AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 8, Number 2, 1992 Mary Ann Liebert, Inc., Publishers

An Antigenic Structure of the Trans-Activator Protein Encoded by Human T-Cell Leukemia Virus Type-I (HTLV-I), as Defined by a Panel of Monoclonal Antibodies YUETSU TANAKA,1 MIWAKO MASUDA,1 ATSUSHI YOSHIDA,1 HISATOSHI SHIDA,2 HIROSHI NYUNOYA,3 KUNITADA SHIMOTOHNO,3 and HIDEKI TOZAWA1

ABSTRACT In order to study an antigenic structure of the irans-activator protein encoded by human T-cell leukemia virus type-I (HTLV-I), tax, antigen, we generated and characterized a panel of rat anti-fax, monoclonal antibodies (MAbs) designated WATM-1, WATM-2, WATM-3, and WATM-4. These MAbs were derived from WKA rats immunized with HTLV-I-transformed (HTLV-I+ ) syngeneic T cells. Immunoblot assays showed that: (1) All the MAbs reacted with the tax, antigen in HTLV-I+ cell lines and a recombinant tax, antigen, PX141 (containing entire tax, polypeptide); (2) WATM-3 and WATM-4, but not WATM-1 or WATM-2, reacted with a truncated tax, antigen, XD59 (tax, amino acids 180-338); (3) None of them reacted with another truncated tax, antigen, XD128 (tax, amino acids 1-47 and 286-353); and (4) each of the four MAbs had different reactivity with tax, -related antigens in the range 38-41 kDa expressed in simian cell lines infected with various HTLV-I-related simian retroviruses (STLV-I). None of the MAbs reacted with HTLV-II tax antigen. Human sera containing anti-tax, antibodies interfered specifically with the antigen-specific binding of all the MAbs. These results suggest that the present rat MAbs are directed against various epitopes on the tax, antigen. An antigenic structure of the tax, antigen deduced from reactivity of a panel of anti-tax, MAbs including the present rat MAbs is discussed.

INTRODUCTION

least five distinct epitopes. The present study was designed in order to extend the analysis of an antigenic structure of the tax, at

is A C-TYPE RETROVIRUS etiologically associated with a human T-cell leukemia, adult T-cell [~* leukemia and some neurologic disorders.5'' HTLV-I transforms T cells from wide variety of species,7-9 probably by means of a unique gene product called fra«.ç-activator (tax,) antigen (for review see ref. 10), which activates not only in trans the HTLV-I promoter, long-terminal repeat (LTR), but also several cellular genes."'5 For immunological and biochemical studies of the tax, antigen, we had generated a panel of MAbs against the tax, antigen from mice immunized with either partially purified or recombinant tax, antigen preparations.I617 With these MAbs, it was revealed that the tax, antigen expressed

HTLV-I(ATL),malignant

antigen.

Because rat T cells

tion7 and

were susceptible to HTLV-I-transformagenerally are used to generate MAbs, we used to produce a panel of anti-fax, MAbs in this study.

rats

inbred rats The rat strain chosen was WKA, because this strain has been shown to produce anti-far, antibodies upon immunization with HTLV-I * syngeneic cells.Ix We have succeeded in generating four distinct anti-fax, MAbs and examined their reactivity. The results indicate that there exist additional epitopes on the tax, antigen which had not been revealed with the previous mouse anti-fax, MAbs, and that some of the epitopes are also expressed on antigens encoded by several STLV-I strains.

'Department of Immunology, School of Hygienic Sciences, Kitasato University, Kitasato

institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto.

^Virology Division, National Cancer Center Research Institute, Tsukiji, Tokyo, Japan. 227

1-15-1, Sagamihara, Kanagawa228.

TANAKA ET AL.

228

?

** i í

ê

/ i

g

í

"

REY-7

WATM-2

WATM-1

WATM-4

WATM-3

Mo-B,29 was obtained from the American Culture Collection (Bethesda, MD), and maintained in Type Iscove's modified Dulbecco's medium supplemented with 20% FCS. Another HTLV-II tax antigen-positive cell line, Wil-2/ 43,30 was cultured in RPMI-1. HTLV-II+ cell line,

MATERIALS AND METHODS Animals Six-weeks-old female WKA rats were purchased from SLC and maintained at the Institute of Laboratory Animal Science, School of Hygienic Sciences, Kitasato University.

Japan

Cells HTLV-I+ rat T-cell lines used were W7TM-1 andD9TM-l of WKA rat and DA rat origin, respectively.18 HTLV-I+ human T-cell lines used were MT-2,'9 HUT-102,' F-Taj and F-Aki,20 and ILT-MOR-1I.21 HTLV-I-negative (HTLV-H) cell lines used were Molt-4,22 CCRF-CEM,23 HPB-ALL and HPBMLT,24 TALL-1,25 Daudi,26 Raji,27 a SV40-transformed kidney cell line of WKA rat origin (W7KSV),18 and HeLa cells. The myeloma cell line used was Sp2/0-Agl4. STLV-I-infected cell lines used were ChM114-l, GM0605, PtM3, JM86, RÍM26-2, andBM5, which were isolated from a chimpanzee, an African green monkey, a pig-tailed monkey, a Japanese monkey, a red-face macaque, and a bonnet monkey, respectively.28 These cell lines were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin, and 100 p-g/ml streptomycin (referred to as RPMI-1), ILTMOR-II was cultured in RPM1-1 supplemented with 40 units/ml of interleukin-2 (IL-2) (Shionogi, Osaka, Japan). HeLa cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FCS and 20 p-g/ml kanamycin (referred to as DMEM). HeLa and CCRF-CEM cells were obtained from the Japanese Cancer Research Resources Bank (JCRB, Tokyo). An

Immunization and preparation

of hybridomas

Four rats were injected i.p. with two doses of W7TM-1 cells x 107 cells per animal) emulsified in Freund's incomplete adjuvant at an interval of 2 weeks. After 2 weeks, each rat was boosted by injection i.v. with 1 x 107 W7TM-1 cells in phosphate-buffered saline (PBS). Immune spleen cells were isolated from the four rats 3 days after the final immunization, and were fused with Sp-2/0-Agl4 myeloma cells at a spleen cell to myeloma cell ratio of 4:1 in the presence of 50% polyethyleneglycol 2000. After HAT selection, hybridomas producing antibodies reactive with the fax, antigen were screened and cloned by limiting dilution.

(1

Immunofluorescence (IF)

assay

Cells smeared onto glass slides and fixed with methanol for 5 min at 20°C were stained with MAbs by an IF method using FITC-labeled goat IgG anti-rat IgG (Cappel, PA) as the secondary reagent as described previously.31 Fluorescent cells were examined by fluorescence microscopy. —

MAbs Four anti-fax, MAbs of mouse origin used were Lt-4, which generated by immunization with native tax, antigen purified

was

Reactivity of MAbs with HTLV-I+ and STLV-I cell lines. Lysates from (A) HTLV-I+ cells (MT-2, HUT102, F-Taj and W7TM-1) and STLV-I+ cells derived from a Bonnet monkey (BM5), a Japanese macaque (JM86) and a Red face macaque (RÍM26-1), and (B) STLV-I+ cells derived from a chimpanzee (ChMl 14-1), a pig-tailed monkey (PtM3) and an African green monkey (GM0605), were separated by SDS-PAGE on either 10% (A) or 12.5% (B) gel and subjected to immunoblot analysis. Lt-4 and NOR-1 were mouse MAbs specific for the tax, and HTLV-I gag p24, respectively.

FIG. 2.

+

ANTIGENICITY OF THE HTLV-I tax ANTIGEN

229

tff/i*

£

-gp68

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•gp68

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31 Kd-

WATM-1

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WATM-3

WATM-2

to

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,

.

¿iffa

-gp68

H .p40tax 31 Kd

31Kd-

B

NOR-1

Lt-4

WATM-4

1 23 97Kd67Kd-

123

123

123

1 23

„5

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31 Kd-

22Kd-

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WATM-1 WATM-2 WATM-3 WATM-4

1:ChM114-1

2: PtM3

3: GM0605

230

TANAKA ET AL.

Table 1. Molecular Weight of Specific Antigens Detected with Anti-íox i MAbs by IMMUNOBLOT IN HTLV-I+ AND STLV-I + CELL LINES

Cell line

Infected with

MT-2 HUT-102

F-Taj

ILT-Aki ILT-MOR-II ChM114-l GM0650 PtM-3 JM86 RÍM26-1 BM5 "Weak

HTLV-I HTLV-I HTLV-I HTLV-I HTLV-I STLV-I STLV-I STLV-I STLV-I STLV-I STLV-I

Lt-4

WATM-1

68,40

68,40

39 40 40 40 41 40 36 None None None

39 40 40 40 41 40

column

chromatography

(RWs)

containing HTLV-I gag gene (WR-gag) and pX gene (WR-27* containing fax,, rex,, and p21jr genes) were reported previously.32'33 For the expression of HTLV-I antigen by RVV, HeLa cells were adsorbed with RVV for 1 h at a multiplicity of RVVs

infection (MOI) of 10 and incubated for 16-18 h in DMEM at 37°C. After washing with phosphase-buffered saline (PBS), the infected cells were lysed in a low salt extraction buffer at cell concentration of 2 x 107 cells/ml as described previously.21 Cell lysates were obtained by centrifugation at 12000 g for 10 min.

40 40 40 41

(40)a

(36)a

36 38 None None

38 None None

68,40 39,38

40 40 40 41 40 36 None 38 39

preparation of crude and purified recombinant fox, protein, PX141 (containing entire tax, polypeptide), and truncated tax, proteins, XD59 (the amino acids 180-338) and XD128 (the amino acids 1-47 and 286-353),

was

described

'7

¡mmunoblot

Samples boiled for 3 min in the sample buffer were subjected sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to blotting sheets, and the sheets then were incubated with each MAb. The binding of MAb was visualized by "PAP method," as described previously.34

to

Antibody binding competition

the tax, antigen to the wells. After 1 h, the washed with T-PBS and preincubated with 100 p-l/well of human serum, which was diluted serially, for 1 h, and then 100 p-l/well of appropriately diluted MAb was added without washing. After 30 min, the wells were washed and further reacted with POD-labeled anti-rat IgG (Cappel, USA) for 30 min. Binding of MAb was visualized as described

cells/ml) wells

to adsorb

were

previously.21

IF: Fixed MT-2 cells on wells of glass slides were pretreated with each competitor mouse MAb (100 p,g/ml) for 30 min, and then reacted with each rat MAb diluted to give half maximum fluorescence intensity for additional 30 min. The wells were then washed and incubated with FITC-conjugated goat IgG anti-rat IgG which was preabsorbed with mouse IgG. Intensity of fluorescence was determined by fluorescence microscopy.

RESULTS Generation

antigens

The

previously.

68,40 39,38

68,40

WATM-4

reactivity.

Recombinant vaccinia viruses

Recombinant tax,

WATM-3

39 40 40 40 41 40 36 None None None

from HTLV-I + human T cells, and TAXY-6, TAXY-7, and TAXY-8, which were generated from mice immunized with a recombinant fax, MAbs used were a mouse antiantigen.17 Negative control ' ' HTLV-I p24 (NOR-1 )3 and a rat anti-HTLV-I gp46 (REY-7).2

by antibody affinity '6

WATM-2

assay

ELISA : Wells of 96-well-plates (Corning 25802) were coated with 50 p-l/well of Lt-4 MAb dissolved in PBS at concentration of 1 p-g/ml at 4°C overnight. Afterblocking with 20% Block Ace (Dainippon Pharmaceutical, Osaka, Japan), wells were washed with PBS containing 0.05% Tween-20 (T-PBS), and then incubated with 100 p-l/well of MT-2 cell lysates (2 x 106

Fusion of

of four MAbs against the tax, antigen spleen

cells from four WKA rats immunized with

HTLV-I+ syngeneic T cells with mouse myeloma cells resulted in growth of 1972 hybridomas in culture plates. Screening of antibodies reactive to HTLV-I+ but not HTLV-I" cells by IF assays showed that about 10% hybridomas produced such antibodies. We selected and expanded 100 hybridomas of which antibodies stained the nucleus of MT-2 cells, where the tax, protein is located, and then the supernatants were tested for reactivity with the tax, antigen by immunoblot assays. There were 14 hybridoma supernatants reacting specifically with the tax, antigen expressed in HeLa cells by infection with tax,expressing RVV, WR-27V. These fax,-specific antibody-producing hybridomas were expanded, and four of them that showed continuous antibody production and strong reactivity to tax, antigen were cloned. These new MAbs were designated WATM-1, WATM-2. WATM-3, and WATM-4, and were of IgG2b, IgG2b, IgG2b, and IgG2a, respectively, as determined

by an Ouchterlony analysis (data not shown). Immunoblot assay

The reactivity of these MAbs with the tax, antigen, as determined by immunoblot assays, is shown in Figure 1. The

ANTIGENICITY OF THE HTLV-I tax ANTIGEN Table 2. Reactivity of Rat

Infected

Cell line

with

MT-2 HUT-102

231

Anti-íoxi MAb WATM-1

HTLV-I HTLV-I HTLV-I HTLV-I HTLV-I HTLV-I HTLV-I

F-Taj

ILT-Aki ILT-MOR-II W7TM-1 D9TM-1 HPB-MLT TALL-1 Molt-4 HPB-ALL CCRF-CEM

with

Various Cells in IF Assay

WATM-2

WATM-3

WATM-4

+-P

+•+

++

++ +

++ ++ + + + + +

++ ++ + + + + +

+

±

+ + + + +

+ + + +

±

Raji

Daudi W7KSV HeLa cellsb

SV40

WR-27X

Mo-B Wil-2/43

"Cell

+

+

+

+

WR-gag

HTLV-II

(HTLV-II)C

smears

fixed with methanol

were

reacted with MAbs (1:10 diluted culture

supernatants) and then with FITC-labeled anti-rat IgG. Fluorescent cells were examined by fluorescence microscopy. Fluorescence intensity was scored as; ++, strongly positive; +, positive; ±, weakly positive; and —, negative. bHeLa cells infected with recombinant vaccinia viruses expressing HTLV-I tax, gene

(WR-27")

or

HTLV-I gag gene

(WR-gag) for 16 h.

cTransfected with HTLV-II genome.

four MAbs reacted with the tax, antigen expressed in both syngeneic HTLV-I+ cells and HeLa cells infected with WR-27* and with the recombinant tax, antigen, PX141 (containing the entire tax, polypeptide). WATM-3 reacted with tax,-unrelated proteins of about 90 and 30 kD. The reactivity of these MAbs with proteins in the various cell

lines infected with HTLV-I and related STLV-I is shown in Figure 2. All the MAbs reacted with the tax, antigens in HTLV-I+ human and rat cell lines, and with tax, -related gp68 containing the tax, amino acids 94-353, which was found only in MT-2 and related cells.35 We showed that the HUT-102 cell line maintained in our laboratory expressed aberrant fax, molecules of 39 and 38 kD, which were detected with mouse anti-fax, MAbs.17'34 WATM-1 and WATM-2 reacted only with the 39 kD protein, but WATM-3 and WATM-4 reacted with both the 39 and 38 kD proteins of HUT-102 cells. Three STLV-1-infected cell lines used in Figure 2a had been shown to be negative for one tax, epitope recognized by a mouse anti-fax, MAb, Lt-4.34 Thus, these cell lines were used to distinguish antibody reactivity among the new MAbs. Figure 2 shows that: (1) WATM-1 did not react with the three cell lines; (2) WATM-2 and WATM-3 reacted with 38 kD antigen of JM86 cell line; and (3) WATM-4 reacted with 39 kD antigen of BM5 cells and 38kD antigen of RfM26-1 cells. We also examined the reactivity of the MAbs with the other panel of STLV-I+ cell lines (Fig. 2b) and HTLV-I+ cell lines derived from HTLV-Iinfected humans (data not shown), and the results are summarized in Table 1.

IF assay The reactivity of these rat MAbs with various cells, as determined by IF assays, is shown in Table 2. These MAbs preferentially stained the nucleus of the WR-27 "-infected HeLa cells and the HTLV-I+ cell lines except for MT-2 cells, as was the case in the mouse anti-fax, MAb, Lt-4.16 Both the nucleus and cytoplasm of MT-2 cells were stained with these MAbs. None reacted with HTLV-II+ cell lines. WATM-1 and WATM-2 were specific for the tax, -expressing cells. However, WATM-3 and WATM-4 stained faintly and diffusely the fixed cells of some HTLV-I" T cell lines.

Epitope mapping epitopes recognized by these MAbs, we have truncated tax, recombinant proteins, XD59 and XD128, which contained the fox, amino acids 180-338 and both 1-47 and 286-353, respectively, as antigens in immunoblot analysis. Figure 3 shows that WATM-1 and WATM-2 did not react with either antigen, and that WATM-3 and WATM-4 reacted with XD59 but not XD128. Several specific bands detected in the crude preparations of PX 141 and XD59 might be degraded or aggregated materials of the intact proteins. The presence of XD128 on the blotting sheets was demonstrated by the positive reaction with a mouse anti-fax, MAb, TAXY-7, which was shown to react with XD128.'7 To localize

prepared

two

232

TANAKA ET AL.

,vVV

///

97Kd

-i

97Kd-,_

WATM-1

WATM-2

///

y>V

WATM-3

66Kd-

43K0-

31Kd-

22Kd