(~) INSTITUTPASTEUR/ELsEVIER Paris 1990

Res. MicrobioL 1990, 141, 859-871

PROTEIN ANTIGENS OF M Y C O B A C T E R I U M L E P R A E J . E . Clark-Curtiss (i. 2), J . E . R . Thole (2) (,), M. Sathish (2) (**),

B.A. Boseeker (2), S. Sela (2), E.F. de Carvalho (2) and R . E . Esser (2) (***)

(t) Departments of Molecular Microbiology and of (2) Biology, Washington University, St. Louis, MO (USA) 63130

Summary. Protein antigens of Mycobacterium leprae have been identified by screening the ),gtl 1, pYA626 and pHC79::M, leprae genomic libraries with pooled sera from leprosy patients and with antiserum to M. leprae cell wall protein (CWP) aggregate. Immunological screening of the ),gtl I library with pooled sera from 21 lepromatous (LL) leprosy patients resulted in the identification of 19 antigens that are apparently different from previously identified M. leprae antigens. Five additional antigens were identified by screening the ),gt I 1 library with pooled sera from 30 borderline tuberculoid or tuberculoid patients. Four other antigens were identified by screening the ),gtl 1 library with anti-CWP. Two groups of recombinant cosmids were identified by screening the pHC79 library with LL patients' sera: one group specified proteins that reacted with monoclonal antibodies (mAb) against the 65-kDa protein and against the 18-kDa protein; the other group specified a 15-kDa protein that did not react with any of the mAb that were tested. One pYA626 clone also specified a 15-kDa protein that reacted with LL patients' sera, but did not react with any mAb. Genes specifying several of these antigens have been subcloned into the Asd + plasmid vector pYA292 and have been introduced into a Acya Acrp Aasd Salmonella typhimurium strain to evaluate the ability of individual M. leprae proteins to elicit immune responses against M. leprae infection. KEY-WORDS:Mycobacterium leprae, Protein antigen, Leprosy, Salmonella; Plasmid stability: Immunological screening, Recombinant avirulent live vaccine.

Introduction. The disease of leprosy has plagued humankind since the beginning of recorded history (Binford et ai., 1982; Jopling, 1984; Bloom, 1986). Although leprosy is un-

(*) Presentaddress: Dept.of lmmunohaemotologyand BloodBank,UniversityHospital,PO Box 9600, 2300 RC Leiden(The Netherlands). (**) Presentaddress: Hansen'sDiseaseLaboratory,Centersfor DiseaseControl.Atlanta, GA 30333 (USA). (***) Present address: Marion Laboratories,Kansas City, MO 64134-0627(USA).

J.E. C L A R K - C U R T I S S E T A L .

860

common in many parts of the world today, there are still approximately 12 million people afflicted with the disease, mostly in the tropical and subtropical nations of the world (Bloom, 1986). The disease is caused by the obligate intracellular parasite Mycobacterium leprae, which is believed to enter the body via inhalation of nasal droplets from heavily infected individuals, although the mechanism of entry is not understood (Binford et a'., 1982; Jopling, 1984). Once inside the body, M. leprae is taken up by macrophages, the primary cells in which the bacilli multiply (Binford et al., 1982; Jopling, 1984). M. leprae is also able to invade and multiply in Schwann cells (Binford et al., 1982; Jopling, 1984). In humans, the severity of the ensuing disease is determined by the immunological competence of the infected individual (Bloom and Godal, 1983; Jopling, 1984; Bloom, 1986; Kaplan and Cohn, 1986). Initially, individuals develop what is termed indeterminate or borderline leprosy. The majority of infected humans mount a sufficient immune response to clear the bacilli before few, if any, symptoms of leprosy are manifested. However, a small percentage of infected individuals progress to one of two extreme disease states, lepromatous leprosy or tuberculoid leprosy (Bloom and Godal, 1983; Jopling, 1984; Kaplan and Cohn, 1986). In persons who develop lepromatous (LL) leprosy, the cell-mediated immune response to M. leprae is strongly suppressed, the bacilli multiply freely and the patients have high titres of circulating antibodies against a diversity ofM. leprae antigens (Bloom and Godal, 1983; Jopling, 1984; Kaplan and Cohn, 1986). In contrast, tuberculoid (TT) leprosy patients are able to mount at least a partial cell-mediated response against M. leprae, have very low titres of circulating antibodies and have few detectable bacteria in tissue biopsies and secretions (Bloom and Godal, 1983; Jopling, 1984; Kaplan and Cohn, 1986). Although M. leprae was the first bacterial species to be implicated as the probable cause of an infectious disease (Hansen, 1874), cultivation of the bacilli in conventional laboratory media or by conventional tissue culture techniques has been unsuccessful thus far, due partly to the fact that the generation time for M. leprae (in vivo) has been estimated to be approximately 14 days (Jopling, 1984). However, the hind footpads of mice can" be infected with 103 to 104 M. leprae and sustain a limited multiplication of the bacilli (approximately 2 to 3 logs) over a 9- to 12-month period (Shepard, 1960). The nine-banded armadillo (Dasypus novemcinctus) has proven to be the best host for propagation of M. leprae cells in that, 18 to 24 months after experimental inoculation of the armadillo, the liver and spleen contain more than 109 bacilli per gram of tissue (Kirchheimer and Storrs, 1971 ; Storrs, 1971). The ability to purify large quantities o f M . leprae from infected armadillo tissue has enabled researchers to characterize and identify components of the M. leprae cell, and in conjunction with recombinant DNA technologies, to elucidate some of the molecular genetic attributes of this organism. Although there are antimicrobial agents that are effective in treating leprosy (Waters, 1989), an alternative or complementary approach to controlling leprosy would be the development of a safe, effective vaccine. The availability of M. leprae cells from infected armadillo tissue has also permitted significant progress in the identification and characterization of antigens. Early studies made use of the technologies of immunoelectrophoresis or sodium dodecyl sulfate (SDS)-polyacrylamide gels to identify antigens on the basis of molecular mass and reactivity with sera from leprosy patients or infected animals (Harboe et al., 1978; Closs et al., 1979; Klatser et al., 1984; Ehrenberg and Gebre, 1987; Britton et al., 1988; Vega-Lopez et al., 1988).

BT CFU CWP DAP

= = = =

borderlinetuberculoid. colony-formingunit. cell wall protein, diaminopimelicacid.

DTH LL mAb TT

= = = =

delayed-typehypersensitivity. lepromatousleprosy. monoclonalantibody. tuberculoidleprosy.

PROTEIN ANTIGENS

O F MYCOBACTERIUM LEPRAE

861

These studies showed that the major protein antigens of M. leprae that were recognized by LL patients' sera were of low molecular mass: proteins of 33, 22-27, 15 and 12 kDa were most frequently recognized by antibodies in the sera of leprosy patients analysed by Klatser et al. (1984), Britton et al. (1988) and Vega-Lopez et aL (1988). Monoclonal antibodies (mAb) have been prepared against sonic extracts of M. leprae and have been used to identify and partially characterize several protein antigenic determinants (Gillis and Buchanan, 1982; Ivanyi et al., 1983; Engers et al., 1985; Atlaw et al., 1985). The elegant dissection of the cell wall structures by thorough chemical analyses of M. leprae by Brennan, Hunter and their colleagues has revealed the complexity of these structures, but has also identified a number of components that are highly immunogenic, including phenolic glycolipid I (Hunter et al., 1982), lipoarabinomannan (Hunter et aL, 1986) and proteins that are integral components of the M. leprae cell wall (Hunter et aL, 1989). However, the supply of M. leprae cells from infected armadillo tissue was inadequate to permit large-scale purification and chemical analysis of many of the protein antigens. Recombinant DNA technology has also aided investigators in the identification of protein antigens of M. leprae. Young et al. (1985) screened a kgtl I::M. leprae genomic library with mAb and identified recombinant clones that specified five protein antigens. Several of these have been further characterized and have been shown to be highly homologous to the heat-shock or stress proteins of Escherichia coli and other bacteria (Shinnick et al., 1988; Young et al., 1988; Garsia et al., 1989). Thangaraj et al. (1989) have recently deduced that the 28-kDa protein identified by mAb screening i.q the superoxide dismutase of M. leprae. We have screened the ).gt1I::M. leprae library with pooled sera from leprosy patients and have identified 19 antigens that are recognized by antibodies in LL patients' sera and five antigens that are recognized by antibodies in TT patients' sera (Sathish et al., 1990). More recently, Hartskeel et al. (1990) have screened a pEX2::M, leprae genomic library with sera from household contacts of leprosy patients and have identified a number of clones that specified proteins that reacted with antibodies in the contacts' sera but not with any mAb to a variety of M. leprae antigens. Since stimulation of cell-mediated immunity to M. leprae appears to be the mechanism whereby a protective immune response occurs, it is important to identify antigens that stimulate a T-cell response (Jopling, 1984; Bloom, 1986; Kaplan and Cohn, 1986). Antigens identified by their reactivity with antibodies may or may not have epitopes that stimulate T-cell proliferation; therefore, several groups have been trying to define M. leprae antigens that elicit a T-cell response. Mendez-Sampiero et al. (1989) studied the reactivity of SDS-PAGE-separated proteins ofM. leprae with T cells from household contacts and leprosy patients and determined that the main immunogenic fractions for both contacts and patients that caused lymphocyte stimulation were in three size ranges: proteins of 12-22 kDa and of 35-40 kDa stimulated lymphocytes from TT leprosy patients and their contacts to proliferate, whereas lymphocytes from LL patients and their contacts responded more frequently to the 65-kDa protein fraction. These findings were corroborated by the studies of Ottenhoff et al. (1989). In a similar study of lymphocyte proliferation patterns among staff members with extensive occupational exposure to leprosy patients, Converse et al. (1988) found that SDS-PAGE-separated proteins from M. leprae in the range of 22 to 26 kDa elicited the most frequent stimulatory responses. Several other groups have studied the T-cell-stimulating activity of specific protein antigens. Mehra et al. (1989) used a cell wall protein-peptidoglycan complex and a cell wall protein preparation devoid of lipids and carbohydrates to determine that proteins with molecular masses of 28, 16, and 7 kDa elicited the greatest reactivity from T-cell clones derived from leprosy patients. Thole, Klatser, Ottenhoff and their colleagues have demonstrated that a proline-rich 36-kDa protein carries antigenic determinants both common to mycobacteria and specific for M. leprae~ some of these determinants are recognized by antibodies (Thole et ai., 1990; de Wit and Klatser,

862

J.E. CLARK-CURTISS

ET AL.

1988) and others are recognized by T ,:ells (Ottenhoff et 6.:., 1986; van Schooten et al., 1988) from leprosy patients. Mohagheghpour et al. (1990) have isolated a 35-kDa protein from M. leprae cells which they have shown to react with antibodies in LL patients' sera, with mAb ML03-A I and with T cells from leprosy patients. Gelber et al. (1990) used this protein to vaccinate mice via intradermal injection and showed that the mice were protected against growth of M. leprae cells in the mouse footpad assay. The 18-kDa antigen, originally identified by screening the ;~gtl 1::M. leprae library with mAb MC8026-S (Engers et al., 1985), has recently been shown to elicit proliferative responses by T cells from both untreated TT leprosy patients and by long-term leprosy contacts (Dockrell et al., 1989). Therefore, within the past three years, a variety of approaches have been used to successfully identify protein antigens of M. ieprae that stimulate both B- and T-cell reactivity. Which of these proteins are most important in inducing a protective response against M. leprae remains to be determined. Materials and methods.

Recombinant D N A libraries. - - We used the ~.gtI I::M. leprae library (Young et al., 1985) and the pYA626::M, leprae and pHC79::M, leprae libraries (Clark-Curtiss et al., 1985). Antisera. m The origins and preparation of the pooled sera from leprosy patients were described by Sathish et al. (1990). Antiserum against the M. leprae cell wall aggregate obtained after muramidase treatment of the cell wail protein-peptidoglycan complex was the generous gift of P.J. Brennan (Mehra et al., 1989). Immunological screening o f the genomic libraries. ~ The methods for immunological screening of the ),gt 1I::M. leprae library were described by Young et al. (1985) and Sathish et al. (1990). The pYA626 and pHC79 libraries were screened with the same pooled sera from LL leprosy patients by colony immunoblotting (Sambrook et al., 1989). Recombinant techniques and molecular biology methods. ~ Standard molecular biology methods (Sambrook et al., 1989) have been employed, with minor modifications, as described by Sathish et al. (1990). Measurement o f recombinant plasmid stability in Acya Acrp Aasd S. typhimurium strains. - - Subclones of M. leprae DNA insert fragments in Asd + plasmid pYA292 (Galan et al., 1990) were introduced into S. typh#nurium strains X3987 (Curtiss et al., 1990) or X4072 (Nakayama et al., 1988) by transformation or electroporation. S. typhimurium strains containing the pYA292 subclones were grown and used for oral immunization of eight week-old BALB/c mice by the methods described by Curtiss and Kelly (1987). At 1, 4, 7, 10, 14 and 21 days after inoculation, two or three mice were sacrificed and the titres of S. typhimurium in Peyer's patches, spleen and liver were determined by plating on MacConkey-lactose agar plus diaminopimelic acid (DAP) and, when ;~4072 subclones were used, nalidixic acid to preclude growth of other bacteria (Curtiss and Kelly, 1987). The colonies were replica-plated to MacConkey agar without DAP to determine the number of colonies that were asd + due to the presence of pYA292. Twenty colonies from each site were used for extraction of plasmid DNA, which was spotted onto nitrocellulose filters and hybridized with the M. leprae insert to determine whether or not the M. leprae genes were stably maintained in S. typhimurium.

PROTEIN ANTIGENS OF MYCOBACTERIUM

LEPRAE

863

Results.

Protein antigens identified by immunological screening of genomic libraries with sera fro~., leprosy patients. As we described in greater detail previously (Sathish et al., 1990), we have screened the ),gtl I::M. leprae genomic library with two pools o f sera from leprosy patients. Using a pool composed o f sera from 21 lepromatous (LL) leprosy patients, a total o f 45 plaques (out o f approximately 5 x 105 plaques screened) contained proteins that reacted with antibodies in the pool. These clones were further characterized to determine the size o f the insert D N A fragments, the size o f the proteins specified b y the clones a n d the reactivity o f the proteins with the T T patients' serum pool and 30 m A b against a variety o f M. leprae proteins. The clones were placed into eight multiple clone groups based on hybridization experiments between the M. leprae insert D N A fragments. In a d d i t i o n to these groups, there were eleven recombinant molecules whose insert fragments did not hybridize to any other insert and thus appear to contain unique D N A fragments. The results o f the characteristics o f the clones are summarized in table I. Most o f the clones specified fusion proteins with [~-galactosidase, with molecular masses o f 117 to 175 kDa. However, there were a few clones that specified non-fusion proteins o f 38, 42, 45 and 75 kDa. The reactivity o f the recombinant proteins with the L L patients' sera varied from very weak ( + / - ) to very strong ( + + + + ) (fig. 1), sometimes even a m o n g members o f the _~ame hybridization group. F o r example, the reactivity o f proteins specified by four clones from group I can be compared by looking at lanes 4, 9, 13 a n d 14 o f figure 1. Since the insert D N A fragments and the proteins from these clones varied in size, one explanation for these results is that the different clones contained variable amounts o f the genes or different parts o f the genes, so that the proteins specified by the clones contained different epitopes, some o f which were more immunoreactive than others. When the proteins specified by these clones were reacted with the TT patients' sera, variable reactivity.was observed again.

TABLE I. - - A n t i g e n i c d e t e r m i n a n t s reactive w i t h L L patients' sera.

Number of clones G r o u p (*) identified I II III IV V VI VII VIII Others

6 7 2 2 2 3 1 3 2 2 2 11

Range o f molecular mass o f recombinant proteins (kDa) 117-164 120-175 128-155 119/45 (**) 45 120-129 38/42 (**) 125-140 154-155 155/160 (**) 135/145 (**) 75/165

Reactivity with: L L sera TT sera

mAb

+ + + to + + + + - to + MC0401A + / - to + + + - to + + + + to + + + + + / - to + + + / - to + + - to + + / - to + + + - to + + + + + + +/+ + + + + - to + + + + + + + / - to + + + + - to + -

(*) Determined by DNA hybridizations between M. lepraeDNA insert fragments; those classified as "Others" did not hybridize to any insert DNA other than their own. (**) More than one protein specified by the recombinant molecule was observed on Western blots.

864

J.E. C L A R K - C U R T I S S

ET AL.

I 2345 6 7 8 9 1011121314151617181920 IV.'-

200-

t

l=

-

.g

"1,

116-

84-

"

5848.5 -

FIG. 1. - - Western blot o f proteins specified by ~gtl I::M. leprae clones which reacted with sera from LL patients. Lanes: 1, molecular size markers; 2, E. coil Y I090 infected with ).gtl I (control); 3, blank; 4, clone LI6; 5, clone LI5; 6, clone LI4; 7, clone LI3; 8, clone LI2; 9, clone LI1; 10, clone LI0; 11, clone L9; 12, clone LS; 13, clone L6; 14, clone L5; 15, clone L4; 16, clone L3; 17, clone L2; 18, clone LI ; 19, molecular size markers; 20, E. coli YI090 without ).gtl 1. The numbers on the left of the blot indicate molecular sizes in kilodaltons (after Sathish et al., 1990).

Most of the proteins that reacted with the LL patients' sera reacted weakly or not at all to the T T patients' sera, except for the proteins from one clone of hybridization group I (clone L30) and one from group VII (clone L32), which reacted as well or better than the proteins from clones identified by their reactivity with T T patients' sera (see below and figure 2). Only members of hybridization group I specified proteins that reacted with any of the 30 m A b against which they were tested; these recombinant proteins reacted with one m A b directed against an epitope of the 65-kDa protein. However, since ).gt I 1 clone Y3178, which contains the entire coding sequence for the 65-kDa protein (Mehra et al., 1986), did not hybridize to clones in this group, and since the group I proteins did not react with any of the other m A b ~.~ainst the 65-kDa protein, we hypothesize that the gene present in clones of gloup I specifies a protein with a crossreactive epitope to YI.2 (Sathish et al., 1990).

P R O T E I N A N T I G E N S OF MYCOBACTERIUM LEPRAE

865

The proteins specified by at least one member of each hybridization group were tested by Western blot analysis for their reactivity against the individual sera that were used to prepare the LL patients' pool. These studies revealed that the proteins specified by the members of groups I through IV and two of the apparently unique clones were recognized by antibodies in 50 to 90 % of the individual patient's sera. In contrast, proteins specified by clones from groups V through VIII were recognized by only a small percentage (5 to 30 %) of the individual patient's sera. The ),gtll::M. leprae library was also screened with a pool of sera from 30 tuberculoid (TT) and borderline tuberculoid (BT) patients (Sathish et al., 1990). Five out of approximately 10s plaques screened contained proteins that reacted with antibodies in the pooled sera, although none of these recombinant proteins reacted as strongly with the B T / T T serum pool as the stronger reacting proteins discussed above reacted with the LL serum pool (figure 2). Hybridization experiments demonstrated that none of the insert fragments from the TT-reactive clones hybridized to any other TT-reactive or LL-reactive clone; thus each clone appears to contain a unique DNA fragment. The proteins specified by these clones did not react with the LL serum pool nor with any of the mAb (Sathish et al., 1990). Recently, the pYA626 and pHC79:~M, leprae genomic libraries (Clark-Curtiss et al., 1985) were screened with the LL patients' pooled sera by colony immunoblot.

I

2

:3

4

5

6

7

8

9

I0 II

12 13 14

200-

116- e ~ 97~,.

66.2 -

~

42.6 ~

~

~

~

~,

.........

9

FIG. 2. - - Western blot of proteins specified by )~gtll::M. [eprae clones which reacted with sera from B T / T T patients• Lanes: 1, clone L30; 2, clone L31 ; 3, clone L32; 4, clone L41 ; 5, clone L42; 6, clone L43; 7, clone L44; 8, clone L45; 9, clone TI; 10, clone T2; 11, clone T3; 12, clone T4; 13, nonrecombinant ).gtl I (control); 14, clone T5. The numbers on the left of the blot indicate molecular sizes in kilodaltons (after Sathish et al., 1990).

866

J.E. C L A R K - C U R T I S S E T A L .

Two recombinant plasmids and several hundred cosmids specified proteins that reacted with antibodies in the pooled sera. Fourteen cosmids were chosen for further analysis: these could be divided into two groups. One group consisted of 10 cosmid clones that had a 10-kb Pstl fragment and a 3-kb PstI fragment in common (as well as a number of PstI fragments of various sizes that were unique to each cosmid). The 10-kb PstI fragment carried a copy of the M. leprae repetitive sequence (Clark-Curtiss and Docherty, 1989) determined by hybridization experiments using the repetitive DNA sequence probe. This group of cosmid clones also specified proteins that reacted with two pools of mAb by colony immunoblot assays: one composed of mAb against high molecular weight M. leprae proteins (65 kDa and larger) and the other of mAb against low molecular weight M. leprae proteins (12 kDa to 43 kDa). The proteins specified by three of these cosmids were tested against the individual mAb by ELISA (Vos et al., 1979). Recombinant proteins from all three cosmids reacted strongly with mAb against the 65-kDa protein and the 18-kDa protein and weakly with mAb in the 33-35-kDa range. It is likely that these clones contain the gene for the 65-kDa protein and hybridization experiments currently underway with Y3178 will resolve this question. However, since the clones specify proteins that react with several other mAb, we will localize the genes for these proteins on the cosmid fragments in order to obtain information about possible spatial relationships among genes for immunologically reactive proteins on the M. leprae chromosome. The other four cosmids that comprised the second group of cosmids specifying LL serum-reactive proteins all contained a common 1.2 kb Pstl fragment. Moreover, one of the pYA626 clones also contained a 1.2 kb Pstl fragment. All five of these recombinants specified a 15-kDa protein that reacted with antibodies in the LL serum pool, but this 15-kDa protein did not react with either of the mAb antibody pools described above. The 1.2 kb PstI fragment from the pYA626 clone was subcloned into pUC13 (Messing, 1983) and was shown to express the 15-kDa protein that reacted with LL patients' scra. The second pYA626 clone has not yet been further characterized.

Protein antigens identified by immunological screening of the kgt I 1::M. leprae library with anti-cell wall protein (CHIP) aggregate serum. In conjunction with their studies on the immunoreactivity of various components of the M. leprae cell wall, Brennan and his colleagues prepared polyclonal antisera against several preparations of cell wall components, including CWP (Mehra et al., 1989). Anti-CWP was used to screen approximately 105 plaques of the ~,gtl 1::M. leprae library and seven clones that expressed proteins that reacted with the serum were recovered. The three strongest-reacting plaques probably specified the 65-kDa protein, since these plaques also reacted with a pool of nine mAb against the 65-kDa protein (Sathish et al., 1990). Proteins from the other four clones reacted less strongly to anti-CWP serum, but did not react with any of the four mAb pools used to test the LL serum-reactive clones (Sathish et al., 1990). By Western blot analysis, it was determined that the recombinant proteins specified by these four clones ranged in size from 125 to 150 kDa; the proteins did not react with the LL patients' serum pool, but reacted weakly with the B T / T T patients' serum pool.

Construction of avirulent S. typhimurium strains expressing M. leprae antigens. In order to evaluate the potential for elicitation of a protective immune response by the recombinant proteins described above, we have subcloned several of the M. leprae DNA insert fragments (clones L6, LI4, and LI7) into one of the Asd + plasmid vectors developed by R. Curtiss III and colleagues for use in their balancec~lethal avirulent S. typhimurium vaccine delivery system (Curtiss et al., 1989; Curtiss

PROTEIN ANTIGENS OF MYCOBACTERIUM LEPRAE

867

TABLEII. ~ Recovery of S. typhtmurlum X4072 with pYA1077 from BALB/e mice. Day of sacrifice CFU from post-inoculation (*) Peyer's patches Asd+ 1 4

< 10 2.8 x 102

ND ND

7 10 14 21

6.0× 103 6.1×103 3.7x 103 5.0× 103

233/233 117/117 65/65 97/99

CFU from spleen

Asd +

CFU from liver

Asd+

< 10 < 10

ND ND

ND 9.3 × 10 i

ND ND

2.8× 102 47/47 3.5× 102 52/52 3.5×103 722/722 2.6×103 395/395 3.5× 102 39/39 5.3×102 83/83 9.2× 102 184/184 1.1 × 102 8/8

(*) Inoculatedper orally with 9.6× 10s cfu/ml by methods describedby Curtiss and Kelly(1987). ND = not determined.

et al., this volume). In addition, we are isolating the whole genes specifying several other antigens from the pHC79::M, lepra¢ libra: y, using the ~,gtl I::M. leprae DNA insert fragments as probes. When the whole ge~es are identified, they will be subcloned into pYA292 (Galan et al., 1990). The pYA292 subclones have been or will ~.e ~.roduced into Acya Acrp Aasd •S. typhimurium strains X3987 or x4072. Experiments have been conducted to measure the stability of one of the pYA292 subclones, designated pYA1077, in S. typhimurium X4072. This subclone contv;ns the M. ieprae insert fragment from ~,gtl I clone L14 and specifies a protein of ~vproximately ~3 kDa, which reacts strongly with LL patients' sera by Western blot ~n~!y~i~. The data from the plasmid stability experiment with X4072 (pYAI077) are presented in table II. S. typhimurium X4072 (pYA1077) colonies were recovered from Peyer's patches at detectable frequencies from 4 days after inoculation through the duration of the experiment (21 days) and from the spleen and liver from 7 days after inoculation through the end of the experiment. A total of 2,042 colonies out of 2,044 tested (99.9 %) were Asd+, indicating that the~ had ~etained pYA292. All of the plasmids tested for the presence of the M. leprae insert were positive. Thus, as had been shown previously for Streptococcus mutam gene~ cloned into Asd + vectors (Nakayama et al., 1988; Curtiss et ai., 1989), pYA1077 w~.s very stable in the Acya Acrp Ausd S. typhimurium strain X4072. The second type of experiment to be done ~:ith the recombinant S. typhimurium strains is to determine whether or not the M. leprae proteins can elicit a delayed-type hypersensitivity (DTH) reaction in mice. Thus, the recombinant S. typhimurium strains have been or will be used to orally vaccinate mice; two weeks later, the mice have been or will be tested for DTH reactions to M. leprae sonicates, to 5. typhimurium sonicates and to the partially purified recombinant proteins specified by the M. leprae genes being studied. DT~-I responses will be determined by measuring the amount of footpad enlargement o~served 24 to 48 h after challenge with the sonicates and proteins compared to the -~mount of footpad enlargement observed 3 to 6 h after challenge (which would be indicative of an immediate hypersensitivity reaction). The pYA292 subclones that elicit a positive DTH response to M. leprae sonicates and to the recombinant proteins will then be used to immunize other mice to evaluate the ability of the M. leprae recombinant protein to prevent growth of live M. leprae cells in the footpads of the immunized mice as compared to the growth of M. leprae cells in the footpads of unimmunized mice (Shepard, 1960).

868

J.E. C L A R K - C U R T I S S E T A L .

DTH experiments with g4072 (pYAI077) have shown that this recombinant protein does not elicit a DTH response against M. leprae (data not shown). Moreover, gt~e protein specified by the LI4 insert DNA fragment does not stimulate T-cell proliferation in in vitro assays (data not shown). Discussion. We have identified a number of protein antigens of M. leprae by screening the ~.gt11, pYA626 and pHC79::M, leprae genomic libraries with sera from leprosy patients and with antiserum against M. leprae cell wall proteins. Most of these antigens are different from the antigens identified earlier by immunological screening of the ).gtl 1::M. leprae library with mAb. The important question that still remains to be answered is which, if any, of these antigens is(are) important in eliciting a protective immune response against M. leprae infection. Although protection against M. leprae in humans is governed by the cell-mediated branch of the immune system rather than the humeral branch, our rationale for studying antigens identified by screening recombinant libraries with antiserum is that many antigens, including mycobacterial antigens (Dockrell et al., 1989; Thole et al., 1990; Mohagheghpour et al., 1990), have been shown to carry different epitopes, some of which are involved in B-cell recognition and others which are involved in T-cell recognition. Thus, we anticipate that the antigens identified by their B-cell epitopes will contain T-cell epitopes that we can ultimately define by subcloning and ~n~lysis, by immunization and by reactivity with T-cell clones from leprosy patients and/o~ household contacts. In this way, it should ultimately be possible to construct a vaccine with multiple important T-cell epitopes capable of inducing a protective immune response against M. leprae infection. The use of the balanced-lethal avirulent S. typhimurium vaccine delivery system permits an in vivo evaluation of DTH responses to specific M. leprae antigens. When M. leprae antigens that elicit a protective response are identified, use of the avirulent S. typhimurium vaccine delivery system may prove to be very effective in protecting mice against M. leprae infection, since all three branches of the immune system are stimulated (see review by Curtiss, 1990). While stimulation of the cell-mediated response against M. leprae will be the primary means of protection, stimulation of a mucosal immune response against surface antigens of M. leprae might lessen the likelihood of contagious spread of M. leprae by aerosol inhalation. If this pr:. :es to be the case, introduction of pYA292 subclones carrying M. leprae genes for such protective antigens into avirulent Salmonella typhi may very well result in an effective vaccine against leprosy and typhoid in humans. Acknowledgements.

The researchwas supported by postdoctoralfellowshipsfrom the HeiserProgram for Researchin Leprosyawardedto R.E.E., M.S. and S.S., by a postdoctoralfellowshipfromthe Immunologyof Leprosy (IMMLEP)componentof the United Nations DevelopmentProgram/WorldHealth OrganizationSpecial Programmefor Researchand Trainingin TropicalDiseasesawardedto E.F.C. and by PublicHealth Servicegrants A126186and A123470from the National Institutes of Health, References.

ATL^W,T., KOZBOR,D. &RODER,C. (1985), Human monoclonal antibodies against Mycobacterium leprae. Infect. lmmun., 49, 104-110. BLOOM,B.R. (1986), Learning from leprosy: a perspective on immunology and the Third World. J. Immunol., I37, i-x.

PROTEIN ANTIGENS OF MYCOBACTERIUM LEPRAE

869

BLOOM, B.R. & GODAL,T. (1983), Selective primary health care: strategies for control of disease in the developing world. - - V. Leprosy. Rev. infect. D/s., .4;,765-780. BINFORD,C.H., MEYERS,W.M. & WALSH,G.P. (1982), Leprosy. J. Arner. Med. Ass., 247, 2283-2292. BRITTON,W.J., HELLQVIST,L., GARSIA,R.J. & BASTEN,A. (1988), Antigens of Mycobacteriurn leprae identified by immunoprecipitation with sc~'afrom leprosy and tuberculosis patients. Clin. exp. Imrnunol., 71, 394-398. CLARK-CURTISS,J.E., JACOBS,W.R., DOCHERTY,M.A., RITCHIE,L.R. & CURTISS,R. III (1985), Molecular analysis of DNA and construction of genomic libraries of Mycobacteriurn leprae. J. Bact., 161, 1093-1102. CLARK-CURTISS,J.E. & DOCHeRTY,M.A. (1989), A species-specific repetitive sequence in Mycobacteriurn leprae DNA. J. infect. D;.s., 159, 7-15. CLOSS,O., MSHANA,R.H. & HARBOE,M. (1979), Antigenic analysis of Mycobacteriurn leprae. Scand. J. Irnrnunol., 26, 673-681. CONVERSE,P.J., OTTENHO~,T.H.M., GENRE,N., EHRENSERC,J.P. & KIF.SSLI~3,R. (1989), Cellular, humoral and gamma interferon responses to Mycobaeterium leprae and BCG antigens in healthy individuals exposed to leprosy. Scand. J. Immunol., 27, 515-521. CURTISS,R. Ill (1990), Attenuated Salmonella as live vectors for expression of foreign antigens, Chap. 13, in "New generation vaccines: the molecular approach" (G.C. Woodrow and M.M. Levine). Marcel Dekker, Inc., New York. CURTISS,R. III, GALAN,J., NAKAYAMA,K. & KELLY,S.M. (1990), Stabilization of recombinant avirulent vaccine strains in vivo. Res. Microbiol., 141 (in press). CURTISS, R. I I I & KELLer,S.M. (1987), Salmonella typhirnuriurn deletion mutants lacking adenylate cyclase and cyclic AMP receptor protein are avirulent and immnnogenic. Infect. Irnmun., 55, 3035-3043. CURTmS,R. III, KELLY,S.M., Guuo, P.A. & NAKAYAMA,K. (1989), Selective delivery of antigens by recombinant bacteria. Curt. Top. Microbiol. IrnrnunoL, 146, 35-49. CURTISS,R. III, PORTER,S.B., MUNSON,M., TINGE,S.A., HASSAN,J.O., GENTRY-WEEKS,C. & K~LL~f, S.M. (1990), Nonrecombinant and recombinant avirulent Salmonella live vaccines for poultry, in "Colonization control of human bacterial enteropathogens in poultry" (L.C. Blankenship, J.S. Bailey, N.A. Cox, N.J. Stern, R.J. Meinersmann). Academic Press, New York, London (in press). De WIT, M.Y.L. & KLATSER,P.R. (1988), Purification and characterization of a 36-kDa antigen of M. leprae. J. gen. Microbiol., 134, 1541-1548. DOCKRELL,H.M., STOKER,N.G., LEE, S.P., JACKSON,M., GRANT,K.A., JONO, N.F., LUCAS,S.B., HASAN,R., HUSSAIN,R. & McAD~, K.P.W.J. (1989), T-cell recognition of the 18-kilodalton antigen of Mycobacteriurn leprae. Infect. Irnrnun., 57,1979-1983. El~oEas, H., Ace, M., BLOOM,B.R., MEHP.A,V., BmYrON,W., BUCHANAN,T.M., KHANOLKAR, S.R., YOUNG,D.B., CLOSS,O., GJLUS,T., HAReOE,M., IVANVi,J., KOLK,A.H.J. & SHEPARD,C.C. (1985), Results of a World Health Organization-sponsored workshop on monoclonal antibodies to Mycobacteriurn ieprae. Infect. lrnrnun., 48, 603-605. GALAN,J.E., NAKAYAMA,K. & CURTISS,R. Ill (1990), Cloning and molecular characterization of the asd gene of Salmonella typhirnuriurn and its use for stable maintenance of recombinant plasmids in Salmonella vaccine strains. Gene, 94, 29-35. GARSlA, R.J., HELLQVlST,L., BOOTH, R.J., RADFORD,A.J., BRITTON,W.J., ASTBURY,L., TRarcr, R.J. & BAST~N,A. (1989), Homology of the 70-kilodalton antigens from iMoYCObacteriurnleprae and Mycobacterium boris with the Myrobacterium tubercuis 7 l-kilodalton antigen and the conserved heat shock protein 70 of eukaryotes. Infect. Irnrnun., 57, 204-212. GELEER,R.H., BRENNAN,P.J., HUNTER,S.W., IVIUNN,M.W., MONSON,J.M., MURRAY,L.P., SIu, P., TSANG,M., ENGLEMAN,E.G. & MOHAGHEGHPOUR,N. (1990), Effective vaccination of mice against leprosy bacilli with subunits of Mycobacleriurn leprae. Infect. Irnrnun., 58, 711-718. GILLIS,T.P. & BUCHANAN,T.M. (1982), Production and partial characterization of monoclonal antibodies against Mycobacterium leprae. Infect. Immun., 37, 172-178. HANSeN,G.A. (1874), Causes of leprosy. Norsk. MEg. Laegervidenskaben, 4, 76-79. HARBOE,M., CLOSS,O., BJUNE,G., KRONVALL,G. & AXELSON,N.H. (1979), Mycobacteriurn leprae-specific antibodies detected by radioimmunoassay. Scand. J. Irnrnunol., 26, 673-681. HARTSKEEL,R.A., VAN RENS, R.M., STABEL,L.F.E.M., DE WIT, M.Y.L. & KLATSER,P.R. (1990), Selection and characterization of recombinant clones producting Mycobacteriurn leprae antigens recognized by antibodies in sera from household contacts of leprosy patients. Infect. Irn.,nun., 58, 2821-2827.

870

J.E. C L A R K - C U R T I S S E T A L .

HUNTER,S.W., FUJIWARA,T. & B~NNAN,P.J. (1982), Structure and antigenicity of the major specific glycofip~dantigen of Mycobacterium leprae. J. biol. Chem., 2,57,15072-15078. HUNTER,S.W., GAYLORD,H. & BRENNAN,P.J. (1986), Structure and antigenicity of the phosphorylated lipopolysaceharide antigens from the leprosy and tubercle bacilli. J. biol. Chem., 261, 12345-12351. HUNTER,S.W., McNEIL, M., MODLIN,R.L., MEHBA,V., BLOOM,B.R. & BI~NNAN,P.J. (1989), Isolation and characterization of the highly immunogenic cell wall-associated proteins of Mycobacterium ieprae. J. lmmunol., 142, 2864-2872. IVANYi,J., SINHA,S., ASTON,R., CASSELL,D., KEEN,M. & SENGUPTA,U. (1983), Definition of species-specific and cross-reactive antigenic determinants of Mycobacterium leprae using monoclonal antibodies. Clin. exp. lmmunol., 52, 528-536. JOPLING,W.H. (1984), "Handbook of leprosy." William Heineman Medical Books, Ltd., London. KAPLAN,G. & COHN,Z.A. (1986), The immunobiology of leprosy. Int. Rev. exp. Fath., 28, 45-78. KIRCHHEIMER,W.F. & STORRS,E.E. (1971), Attempts to establish the armadillo (Dasypus novemcinctus, Linn.) as a model for the study of leprosy. Int. J. Leprosy, 39, 693-703. KLATSER,P.R., VAN RENS,M.M. & EGGELTE,T.A. (1984), Immunochemical characterization of Mycobacterium leprae antigens by the SDS-polyacrylamide gel electrophoresis immunoperoxidase technique (SGIP) using patients' sera. Clin. exp. Immunol., $6, 537-544. MEHRA,V., BLOOM,B.R., TORIOIAN,V.K., MANDICH,D., REICHEL,M., YOUNG,S.M.M., SALGAME, P.. CONVIT,J., HUNTER,S.W., McNE,L, M., B~NNAN, P.J., REA, T.H. & MODLIN,R.L. (1989), Characterization of Mycobacterium leprae cell wall-associated proteins with the use of T-lymphocyte clones. J. Immunol., 142, 2873-2878. MEHRA,V., SWEETSER,D. & YOUNG,R.A. (1986), Efficient mapping of prow "~' antigenic determinants. Proc. nat. Acad. Sci. (Wash.), 83, 7013-7017. MENDEZ-SAMPIERO,P., LAMB,J., BOTNAMIEY,G., STANLEY,P., ELLIS,C. & IVANYI,J. (1989), Molecular study of the T-cell repertoire in family contacts and patients with leprosy. J. !mmunoL, 142, 3599-3604. MESSING,J. (1983), New MI3 vectors for cloning. Meth. EnzymoL, 101, 20-78. MOHAGHEOH~OUR,N., MUNN, M.C., GELBER,R.H. & ENGLEM^N,E.G. (1990), Identification of an immunostimulating protein from Mycobacterium leprae. Infect. lmmun., 58, 703-710. NAr~YAMA,K., KELLY,S.M. & CVRTISS,R. III (1988), Construction of an Asd + expressioncloning vector: stable maintenance and high level expression of cloned genes in a Salmonella vaccine strain. Bio/Tech., 6, 693-697. OTTENBOFF, T.H.M., CONVERSE, P.J., GEBRE, N., WONDIMO, A., EHRENBERG, J.P. & KI~SSUNO,R. (1989), T-cell responses to fractionated Mycobacterium leprae antigens in leprosy. The lepromatous nonresponder defect can be overcome in vitro by stimulation with fractionated M. leprae components. Europ. J. Immunol., 19, 707-713. OrrENHOFF,T.H.M., ELFERINK,D.G., KLArSER,P.R. & DE VRIES,R.R.P. 0986), Cloned suppressor T cells from a lepromatous leprosy patient suppress Mycobacterium leprae reactive helper T cells. Nature (Lond.), 322, 462-464. SAMBROOX,J., FRITSCH,E.F. & MAN,^T,S, T. (1989), "Molecular Cloning: A Laboratory Manual". Cold Spring Harbor Laboratory, New York. SATHISH,M., ESSER, R.E., THOLE, J.E.R. & CLARK-CURTIS.S,J.E. (1990), Identification and characterization of antigenic determinants of Mycobacterium leprae that react with antibodies in sera of leprosy patients. Infect. Immun., 58, 1327-1336. SHEPARD,C.C. (1960), The experimental disease that follows the injection of human leprosy bacilli into the footpads of mice. J. exp. Med., 112, 445-454. SmNNICK,T.M., VODKIN,M.H. & WILUAMS,J.C. (1988), The Mycobacterium tuberculosis 65-kilodalton antigen is a heat shock protein which corresponds to common antigen and to the Escherichia coil GroEL protein. Infect. Immun., 56, 446-451. STORRS,E.E. (1971), The nine-banded armadillo: a model for leprosy and other biomedical research. Int. J. Leprosy, 39, 703-714. TH~,NGAn~, H.S., LAMB,F.I., DAVIS,E.G. & COLSTON,M.J. (1989), Nucleotide and deduced amino acid sequence of Mycobacterium leprae manganese superoxide dismutase. Nuc. Acids Res., 17, 8378. THOLE,J.E.R., STABEL,L.F.E.M., SUYKERBUYK,M.E.G., DE WIT, M.Y.L., KLATSER,P.R., KOLK,A.H.J. & HARTSKEEL,R.A. (1990), A major immunogenic 36,000-molecular weight antigen from Mycobacterium leprae contains an immunoreactive region of proline-rich repeats. Infect. Immun., $8, 80.87.

PROTEIN ANTIGENS OF MYCOBACTERIUM LEPRAE

871

VANSCHOOTEN,W.C.A., OTrENHOFP,T.H.M., KLATSER,P.R., THOLE,J.E.R., DE VRIES,R.R.P. 86 KOLK,A.H.J. (1988), T cell epitopes on the 36 K and 65 K Mycobacterium leprae antigens defined by human T cell clones. Europ. J. Immunol., 18, 849-854. VEoA-LoPEZ, F., STOKER,N.G., LOC'NlSKAR,M.F., DOCKRELL,H.M., GRANT,K.A. & McADAM,K.P.W.J. (1988), Recognition of mycobacterial antigens by sera from patients with leprosy. J. din. MicrobioL, 26, 2474-2479. Vos, J.G.! BUYS,J., HANDSTEDE, J.G. & HAGENAARS,A.M: (1979), Compari.son of en.z~elinKeo immunosoroent assay anu passive nemaggmtinaUon methoO for quantincation of antibodies to lipopolysaccharide and tetanus toxoid in rats. Infect. Immun., 24, 798-803. W^~RS, M.F.R. (1989), The chemotherapy of leprosy, in "The biology of the mycobacteria" (C. Ratledge, J. Stanford & J.M. Grange) Vol. 3 (pp. 405-474). Academic Press, New York, London. YouNo, D.B., L^TXRZOA,D., HENDXlX,R., SWEETS~R,D. & YOUNO,R.A. (1988), Stress proteins are immune targets in leprosy and tuberculosis. Proc. nat. Acad. Sci. (Wash.), 85, 4267-4270. YOUNO, R.A., MEHRA, V., SW~ETSER,D., BUCHANAN,CLARK-CURTISS,J.E., DAVIS,R.W. & BLOOM,B.R. (1985), Genes for the major protein antigens of the leprosy parasite Mycobacterium ieprae. Nature (Lond.), 316, 450-452.

Protein antigens of Mycobacterium leprae.

Protein antigens of Mycobacterium leprae have been identified by screening the lambda gt11, pYA626 and pHC79::M. leprae genomic libraries with pooled ...
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