Vol. 58, No. 12

INFECTION AND IMMUNITY, Dec. 1990, p. 4089-4098 0019-9567/90/124089-10$02.00/0 Copyright C 1990, American Society for Microbiology

Expression of Mycobacterium tuberculosis and Mycobacterium leprae Proteins by Vaccinia Virus JANET LYONS, CHRISTOS SINOS, ANTONIA DESTREE, TERRI CAIAZZO, KELLY HAVICAN, SARA McKENZIE, DENNIS PANICALI, AND ANNA MAHR* Applied bioTechnology, 80 Rogers Street, Cambridge, Massachusetts 02142 Received 12 July 1990/Accepted 10 September 1990

Eight Mycobacterium tuberculosis and M. leprae genes were inserted into the vaccinia virus genome by in vivo recombination. The resulting virus recombinants were shown to express five different M. tuberculosis proteins (71, 65, 35, 19, and 12 kDa) and three M. leprae proteins (65 and 18 kDa and a biotin-binding protein) by Western immunoblot analysis, radioimmunoprecipitation, or black-plaque assay. When injected into BALB/c mice, the recombinants expressing the M. tuberculosis 71-, 65-, or 35-kDa protein and the M. leprae 65-kDa protein or the biotin-binding protein elicited antibodies against the appropriate M. tuberculosis or M. keprae protein. These vaccinia virus recombinants are being tested for the ability to elicit immune protection against M. tuberculosis or M. leprae challenge in animal model systems. The recombinants are also useful in generating target cells for assays aimed at elucidating the cellular immune responses to mycobacterial proteins in leprosy and tuberculosis. Furthermore, the M. tuberculosis 65-kDa protein and four of the other mycobacterial proteins share homology with known eucaryotic and procaryotic stress proteins, some of which may play a role in autoimmunity.

The etiological agents of tuberculosis and leprosy, Mycobacterium tuberculosis and M. leprae, respectively, were each identified over 100 years ago. These slow-growing, intracellular parasitic organisms remain difficult to study, and M. leprae still has not been successfully cultured in vitro. The incidence of leprosy is 15 million worldwide, with approximately 30% of its victims suffering severe disfiguration (5). Because of the stigma attached to leprosy, this disease exacts a tremendous social cost from its victims. Tuberculosis is a highly contagious disease with over 8 million new cases and 3 million fatalities occurring annually (14). Immunosuppression by human immunodeficiency virus type 1, the causative agent of AIDS, could have a marked impact on these numbers by allowing endogenous reactivation of dormant M. tuberculosis. Although drug therapy can be effective, vaccines offer the best hope of controlling both diseases. Mycobacterium bovis bacillus Calmette-Gudrin (BCG), the current vaccine against tuberculosis, is not consistently efficacious (52). Experimental vaccines against leprosy, including BCG, killed M. leprae, BCG plus killed M. leprae or several cultivable mycobacteria related to M. leprae, are unproven. To develop better vaccines, we have generated a series of vaccinia virus recombinants that express M. leprae and M. tuberculosis antigens. Vaccinia virus has been used extensively as a vector to express foreign proteins derived from RNA viruses, DNA viruses, and protozoa (see 31; see reference 35 for a review). When used as live vaccines, vaccinia virus recombinants expressing foreign proteins have been shown to elicit humoral and/or cellular immune responses that are often protective. Little work, however, has been done to show that vaccinia virus recombinants that express bacterial antigens, presumably with an intracellular localization, can elicit protective immune responses. The only report of a recombinant vaccinia virus protecting against bacterial disease showed that a vaccinia virus recombinant expressing *

the streptococcus M protein was able to reduce pharyngeal colonization in vaccinated mice challenged with group A streptococci (15). We inserted the genes that encode the M. tuberculosis 71-, 65-, 35-, 19-, and 12-kDa proteins; the M. leprae 65- and 18-kDa proteins; and an M. leprae-encoded biotin-binding protein (BBP) into vaccinia virus. These vaccinia virus recombinants were shown to express the mycobacterial proteins, and when injected into mice, most of these recombinants elicited a humoral immune response to the respective mycobacterial antigens. Experiments intended to assess the abilities of these recombinants to elicit a cellular immune response and protect animals against a live M. tuberculosis or M. leprae challenge are in progress. MATERIALS AND METHODS Mycobacterial genes. Clones containing the genes for the M. tuberculosis 71-, 65-, and 19-kDa proteins and the M. leprae 65- and 18-kDa proteins and BBP (60) were obtained from R. Young, Whitehead Institute for Biomedical Research, Cambridge, Mass. The gene for the M. tuberculosis 35-kDa protein was obtained from H. Rumschlag and M. Cohen, Centers for Disease Control, Atlanta, Ga. The gene that encodes the M. tuberculosis 12-kDa protein was obtained from T. Shinnick, Centers for Disease Control. Genes were originally cloned into either Xgtll or EMBL3 bacteriophage or into bacterial plasmids. Some of the genes were modified for expression in vaccinia virus because they lacked ATG initiation codons or convenient restriction sites immediately upstream of their initiation codons. Modifications were made by in vitro mutagenesis (53) by using a kit purchased from Amersham Corp., Arlington Heights, Ill., or by addition of synthetic oligonucleotides. The modifications are described briefly below and summarized in Table 1. Cloning of M. leprae genes into vaccinia virus expression vectors. The three M. leprae genes were cloned into insertion vectors for in vivo recombination into vaccinia virus. The M. leprae 65-kDa protein-encoding gene (34, 60), which con-

Corresponding author. 4089

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LYONS ET AL.

INFECT. IM4MUN. TABLE 1. Construction of vaccinia virus recombinants

Virus

Protein'

Original fragment (size

clone Startilng (reference)

[bp])

Vaccinia

virusi

M. leprae 65 kDa

Y3178 (34, 60)

NruI-EcoRV (2,253)

11K

vAbT121

M. leprae 65 kDa

pAbT6007e (this report) BglII (2,253)

7.5K

M. leprae 65 kDa vAbT152h M. leprae 65 kDa vAbT152h M. Ieprae BBP vAbT117 M. leprae BBP

vAbT234 vAbT254

M. leprae BBP M. leprae 18 kDa

pAbT85259 (this report) pAbT8031' (this report) pAbT8502' (this report) Y3184 (60; R. Young, personal communication) pAbT8502 (this report) N3011 (6, 39)

vAbT384 vAbT237

M. tuberculosis 71 kDa M. tuberculosis 65 kDa

Y3111 (16, 28, 58) Y3253 (21, 29, 48, 59)

vAbT383 vAbT235

M. tuberculosis 35 kDa M. tuberculosis 19 kDa

vAbT363

M. tuberculosis 12 kDa

pSOC130 (39, 40) p19K2.8' (1; 21; R. Young, personal communication) pRL35 (21, 49)

Protein encoded by gene at in-

pro-setosi' motersetnsie

vAbT53

vAbT236

5' Modification'

ATG AAT TCA GAT CTT CCC GAT CAG CGA GTGd CG ATG GAT CTT CCC GAT CAG CGA GTGf As in vAbT121 As in vAbT121 As in vAbT117 CG ATG GAA TTC GAG GTC GAA GGC CTG

TK TK

EcoRI (2,253) ClaI (2,253) ClaI (620) EcoRI-ClaI (620)

40K 30K 7.5K 7.5K

ClaI (620) BstEII-ClaI (1,229) NcoI (3 j00)k

40K 40K

As in vAbT117 None

30 kDa 30 kDa

40K 40K

None None

30 kDa 30 kDa

HpaI-SphI (884) AvaI (904)

40K 40K

AGT TAA CTG AI CG ATG GGC AGG GTG AAGf

30 kDa 30 kDa

TaqI-SphI (370)

40K

AAT TCT AGA ATG GCG AAG GTG AAC ATC AAG CCA CT

30 kDa

BspMII-KpnI

(1,700)k

30 kDa TK TK TK

a All M. leprae genes were derived from bacteria grown in armadillos and were originally from a single human patient. The gene for the M. tuberculosis 35-kDa protein is from strain H37RA, and the genes for the M. tuberculosis 71-, 65-, 19-, and 12-kDa proteins are from the Erdmann strain. b Modifications affecting the reading frame, providing an ATG, or creating a new restriction site at the 5' end are included. Underlined codons indicate where vaccinia virus translation initiation occurs, and italicized codons indicate mycobacterial coding sequences. c The gene for TK is located in the vaccinia virus HindIII J fragment, and the gene for the 30-kDa protein is contained within the vaccinia virus HindIII M DNA fragment. d This puts the GTG of the mycobacterial gene in frame with the ATG of the promoter. e Final plasmid used in construction of vAbT53. f The ATG provided by the linker is now in frame with the mycobacterial coding sequence. g Final plasmid used to make vAbT152. h Divalent virus with two M. leprae genes. Intermediate in construction of the plasmid used to make vAbT121. Intermediate in construction of the plasmid used to make vAbT117. Size is approximate because the fragment was not completely sequenced. The new sequence contains a unique HpaI site. mThis plasmid was derived from phage Y3147 (19).

tains a GTG initiation codon, was excised from phage Y3178 with NruI and EcoRV. This fragment was ligated to BglII linkers [d(pGGAAGATCTTCC); New England BioLabs, Beverly, Mass.] and cloned into the BglII site of a vaccinia virus insertion vector such that the coding sequence was in frame with the ATG of the vaccinia virus 11K promoter (4). Subsequently, it was excised from this vector as a BglII fragment and inserted into a second plasmid downstream from the vaccinia virus 7.5K (7) promoter by using a ClaI linker [d(pCATCGATG); New England BioLabs] to provide an ATG initiation codon in frame with the sequence encoding the M. leprae 65-kDa protein. This ClaI linker-treated fragment was then removed from this last vector on an EcoRI fragment and inserted behind the vaccinia virus 40K (44) promoter within a third plasmid vector for recombination. The M. leprae 18-kDa protein-encoding gene (6, 39), sequence analysis of which predicts a protein of 16 kDa, was excised from phage N3011 DNA by BstEII-ClaI digestion and ligated into a plasmid allowing for regulation of expression by the vaccinia virus 40K promoter. The gene for the 20-kDa BBP was isolated from phage Y3184 (60; R. Young, personal communication). The amino acid sequence of BBP has homology to other proteins known to bind biotin. Phage Y3184 contained a partial coding

sequence for BBP; therefore, Clal linkers [d(pCCATC GATGG); New England BioLabs] were added to provide an in-frame ATG initiation codon for expression regulated by

the vaccinia virus 7.5K and 40K promoters. The gene for BBP was isolated from a Agtll library with monoclonal antibody ML-10, which was believed to be specific for an M. leprae 12-kDa protein (60). In all probability, the gene was isolated because of its biotin-binding properties. The gene for BBP has no homology to the gene for the M. tuberculosis 12-kDa protein or the M. leprae gene that encodes the respective 12-kDa homolog to the Escherichia coli GroES

protein. Cloning of M. tuberculosis genes into vaccinia virus expression vectors. Five M. tuberculosis genes (21), encoding the 71-, 65-, 35-, 19-, and 12-kDa proteins, were inserted into plasmid vectors for recombination into and expression by vaccinia virus. The BspMII-KpnI fragment of X phage Y3253 (21, 29, 48, 59), containing the gene that encodes the M. tuberculosis 65-kDa antigen, was cloned into vaccinia virus insertion plasmids with expression regulated by either the vaccinia virus 7.5K or 40K promoter. Plasmid p19K2.8 (1, 21; R. Young, personal communication) was the source of the gene that encodes the M. tuberculosis 19-kDa polypeptide, for which the gene sequence predicts a protein of 18

VOL. 58, 1990

EXPRESSION OF MYCOBACTERIUM PROTEINS BY VACCINIA VIRUS

kDa. The 5' end of an AvaI fragment was modified by in vitro mutagenesis with the oligomer d(pCAA AGG AGC TCA GGG TGA AG) (Research Genetics, Huntsville, Ala.) to create a unique Sacl site. Subsequently, the resulting plasmid was cleaved at the Sacl site and a ClaI linker [d(pCC CATCGATGGG); New England BioLabs] was added to provide an ATG in frame with the mycobacterial GTG codon, and the resulting fragment was then cloned downstream of the vaccinia virus 40K promoter. The gene that encodes the M. tuberculosis 12-kDa antigen (21, 49) was removed from plasmid pRL35, and the GTG initiation codon was changed to ATG by use of a synthetic linker [d(pAAT TCT AGA ATG GCG AAG GTG AAC ATC AAG CCA CT); Research Genetics] and its complementary sequence. This synthetic oligomer contained new XbaI and EcoRI sites at the 5' end, contained an ATG in place of a GTG codon, and also reconstituted the M. tuberculosis 12-kDa gene up to the TaqI site. The modified gene was cloned downstream of the vaccinia virus 40K promoter. For expression of the M. tuberculosis 71-kDa protein (16, 28, 58), the NcoI fragment contained within A phage Y3111 was cloned into a plasmid for expression by the vaccinia virus 40K promoter. In vitro mutagenesis with the oligomer p(dAGT TAA CTG ATG) (Research Genetics) was used to create an HpaI site just upstream of the M. tuberculosis 35-kDa protein-encoding gene derived from pSOC130 (8, 39, 40) and the HpaI-SphI fragment containing the M. tuberculosis 35-kDa proteinencoding gene was then cloned into a vector for expression under control of the 40K promoter. Selection and purification of vaccinia virus recombinants. Vaccinia virus-mycobacterial recombinants were made by in vivo recombination (32, 38) into the NYCBH vaccine strain (ATCC VR325; American Type Culture Collection, Rockville, Md.) of vaccinia virus. Vectors directed insertion of foreign genes into the vaccinia virus gene for thymidine kinase (TK) or into the gene that encodes a 30-kDa protein located within the vaccinia virus Hindlll M DNA fragment. Plasmids facilitating recombination into the TK-encoding site contained a vaccinia virus 7.5K constitutive promoter to regulate expression of the foreign gene, as well as the vaccinia virus BamHI-F promoter that regulates the synthesis of E. coli P-galactosidase (24). Insertion into the vaccinia virus TK-encoding site renders recombinants TK-, enabling them to be selected (32) in the presence of bromodeoxyuridine. They can be further selected and purified on the basis of their blue-plaque phenotype in the presence of Bluo-gal (Bethesda Research Laboratories, Inc., Gaithersburg, Md.). Recombinants with DNA insertions into the 30-kDa protein-encoding gene within the vaccinia virus HindIII M DNA fragment were generated by host range selection. The vaccinia virus 29-kDa protein enables the virus to grow on RK-13 cells, while absence of a functional 29-kDa protein results in a 5-order-of-magnitude reduction of growth on RK-13 cells compared with growth on BSC-40 cells (17). The starting virus was vAbT33, a derivative of the NYCBH strain, which contains the E. coli lacZ gene inserted into the HindIII M fragment. Virus vAbT33 contains an incomplete copy of the vaccinia virus 30-kDa protein-encoding gene, lacks the gene for the 29-kDa protein, and carries lacZ. The plasmid used for in vivo recombination contains a functional copy of the 29-kDa protein-encoding gene. Recombination of this plasmid with vAbT33 results in loss of the lacZ gene

from the recombinant virus and the insertion of the gene for the 29-kDa protein. The gene encoding the 30-kDa protein remains incomplete. Recombinant viruses were isolated and

4091

plaque purified on the basis of the ability to replicate on RK-13 cells. All recombinant viruses were isolated through several rounds of plaque purification and analyzed by RNA dot blot hybridization or black-plaque assay (see below). The genomic structures were verified by Southern hybridization. Analysis of mycobacterial protein expression by vaccinia virus recombinants. Monoclonal antibodies were obtained from a variety of sources. Alternate designations for these are indicated in parentheses. Monoclonal antibodies Y1-2 (0401) and IIC8 (4220), both specific for the M. leprae 65-kDa protein, and TB78 (IT-13), specific for the M. tuberculosis 65-kDa protein, were obtained from R. Young (Whitehead Institute). J. Ivanyi (Medical Research Council, London, England) contributed monoclonal antibodies ML10, which is specific for the M. leprae 12-kDa protein, and TB78. Monoclonal antibody SA12 (IT3), specific for the M. tuberculosis 12-kDa protein, was obtained from P. J. Kelleher (Houston Biotechnology, Inc., The Woodlands, Tex.) and P. Minden (formerly of Scripps Clinic and Research Foundation, San Diego, Calif.). Monoclonal antibody 2B2, specific for the M. tuberculosis 35-kDa protein, was obtained from H. Rumschlag and M. Cohen (Centers for Disease Control). Monoclonal antibodies F29-47-3 (IT10; specific for the M. tuberculosis 19-kDa protein), L7.15 (MC8026; specific for the M. leprae 18-kDa protein), and IT40 (recognizing the M. tuberculosis 71-kDa protein) were obtained from the World Health Organization monoclonal antibody bank. Rapid screening for expression was performed by blackplaque analysis (30), an in situ enzyme-linked immunosorbent assay (ELISA). RK-13 cell monolayers were infected with virus, and after plaque formation at 48 to 72 h, the agarose overlay was removed and the cells were washed with phosphate-buffered saline (PBS) and fixed for 20 min in 3% formaldehyde in PBS. After being washed with PBS, the cells were incubated for 1 h to overnight in 0.5 ml of primary antibody diluted in 50% normal goat serum (NGS). Following removal of the antibody, cells were washed three times with PBS, incubated for 1 h in alkaline phosphatase-conjugated secondary antibody (goat anti-mouse immunoglobulin G; Kirkegaard and Perry Laboratories, Gaithersburg, Md.), diluted in 10% NGS, and washed with PBS and Tris-buffered saline. The chromogenic substrates (Kirkegaard and Perry) Nitro Blue Tetrazolium chloride and 5-bromo-4-chloro-3indolylphosphate were added to visualize viral plaques containing the protein of interest. Radioimmunoprecipitation analysis (RIPA) and Western immunoblotting were done by standard procedures. For RIPA, BSC-40 cells were infected for 1 h with recombinant vaccinia virus at a multiplicity of infection of 10 and subsequently overnight in medium containing [35S]methionine. After 24 h, the medium was removed and the cell monolayer was washed with PBS and lysed with 1 ml of immunoprecipitation buffer (10 mM Tris hydrochloride [pH 7.2], 500 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.1 mg of soybean trypsin inhibitor per ml). Monoclonal antibody was added to the sample and incubated for 2 h at room temperature or overnight at 4°C. Protein A-Sepharose suspended in immunoprecipitation buffer was added, and the samples were incubated with rotation for 1 h at 4°C. The samples were washed four times with immunoprecipitation buffer and once with Tris-buffered saline (pH 8.2). Sepharose pellets were dried, suspended in Laemmli sample buffer, and heated to 100°C for 5 min before sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

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LYONS ET AL.

For Western analysis, BSC-40 cells were infected overnight with vaccinia virus at a multiplicity of infection of 10. Cells were scraped, pelleted by centrifugation, and sonicated in hypotonic lysis buffer (10 mM Tris hydrochloride [pH 8.0], 10 mM KCI, 5 mM EDTA) in the presence of phenylmethylsulfonyl fluoride and soybean trypsin inhibitor. Samples were then heated to 100°C for 5 min in Laemmli buffer before electrophoresis on polyacrylamide gels. Proteins were electrophoretically transferred for 1 to 1.5 h to nitrocellulose, which was subsequently blocked with BLOTTO (3% milk, 2% NGS, 0.1% Tween 20 in PBS) for 1 h. The nitrocellulose filter was incubated with the primary antibody diluted in BLOTTO or 50% NGS for a minimum of 2 h. This incubation was followed by three high salt concentration washes (20 mM Tris hydrochloride [pH 7.5], 1 M NaCl, 0.05% Tween 20) and addition of the secondary antibody in BLOTTO or 10% NGS. After three final high salt concentration washes, the chromogenic substrates Nitro Blue Tetrazolium and 5-bromo-4-chloro-3-indolylphosphate were added and color was allowed to develop. The reaction was stopped with PBS. Hybrid selections were done as previously described (33) to show that infected cells transcribed RNAs that could be translated in vitro in proteins of the expected sizes. RNA was isolated from cells infected with a recombinant virus at a multiplicity of infection of 10. This RNA was then hybridized to cloned mycobacterial DNA, eluted, and translated in the rabbit reticulocyte cell-free system in the presence of [35S]methionine. The protein products were then separated on a sodium dodecyl sulfate-polyacrylamide gel for analysis. Serological analysis of antibodies to vaccinia virus and mycobacterial proteins. Female BALB/c mice 6 to 8 weeks old (Taconic Laboratories, Germantown, N.Y.) were vaccinated intraperitoneally with 107 PFU of recombinant virus. Sera were obtained 4 to 6 weeks postvaccination and tested for the presence of antibodies to vaccinia virus and mycobacterial proteins. Sera from mice vaccinated with recombinant viruses were tested for antibodies to mycobacterial proteins by immunoprecipitation of in vitro-translated proteins or by immunoassay of filter-bound mycobacterial proteins made by recombinant A phage. In vitro translation and analysis of mycobacterial proteins were performed as follows. Plasmids containing the mycobacterial genes under control of the T7 promoter were linearized with a restriction enzyme 3' of the coding region for the mycobacterial antigen and transcribed for 2 h at 40°C by the procedure supplied with the Riboprobe Gemini System (Promega Biotec, Madison, Wis.). All four unlabeled ribonucleotides and a cap analog (m7GpppG) were included in the reaction at a final concentration of 0.5 mM. A parallel reaction was performed which also included 1 ,uCi of [32P]CTP. After transcription, the DNA template was removed by incubation for 15 min at 37°C with RNase-free DNase (Promega) at a final concentration of 1 U/Iug of DNA. Efficiency of transcription was monitored by assessing incorporation of acid-precipitable [32P]CTP. The resulting unlabeled transcribed RNAs were translated in the rabbit reticulocyte lysate system (Promega) in the presence of [35S]methionine. The protein synthesized was immunoprecipitated with mouse antiserum as described above. Mouse sera were also tested for the ability to bind to M. leprae and M. tuberculosis proteins made by recombinant Xgtll (59, 60). Recombinant phage diluted in 10 mM Tris hydrochloride (pH 7.5)-10 mM MgCl2 was mixed with E. coli Y1090 and incubated for 20 min at 37°C to allow the phage to adsorb to the bacteria. The mixture of bacteria and

INFECT. IMMUN.

phage was then incubated for 3.5 h at 42°C on Luria-Bertani agar plates, which were subsequently overlaid with nitrocellulose filters saturated in 10 mM isopropyl-p-D-thiogalactopyranoside, and incubated for an additional 3.5 h at 37°C. After the filters were carefully removed from the plates, they were incubated overnight at 4°C in blocking solution (10% NGS or BLOTTO in PBS). The filters were then tested with mouse antiserum that had been preadsorbed with a Xgtll lysogen. Serum diluted in BLOTTO or 50% NGS was added to the filters and incubated for 1 h at room temperature. Filters were washed three times in high salt concentration wash buffer. After incubation for 1 h at room temperature with alkaline phosphatase-labeled goat anti-mouse immunoglobulin G (Kirkegaard and Perry) diluted 1:1,000 in 10% NGS, the filters were again washed and incubated with a 5-bromo-4-chloro-3-indolylphosphate-Nitro Blue Tetrazolium solution. Anti-vaccinia virus antibody responses of animals vaccinated with vaccinia virus recombinants were determined by ELISA. Antigen for coating plates was made by infecting BSC-40 cells with NYCBH at a multiplicity of infection of 1.0 for 24 h. Cells were scraped off the plate, pelleted, and suspended in 1 mM Tris hydrochloride (pH 9.0). The resulting lysate was diluted to 10 p.g of total protein per ml of coating buffer (50 mM NaHCO3, pH 9.6) and used to coat the wells of Nunc Immuno II ELISA plates for 2 h at 37°C. After removal of the solution, serial dilutions of serum in BLOTTO were added and incubated for 1 h at 37°C or overnight at 4°C. Wells were then washed three times with ELISA wash (0.05% Tween 20 in PBS). The secondary antibody, horseradish peroxidase-labeled goat anti-mouse immunoglobulin G (Jackson Immunoresearch Laboratories Inc., West Grove, Pa.) diluted 1:5,000 in BLOTTO, was added to each well and incubated for 1 h at 37°C. The wells were then rinsed three times with ELISA wash before addition of the developing substrate, 3,3',5,5'-tetramethylbenzidine (Sigma). The reaction was stopped by addition of 2.5 N H2SO4, and A450s were read on an ELISA plate reader. RESULTS

Modification of mycobacterial genes for vaccinia virus expression and construction of vaccinia virus recombinants. The mycobacterial genes were initially obtained from phage or plasmid DNAs and subsequently cloned into insertion vectors for recombination into vaccinia virus. ATG initiation codons were provided for genes which contained GTG start codons or lacked start codons. In addition, new restriction sites were placed immediately upstream of some initiation codons to allow placement of the 5' ends of these genes close to vaccinia virus promoters. Each M. leprae or M. tuberculosis gene was placed under the direction of the vaccinia virus late 11K promoter and/or the constitutive 7.5K, 30K, or 40K promoter.

Mycobacterial genes inserted into the 30-kDa proteinencoding gene of the vaccinia virus HindIll M fragment or into the vaccinia virus TK-encoding site through in vivo recombination are listed in Table 1. Recombinants containing the mycobacterial genes inserted into the HindIII M site were isolated by using a host range selection scheme to separate recombinant virus from the parental virus. These viruses were not exposed to drugs during their purification, and except for the mycobacterial gene encoding the antigen of interest, they contained no other foreign DNA. The recombinants containing mycobacterial genes inserted into

EXPRESSION OF MYCOBACTERIUM PROTEINS BY VACCINIA VIRUS

VOL. 58, 1990

1 2 3

4 5

6

4093

1 2 3 45 6 7

200... 93-

6 5 -_o.

4 6-

3 02 2-

1 8FIG. 2. Western analysis of recombinants expressing the M. 65- or 18-kDa protein under the control of various vaccinia virus promoters. Cells were infected with virus, and cell lysates were fractionated on sodium dodecyl sulfate gels and transferred to nitrocellulose. For expression of the M. leprae 65-kDa protein, in lanes 1 to 5, the primary antibody was IIC8. Lanes: 1, vAbT53 (ilK promoter); 2, vAbT121 (7.5K promoter); 3, vAbT152 (30K promoter); 4, vAbT236 (40K promoter); 5, NYCBH. For expression of the M. leprae 18-kDa protein, lanes 6 and 7 contained primary antibody L7.15. Lane 6, vAbT254 (40K promoter); lane 7, NYCBH. The numbers to the left indicate molecular sizes in kilodaltons. leprae

1 4FIG. 1. RIPA of the M. leprae 65-kDa protein expressed by vaccinia virus. BSC-40 cells were infected with vaccinia virus recombinants in the presence of [355]methionine. Cells were lysed, and monoclonal antibody IIC8, specific for the M. leprae 65-kDa protein, was used for immunoprecipitation. Lanes: 1, molecular weight markers (sizes are indicated in thousands to the left); 2, NYCBH; 3, vAbT236 (40K promoter); 4, vAbT152 (30K promoter); 5, vAbT121 (7.5K promoter); 6, vAbT53 (ilK promoter).

the vaccinia virus TK-encoding site were isolated by bromodeoxyuridine selection or by blue-plaque phenotype in the presence of Bluo-gal. Each of the recombinants listed in Table 1 contained one mycobacterial gene, except for divalent recombinant vAbT152, which contained two different mycobacterial genes. Expression analysis for M. leprae and M. tuberculosis proteins. Expression of mycobacterial proteins was analyzed by using available monoclonal antibodies by a variety of techniques which included black-plaque assays, Western analysis, and RIPAs. When specific monoclonal antibodies were unavailable, hybrid selections were done to demonstrate that virus recombinants directed in vivo synthesis of mRNAs that could be translated in vitro into polypeptides of the predicted sizes. Vaccinia virus-directed expression of the M. leprae proteins is shown in Fig. 1 and 2. RIPA and Western analysis showed that vAbT53 (Fig. 1, lane 6; Fig. 2, lane 1), vAbT121 (Fig. 1, lane 5; Fig. 2, lane 2), vAbT152 (Fig. 1, lane 4; Fig. 2, lane 3), and vAbT236 (Fig. 1, lane 3; Fig. 2, lane 4) expressed the M. leprae 65-kDa protein. The 1lK promoter directed the synthesis of the largest quantity of the M. leprae 65-kDa protein, followed by the 40K promoter, the 7.5K promoter, and the 30K promoter. The M. leprae 65-kDa protein expressed by the 30K promoter was visualized by longer exposure of the autoradiograph shown in Fig. 1 (data not shown). Synthesis of the M. leprae 18-kDa polypeptide by vAbT254 is also shown in Fig. 2, lane 6. Western analysis and RIPA (data not shown) with M. leprae 18-kDa protein-

specific monoclonal antibody L7.15 indicated a protein of the expected size. Hybrid-selected RNA isolated from cells infected with vaccinia virus recombinants expressing M. leprae BBP (vAbT117, vAbT152, or vAbT234) was translated into a polypeptide whose migration was consistent with an apparent molecular mass of 20 kDa (Fig. 3). As expected, RIPA and Western analysis of these same viruses with monoclonal antibody ML-10, specific for the authentic M. leprae 12-kDa protein, were negative (data not shown). Hybrid selection of RNA from divalent recombinant vAbT152 was also translated into a 65-kDa protein (Fig. 3, lane 4). Figures 4 and 5 depict the expression of the five M. tuberculosis proteins. In all cases, NYCBH was negative for each M. tuberculosis protein. Black-plaque assays showed expression of the M. tuberculosis 65-kDa protein (data not shown). RIPAs and Western analysis showed the M. tuberculosis 65-kDa protein being synthesized by vAbT237 (Fig. 4A, lane 2; Fig. SA, lane 2). Expression of the M. tuberculosis 12-kDa antigen by vAbT363 was demonstrated by black-plaque assay (data not shown) and Western blotting (Fig. 5C, lane 3). Western analysis and RIPA (data not shown) indicated that vAbT235 expressed the M. tuberculosis 19-kDa protein (Fig. 5B, lane 1). Expression of the M. tuberculosis 35-kDa protein by vAbT383 was shown by RIPA and Western analysis (Fig. 4B, lane 2; Fig. SD, lane 1). RIPA and Western blotting demonstrated expression of the M. tuberculosis 71-kDa protein by vAbt384 (Fig. 4A, lane 4; Fig. 5C, lane 1). Analysis of sera from vaccinated mice. BALB/c or outbred CFW mice were vaccinated intraperitoneally with 107 PFU of recombinant virus to determine whether the vaccinia virus-expressed mycobacterial proteins were immunogenic. Typical anti-vaccinia virus antibody titers (1:5,000 to 1:20,000) were elicited in both strains of mice vaccinated

4094

LYONS ET AL.

..

INFECT. IMMUN.

1 2 3 4 5 6

B

A 1 2

C 1

2

1

3

2

4

1

2

..

71 K-

65K-

69 4 6-W

... ,

.:

..............

::

35K

::

19K-

:.

_

12K

22

14_ FIG. 3. Hybrid selection of M. leprae RNA. RNA was prepared and selected by hybridization to nitrocellulose-bound DNA from pAbT8525, which contains the genes for the M. leprae 65-kDa protein and BBP. The RNA was translated in the rabbit reticulocyte system in the presence of [35S]methionine. Lanes: 1, no RNA; 2, NYCBH RNA; 3, vAbT234 (40K promoter-M. leprae BBP) RNA; 4, vAbT152 (30K promoter-M. leprae 65-kDa protein and 7.5K promoter-BBP) RNA; 5, vAbT117 (7.5K promoter-M. leprae BBP) RNA; 6, molecular weight markers (sizes indicated to the left in thousands).

with recombinants. Mouse sera were tested for the presence of antibodies to M. leprae and M. tuberculosis antigens by immunoprecipitation of mycobacterial proteins synthesized in vitro and/or by binding to filter-bound mycobacterial proteins synthesized by A phage. Sera from mice vaccinated with vAbT234, vAbT235,

A 1 2 3 4

B 1 2

71K. 65K-

-35K

FIG. 4. Immunoprecipitation of M. tuberculosis antigens expressed by using the vaccinia virus 40K promoter. BSC-40 cells were infected with vaccinia virus recombinants in the presence of [35S]methionine. Cell lysates were immunoprecipitated with the indicated monoclonal antibodies. (A) Lanes: 1, TB78 with NYCBH; 2, TB78 with vAbT237 (M. tuberculosis 65-kDa protein); 3, IT40 with NYCBH; 4, IT40 with vAbT384 (M. tuberculosis 71-kDa protein). (B) Lanes: 1, 2B2 with NYCBH; 2, 2B2 with vAbT383 (M. tuberculosis 35-kDa protein).

-

FIG. 5. Western analysis for expression of M. tuberculosis proteins by vaccinia virus recombinants. Cells were infected with virus, and cell lysates were fractionated on a gel and transferred to nitrocellulose. The primary antibodies were specific for M. tuberculosis proteins, and the secondary antibody was goat anti-mouse antibody conjugated to alkaline phosphatase. (A) TB78 with NYCBH (lane 1) and vAbT237 (M. tuberculosis 65-kDa protein) (lane 2). (B) F29-47-3 with vAbT235 (M. tuberculosis 19-kDa protein) (lane 1) and NYCBH (lane 2). (C) IT40 with vAbT384 (M. tuberculosis 71-kDa protein) (lane 1) and NYCBH (lane 2) and SA12 with vAbT363 (M. tuberculosis 12-kDa protein) (lane 3) and NYCBH (lane 4). (D) 2B2 with vAbT383 (M. tuberculosis 35-kDa protein) (lane 1) and NYCBH (lane 2).

vAbT236, vAbT237, vAbT254, vAbT363, and vAbT384 were tested for the presence of antibodies reactive with filter-bound mycobacterial proteins synthesized by Xgtll recombinants. The results indicated that sera from mice vaccinated with the M. leprae BBP (vAbT234) or 65-kDa protein (vAbT236) or the M. tuberculosis 65- or 71-kDa protein (vAbT384) virus contained antibodies against the appropriate antigens (data not shown). Sera from mice vaccinated with the M. tuberculosis 19-kDa (vAbT235), M. leprae 18-kDa (vAbT254), or M. tuberculosis 12-kDa (vAbT363) protein recombinant exhibited no detectable antibodies to the mycobacterial antigens expressed by the k recombinants. Sera from animals vaccinated with wild-type NYCBH showed no reactivity with any of these mycobacterial antigens. Sera from mice vaccinated with vAbT234, vAbT235, vAbT236, vAbT237, vAbT254, vAbT363, vAbT383, and vAbT384 were also tested by RIPA with mycobacterial proteins synthesized in vitro in the rabbit reticulocyte lysate system (Fig. 6). vAbT234 (Fig. 6A, lanes 7 to 11), vAbT236 (Fig. 6A, lanes 1 to 5), vAbT383 (Fig. 6B, lanes 1 to 4), and vAbT384 (Fig. 6B, lanes 5 to 8) each elicited antibodies to the appropriate mycobacterial antigens (the M. leprae BBP and 65-kDa protein and the M. tuberculosis 35- and 71-kDa proteins, respectively). Recombinant vAbT237 also elicited antibodies to the M. tuberculosis 65-kDa protein (data not shown). Recombinants vAbT235, vAbT254, and vAbT363 elicited no detectable antibodies to the M. tuberculosis 19-kDa, M. leprae 18-kDa, or M. tuberculosis 12-kDa protein. These results are consistent with the inability to detect antibodies reactive to the recombinant 19-, 18-, and 12-kDa proteins immobilized on filters. NYCBH-vaccinated control animals contained no antibodies which recognized any of these mycobacterial proteins. Sera from animals vaccinated with recombinants expressing the 65-kDa proteins did contain antibodies that cross-reacted with the M. leprae and M. tuberculosis 65-kDa proteins. In all other cases, antibody responses were specific for the antigen expressed by the

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B

A 1

65K-

2

_-W

3

4

5

6

*4

7

9

1 0 11 12

1 2 3 4 5678

71 K-

35 K

-

BBP

FIG. 6. Binding of mouse antisera to in vitro-synthesized mycobacterial proteins. The genes that encode the M. leprae and M. tuberculosis proteins were cloned into transcription vector pSP72. RNA transcripts were made and translated in the rabbit reticulocyte system. These proteins made in vitro were then immunoprecipitated with mouse antisera. (A) Lane 1, total M. leprae 65-kDa antigen; lanes 2 to 5, immunoprecipitation of M. leprae 65-kDa antigen with MAb Y1.2 (lane 2), vAbT236 serum (M. leprae 65-kDa antigen) (lane 3), vAbT234 serum (M. leprae BBP) (lane 4), and NYCBH serum (lane 5); lane 6, molecular weight markers; lane 7, total M. leprae BBP antigen; lanes 8 to 11, immunoprecipitations of M. leprae BBP antigen with ML-10 (lane 8), vAbT234 (M. leprae BBP) (lane 9), vAbT236 (M. leprae 65-kDa antigen) (lane 10), and NYCBH (lane 11); lane 12, Endogenous translation. (B) Lanes: 1 to 3, immunoprecipitation of the M. tuberculosis 71-kDa protein with antisera from three mice vaccinated with vAbT384; 4, immunoprecipitation of the M. tuberculosis 71-kDa protein by antiserum from a mouse vaccinated with NYCBH; 5 to 7, immunoprecipitation of the M. tuberculosis 35-kDa protein by antisera from three mice vaccinated with vAbT383; 8, immunoprecipitation of the M. tuberculosis 35kDa protein by antiserum from a mouse vaccinated with NYCBH.

respective vaccinia virus recombinant used for immunization and did not cross-react with other, unrelated, mycobacterial proteins.

DISCUSSION Eight mycobacterial proteins, five derived from M. tuberculosis and three derived from M. leprae, were expressed by vaccinia virus. The antigens expressed, the M. tuberculosis 71-, 65-, 35-, 19-, and 12-kDa proteins and the M. leprae 65and 18-kDa proteins and BBP, were all identified in phage or plasmid expression libraries by immunoassay using the antibodies generated by immunization of animals with mycobacterial extracts, whole killed mycobacteria, or live mycobacteria (8, 12, 13, 21, 45, 59, 60). Although some of these apparently immunodominant proteins (the mycobacterial 65-kDa, M. tuberculosis 71- and 12-kDa, and M. leprae 18-kDa proteins) are homologous with eucaryotic or procaryotic stress proteins (1, 2, 16, 28, 39, 49, 50, 58), little is known about their role in mycobacterial growth and infection, and the significance, if any, of these proteins with respect to the immune response to M. leprae or M. tuberculosis infection is not clear. Expression of the mycobacterial proteins was achieved by insertion of the mycobacterial genes into vaccinia virus through in vivo recombination. Two insertion sites were used, the 30-kDa protein-encoding gene of the vaccinia virus HindIII M fragment and the TK-encoding locus. Insertion at HindIII-M has the dual advantage of not requiring drug selection during virus purification and of containing the gene of interest as the only foreign DNA. Expression by different

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vaccinia virus promoters (the late l1K and the constitutive 40K, 7.5K, and 30K promoters) was compared by using the M. tuberculosis 65-kDa protein. RIPA and Western blot results showed that the highest expression was with the ilK promoter, followed by the 40K, 7.5K, and 30K promoters, respectively. Although late vaccinia virus promoters, such as the l1K promoter, direct the synthesis of large amounts of RNA, generally resulting in a high level of antigen production, it has been shown that recombinants using late promoters elicit little or no cell-mediated immunity to the foreign antigen (9, 56, 57). The vaccinia virus recombinants encoding seven of the eight mycobacterial antigens were all shown to express antigen by Western blot, RIPA, or black-plaque assay. Since there were no monoclonal antibodies specific for M. leprae BBP, expression was shown indirectly by hybrid selecting RNA from infected cells and translating it in vitro into a protein of the predicted size. Two different immunoassays were used to detect murine antibody responses to mycobacterial proteins. Assay 1 involved RIPA of in vitro-expressed mycobacterial proteins using sera from vaccinated mice. In assay 2, mouse serum was incubated with membrane-bound ,-galactosidase fusion proteins produced by Agtll-mycobacterium recombinants. We showed by these two assays that vaccinia virus recombinants expressing mycobacterial proteins can elicit mouse antibodies against the M. tuberculosis 71-, 65-, and 35-kDa antigens. We were also able to detect antibodies to the M. leprae 65-kDa protein and BBP. We were unable to detect mouse antibody responses to the M. leprae 18-kDa protein or the M. tuberculosis 12- or 19-kDa proteins. It is possible that antibodies made against vaccinia virus-produced antigen in vivo do not recognize the 3-galactosidase fusion proteins or the proteins synthesized in vitro by the rabbit reticulocyte lysate used in our assays. We investigated the possibility that vaccination of other mouse strains would be more successful in eliciting antibodies. It is known that certain murine haplotypes recognize the rat neuroblastoma protein expressed by vaccinia virus more readily than do others (3). Because of this observation, mice of differing haplotypes were tested for antibodies to the M. leprae 18-kDa and M. tuberculosis 19-kDa proteins after vaccination with vAbT254 or vAbT235. Sera of BALB/C (H-2d), C57B1/6 (H-2b), C3HIHe (H-2k), DBA (H-2d), FVB (H-2"), and outbred Swiss mice all exhibited normal antivaccinia virus antibody titers but lacked detectable antibodies to the M. leprae 18-kDa and M. tuberculosis 19-kDa proteins (data not shown). This suggests that it was not the haplotype of the animals which caused the lack of response to these mycobacterial antigens. Another possible cause of the lack of humoral response to some of our vaccinia virus recombinants is that the mycobacterial proteins may need to be at the cell surface; it is probable that the mycobacterial proteins expressed by vaccinia virus remain in the cytoplasm. Other investigators have shown that the normally secreted plasmodial S antigen could be anchored to the cell surface by addition of the transmembrane domain of an immunoglobulin (27), thereby enhancing its immunogenicity. We are investigating methods to direct mycobacterial proteins to the surface of infected cells similarly and then anchor them there. Since it is the cellular immune system which mediates immunologic resistance to infection by M. tuberculosis and M. leprae (5, 18, 25), an effective vaccine against tuberculosis or leprosy must elicit a strong cellular immune response. It is therefore important to identify M. tuberculosis and M. leprae proteins that elicit a human T-cell response.

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Human T-cell clones that recognize the M. tuberculosis 65-kDa (42), M. tuberculosis 19-kDa (41), M. leprae 65-kDa (26), M. bovis 65-kDa (11), and M. leprae 18-kDa (36, 43) proteins have been isolated from patients with leprosy or tuberculosis or BCG-vaccinated individuals. It has been reported that over 90% of leprosy contacts thought to be immune to M. leprae exhibited a cell-mediated immune response to the M. leprae 18-kDa protein (10). The ability of our recombinants to elicit a cell-mediated immune response has not been tested. Guinea pig (51) and murine models (22, 47) can be used to test for protective efficacy against M. tuberculosis and M. leprae infection, and animal challenge experiments are under way. M. tuberculosis-vaccinia virus recombinants are being tested in guinea pigs and mice, and M. leprae-vaccinia virus recombinants are being tested in the mouse footpad model system. In addition to their availability for testing as vaccines against leprosy and tuberculosis, these vaccinia virus recombinants can also be used as immunological reagents to study cell-mediated immune responses to mycobacteria. Cells infected by these recombinants can serve as target cells in antibody-dependent cellular cytotoxicity and cytotoxic T-cell assays (23, 24). A method for isolating genes using human T lymphocytes as probes has been reported (37), and this technique, although extremely laborious, may be more relevant in the design of recombinant vaccines expressing mycobacterial antigens which can elicit a protective immune response. Recently, there has been much speculation concerning the role of mycobacterial proteins in autoimmune disease (46). The BCG 65-kDa protein, which is identical to the M. tuberculosis 65-kDa protein, has been implicated in adjuvant arthritis in rats, a model for human rheumatoid arthritis (54, 55). Rat T-cell clones induced by immunization with M. tuberculosis recognized antigens present in human synovial fluids, as well as M. tuberculosis antigens. These T-cell clones induced resistance to adjuvant arthritis. Furthermore, administration of purified 65-kDa antigen resulted in resistance to adjuvant arthritis. Another recent study described the isolation of mycobacterium-specific T-lymphocyte clones lacking CD4 and CD8 markers from the synovial fluid of a patient with rheumatoid arthritis (20). Borrelia burgdorferi, the causative agent of Lyme disease, thought to be an autoimmune disease, also expresses a strongly immunogenic 60-kDa protein (19) homologous with the Pseudomonas aeruginosa common antigen, which has been shown to be homologous to the mycobacterial 65-kDa proteins. The vaccinia virus recombinants described in our report may be useful to immunologists in elucidating the immunological role of bacteria and other infectious organisms in autoimmunity and arthritis in humans.

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3.

4.

5. 6.

7.

8.

9.

10.

11.

12.

13.

14. 15.

ACKNOWLEDGMENTS We thank the following for monoclonal reagents: J. Ivanyi, P. Minden, P. Kelleher, M. Cohen, H. Rumschlag, Richard Young, and the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. We thank Richard Young, Thomas Shinnick, Mitchell Cohen, and Hella Rumschlag for mycobacterial DNA clones and sequence information. LITERATURE CITED 1. Ashbridge, H. R., R. J. Booth, J. D. Watson, and R. B. Lathigra. 1989. Nucleotide sequence of the 19 kDa antigen gene from Mycobacterium tuberculosis. Nucleic Acids Res. 17:1249. 2. Baird, P. N., L. M. C. Hall, and A. R. M. Coates. 1988. A major

16.

17.

18. 19.

antigen from Mycobacterium tuberculosis which is homologous to the heat shock proteins GroES from E. coli and the htpA gene product of Coxiella burnetii. Nucleic Acids Res. 16:9047. Bernards, R., A. Destree, S. McKenzie, E. Gordon, R. A. Weinberg, and D. Panicali. 1987. Effective immunotherapy directed against an oncogene-encoded product using a vaccinia virus vector. Proc. Natl. Acad. Sci. USA 84:6854-6858. Bertholet, C., R. Drillien, and R. Wittek. 1985. One hundred base pairs of 5' flanking sequence of a vaccinia virus late gene are sufficient to temporally regulate late transcription. Proc. Natl. Acad. Sci. USA 82:2096-2100. Bloom, B. R., and T. Godal. 1983. Selective primary health care: strategies for control of disease in the developing world. V. Leprosy. Rev. Infect. Dis. 5:765-780. Booth, R. J., D. P. Harris, J. M. Love, and J. D. Watson. 1988. Antigenic proteins of Mycobacterium leprae. J. Immunol. 140: 597-601. Cochran, M. A., C. Puckett, and B. Moss. 1985. In vitro mutagenesis of the promoter region for a vaccinia virus gene: evidence for tandem early and late regulatory signals. J. Virol. 54:30-37. Cohen, M. L., L. W. Mayer, H. S. Rumschlag, M. A. Yakrus, W. D. Jones, Jr., and R. C. Good. 1987. Expression of proteins of Mycobacterium tuberculosis in Escherichia coli and potential of recombinant genes and proteins for development of diagnostic reagents. J. Clin. Microbiol. 25:1176-1180. Coupar, B. E. H., M. E. Andrew, G. W. Both, and D. B. Boyle. 1986. Temporal regulation of influenza hemagglutinin expression in vaccinia virus recombinants and effects on the immune response. Eur. J. Immunol. 16:1479-1487. Dockrell, H. M., N. G. Stoker, S. P. Lee, M. Jackson, K. A. Grant, N. F. Jouy, S. B. Lucas, R. Hasan, R. Hussain, and K. P. W. J. McAdam. 1989. T-cell recognition of the 18kilodalton antigen of Mycobacterium leprae. Infect. Immun. 57:1979-1983. Emmrich, F., J. Thole, J. van Embden, and S. H. E. Kaufmann. 1986. A recombinant 64 kilodalton protein of Mycobacterium bovis bacillus Calmette-Guerin specifically stimulates human T4 clones reactive to mycobacterial antigens. J. Exp. Med. 163: 1024-1029. Engers, H. D., M. Abe, B. R. Bloom, V. Mehra, W. Britton, T. M. Buchanan, S. K. Khanolkar, D. B. Young, 0. Closs, T. Gillis, M. Harboe, J. Ivanyi, A. H. J. Kolk, and C. C. Shepard. 1985. Workshop: results of a World Health Organization-sponsored workshop on monoclonal antibodies to Mycobacterium leprae. Infect. Immun. 48:603-605. Engers, H. D., V. Houba, J. Bennedsen, T. M. Buchanan, S. D. Chaparas, G. Kadival, 0. Closs, J. R. David, J. D. A. van Embden, T. Godal, S. A. Mustafa, J. Ivanyi, D. B. Young, S. H. E. Kaufmann, A. G. Khomenko, A. H. J. Kolk, M. Kubin, J. A. Louis, P. Minden, T. M. Shinnick, L. Trnka, and R. A. Young. 1986. Results of a World Health Organization-sponsored workshop to characterize antigens recognized by Mycobacterium-specific monoclonal antibodies. Infect. Immun. 51:718-720. Fine, P. E. M., and L. C. Rodrigues. 1990. Mycobacterial diseases. Lancet 335:1016-1020. Fischetti, V. A., W. M. Hodges, and D. E. Hruby. 1989. Protection against streptococcal pharyngeal colonization with a vaccinia:M protein recombinant. Science 244:1487-1490. Garsia, R. J., L. Hellqvist, R. J. Booth, A. J. Radford, W. J. Britton, L. Astbury, R. J. Trent, and A. Basten. 1989. Homology of the 70-kilodalton antigens from Mycobacterium leprae and Mycobacterium bovis with the Mycobacterium tuberculosis 71-kilodalton antigen and with the conserved heat shock protein 70 of eucaryotes. Infect. Immun. 57:204-212. Gillard, S., D. Spehner, R. Drillien, and A. Kirn. 1986. Localization and sequence of a vaccinia virus gene required for multiplication in human cells. Proc. Natl. Acad. Sci. USA 83:5573-5577. Hahn, H., and S. H. E. Kaufmann. 1981. Role of cell mediated immunity in bacterial infection. Rev. Infect. Dis. 3:12211250. Hansen, K., J. M. Bangsborg, H. Fjordvang, N. S. Pedersen, and

VOL. 58, 1990

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31. 32. 33. 34. 35.

36.

37.

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P. Hindersson. 1988. Immunochemical characterization of and isolation of the gene for a Borrelia burgdorferi immunodominant 60-kilodalton antigen common to a wide range of bacteria. Infect. Immun. 56:2047-2053. Holoshitz, J., F. Koning, J. E. Coligan, J. De Bruyn, and S. Strober. 1989. Isolation of CD4- CD8- mycobacteria-reactive T lymphocyte clones from rheumatoid arthritis synovial fluid. Nature (London) 339:226-229. Husson, R. N., and R. A. Young. 1987. Genes for the major protein antigens of Mycobacterium tuberculosis: the etiologic agents of tuberculosis and leprosy share an immunodominant antigen. Proc. Natl. Acad. Sci. USA 84:1679-1683. Ivanyi, J., K. Sharp, P. Jackett, and G. Bothamley. 1988. Immunological study of the defined constituents of mycobacteria. Springer Semin. Immunopathol. 10:279-300. Koup, R. A., J. L. Sullivan, P. H. Levine, D. Brettler, A. Mahr, G. Mazzara, S. McKenzie, and D. Panicali. 1989. Detection of major histocompatibility complex class I-restricted HIV-specific cytotoxic T lymphocytes in the blood of infected hemophiliacs. Blood 73:1909-1914. Koup, R. A., J. L. Sullivan, P. H. Levine, F. Brewster, A. Mahr, G. Mazzara, S. McKenzie, and D. Panicali. 1989. Antigenic specificity of antibody-dependent cell-mediated cytotoxicity directed against human immunodeficiency virus in antibodypositive sera. J. Virol. 63:584-590. Lagrange, P. H. 1984. Cell mediated immunity and delayed-type hypersensitivity, p. 682. In G. P. Kubica and G. W. Lawrence (ed.), The mycobacteria-a source book, part V. Microbiology series. Marcel Decker, Inc., New York. Lamb, J. R., J. Ivanyi, A. D. M. Rees, J. B. Rothbard, K. Howland, R. A. Young, and D. B. Young. 1987. Mapping of T cell epitopes using recombinant antigens and synthetic peptides. EMBO J. 6:1245-1249. Langford, C. J., S. J. Edwards, G. L. Smith, G. F. Mitchell, B. Moss, D. J. Kemp, and R. F. Anders. 1986. Anchoring a secreted plasmodium antigen on the surface of recombinant vaccinia virus-infected cells increases its immunogenicity. Mol. Cell. Biol. 6:3191-3199. Lathigra, R. B., D. B. Young, D. Sweetser, and R. A. Young. 1988. A gene from Mycobacterium tuberculosis which is homologous to the DnaJ heat shock protein of E. coli. Nucleic Acids Res. 16:1636. Lu, M. C., M. H. Lien, R. E. Becker, H. C. Heine, A. M. Buggs, D. Lipovsek, R. Gupta, P. W. Robbins, C. M. Grosskinsky, S. C. Hubbard, and R. A. Young. 1987. Genes for immunodominant protein antigens are highly homologous in Mycobacterium tuberculosis, Mycobacterium africanum, and the vaccine strain Mycobacterium bovis BCG. Infect. Immun. 55:23782382. Mackett, M., and J. R. Arrand. 1985. Recombinant vaccinia virus induces neutralising antibodies in rabbits against Epstein-Barr virus membrane antigen gp340. EMBO J. 4:32293234. Mackett, M., and G. L. Smith. 1986. Vaccinia virus expression vectors. J. Gen. Virol. 67:2067-2082. Mackett, M., G. L. Smith, and B. Moss. 1982. Vaccinia virus: a selectable eukaryotic cloning and expression vector. Proc. Natl. Acad. Sci. USA 79:7415-7419. Mahr, A., and B. E. Roberts. 1984. Organization of six early transcripts synthesized from a vaccinia virus EcoRI DNA fragment. J. Virol. 49:497-509. Mehra, V., D. Sweetser, and R. A. Young. 1986. Efficient mapping of protein antigenic determinants. Proc. Natl. Acad. Sci. USA 83:7013-7017. Moss, B., and C. Flexner. 1987. Vaccinia virus expression vectors. Annu. Rev. Immunol. 5:305-324. Mustafa, A. S., H. K. Gill, A. Nerland, W. J. Britton, V. Mehra, B. R. Bloom, R. A. Young, and T. Godal. 1986. Human T-cell clones recognize a major M. leprae protein antigen expressed in E. coli. Nature (London) 319:63-66. Mustafa, A. S., F. Oftung, A. Deggerdal, H. K. Gill, R. A. Young, and T. Godal. 1988. Gene isolation with human T lymphocyte probes. J. Immunol. 141:2729-2733.

4097

38. Nakano, E., D. Panicali, and E. Paoletti. 1982. Molecular genetics of vaccinia virus: demonstration of marker rescue. Proc. Natl. Acad. Sci. USA 79:1593-1596. 39. Nerland, A. H., A. S. Mustafa, D. Sweetser, T. Godal, and R. A. Young. 1988. A protein antigen of Mycobacterium leprae is related to a family of small heat shock proteins. 1988. J. Bacteriol. 170:5919-5921. 40. O'Connor, S. P., H. S. Rumschlag, and L. W. Mayer. 1990. Nucleotide sequence of the gene encoding the 35-kDa protein of Mycobacterium tuberculosis. Res. Microbiol. 141:407423. 41. Oftung, F., A. S. Mustafa, R. Husson, R. A. Young, and T. Godal. 1987. Human T cell clones recognize two abundant Mycobacterium tuberculosis protein antigens expressed in Escherichia coli. J. Immunol. 138:927-931. 42. Oftung, F., A. S. Mustafa, T. M. Shinnick, R. A. Houghten, G. Kvalheim, M. Degre, K. E. A. Lundin, and T. Godal. 1988. Epitopes of the Mycobacterium tuberculosis 65-kilodalton protein antigen as recognized by human T cells. J. Immunol. 141:2749-2754. 43. Ottenhoff, T. H. M., P. R. Klatser, J. Ivanyi, D. G. Elferink, M. Y. L. de Wit, and R. R. P. de Vries. 1986. Mycobacterium leprae-specific protein antigens defined by cloned human helper T cells. Nature (London) 319:66-68. 44. Rohrmann, G., L. Yuen, and B. Moss. 1986. Transcription of vaccinia virus early genes by enzymes isolated from vaccinia virions terminates downstream of a regulatory sequence. Cell 46:1029-1035. 45. Rumschlag, H. S., M. A. Yakrus, M. L. Cohen, S. E. Glickman, and R. C. Good. 1990. Immunologic characterization of a 35-kilodalton recombinant antigen of Mycobacterium tuberculosis. J. Clin. Microbiol. 28:591-595. 46. Schoenfeld, Y., and D. A. Isenberg. 1988. Mycobacteria and autoimmunity. Immunol. Today 9:178-181. 47. Shepard, C. C. 1965. Vaccination against experimental infection with Mycobacterium leprae. Am. J. Epidemiol. 81:150-163. 48. Shinnick, T. M. 1987. The 65-kilodalton antigen on Mycobacterium tuberculosis. J. Bacteriol. 169:1080-1088. 49. Shinnick, T. M., B. B. Plikaytis, A. D. Hyche, R. M. Van Landingham, and L. L. Walker. 1989. The Mycobacterium tuberculosis BCG-a protein has homology with the Escherichia coli GroES protein. Nucleic Acids Res. 17:1254. 50. Shinnick, T. M., M. H. Vodkin, and J. C. Williams. 1988. The Mycobacterium tuberculosis 65-kilodalton antigen is a heat shock protein which corresponds to common antigen and to the Escherichia coli GroEL protein. Infect. Immun. 56:446451. 51. Smith, D., G. Harding, J. Chan, M. Edwards, J. Hank, D. Muller, and F. Sobhi. 1979. Potency of 10 BCG vaccines as evaluated by their influence on the bacillemic phase of experimental airborne tuberculosis in guinea-pigs. J. Biol. Stand. 7:179-197. 52. Smith, D. W., E. H. Wiegeshaus, and M. L. Edwards. 1988. The protective effects of BCG vaccination against tuberculosis, p. 341-370. In M. Bendinelli and H. Friedman (ed.), Mycobacterium tuberculosis. Plenum Publishing Corp., New York. 53. Taylor, J. W., J. Ott, and F. Eckstein. 1985. The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res. 13:8764-8785. 54. Van Eden, W., J. Holoshitz, Z. Nevo, A. Frenkel, A. Klajman, and R. Cohen. 1985. Arthritis induced by a T-lymphocyte clone that responds to Mycobacterium tuberculosis and to cartilage proteoglycans. Proc. Natl. Acad. Sci. USA 82:51175120. 55. Van Eden, W., J. E. R. Thole, R. van der Zee, A. Noordzij, J. D. A. van Embden, E. J. Hensen, and I. R. Cohen. 1988. Cloning of the mycobacterial epitope recognized by T lymphocytes in adjuvant arthritis. Nature (London) 331:171-173. 56. Wachsman, M., L. Aurelian, C. C. Smith, M. E. Perkus, and E. Paoletti. 1989. Regulation of expression of herpes simplex virus (HSV) glycoprotein D in vaccinia recombinants affects their ability to protect from cutaneous HSV-2 disease. J. Infect. Dis.

4098

LYONS ET AL.

159:625-634. 57. Wachsman, M., J. H. Luo, L. Aurelian, M. E. Perkus, and E. Paoletti. 1989. Antigen-presenting capacity of epidermal cells infected with vaccinia virus recombinants containing the herpes simplex virus glycoprotein D, and protective immunity. J. Gen. Virol. 70:2513-2520. 58. Young, D., R. Lathigra, R. Hendrix, D. Sweetser, and R. A. Young. 1988. Stress proteins are immune targets in leprosy and tuberculosis. Proc. Natl. Acad. Sci. USA 85:4267-4270.

INFECT. IMMUN.

59. Young, R. A., B. R. Bloom, C. M. Grosskinsky, J. Ivanyi, D. Thomas, and R. W. Davis. 1985. Dissection of Mycobacterium tuberculosis antigens using recombinant DNA. Proc. Natl. Acad. Sci. USA 82:2583-2587. 60. Young, R. A., V. Mehra, D. Sweetser, T. Buchanan, J. ClarkCurtiss, R. W. Davis, and B. R. Bloom. 1985. Genes for the major protein antigens of the leprosy parasite Mycobacterium leprae. Nature (London) 316:450-452.