Mycobacterial disease, immunosuppression, and acquired immunodeficiency syndrome. Clin. Microbiol. Rev. 2:360-377. 8. Eisenbach, K. D., J. T. Crawford, and J. H. Bates. 1986. Genetic relatedness among strains of the Mycobacterium tuberculosis complex. Am. Rev. Respir. Dis. 133:1065-1068. 9. Hawkins, C. C., J. W. M. Gold, E. Whimbery, T. E. Kiehn, P. Brannon, R. Cammarata, A. E. Brown, and D. Armstrong. 1986. Mycobacterium avium complex infections in patients with the acquired immunodeficiency syndrome. Ann. Intern. Med. 105: 184-188. 10. Huynh, T. V., R. A. Young, and R. W. Davis. 1984. Construction and screening cDNA libraries in Xgtl0 and Xgtll, p. 49-78. In D. Glover (ed.), DNA cloning techniques: a practical approach. IRL Press, Oxford. 11. Ivanyi, J., G. H. Bothamley, and P. S. Jackett. 1988. Immunodiagnostic assays for tuberculosis and leprosy. Br. Med. Bull. 44:635-649. 12. Kadival, G. V., S. D. Chaparas, and D. Hussong. 1987. Characterization of serologic and cell-mediated reactivity of a 38-kDa antigen isolated from Mycobacterium tuberculosis. J. Immunol. 139:2447-2451. 13. Kingston, A. E., P. R. Salgame, N. A. Mitchison, and M. J. Colston. 1987. Immunological activity of a 14-kilodalton recombinant protein of Mycobacterium tubercuilosis H37Rv. Infect. Immun. 55:3149-3154. 14. Kohler, G., and C. Milstein. 1975. Continuous culture of fused cells secreting antibody of predetermined specificity. Nature (London) 256:495-497. 15. Macher, A. M., J. A. Kovars, V. Gill, G. D. Roberts, J. Ames,

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C. H. Pack, S. Strans, H. C. Lane, J. E. Parillo, A. S. Fauci, and H. Masur. 1983. Bacteremia due to Mycobacteriurm aviumintrac ellulare in the acquired immunodeficiency syndrome. Ann. Intern. Med. 79:782-785. 16. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular

cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 17. Morris, S. L., D. A. Rouse, D. Hussong, and S. D. Chaparas. 1988. Isolation and characterization of a recombinant Xgtll bacteriophage which expresses an immunoreactive Mycobacte18.

19.

20.

21. 22.

23.

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Immunological characterization of recombinant antigens isolated from a Mycobacterium avium lambda gt11 expression library by using monoclonal antibody probes.

Nontuberculous mycobacteria, particularly Mycobacterium avium, have been isolated from a significant percentage of patients with AIDS. Early detection...
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