Characterization of a B-cell immunodominant epitope of human T-lymphotropic virus type 1 (HTLV-I) envelope gp46

11

Cancer Letters, 66 (1992) 11 - 20 Elsevier Scientific Publishers Ireland Ltd.

Characterization of a B-cell immunodominant epitope of human T-lymphotropic virus type 1 (HTLV-I) envelope gp46 Michael D. Lairmore”, and Renu B. Lal” ‘Retrouirus Diseases Branch, 30333,

bDepartment

Donna

L. Rudolph”,

Beverly D. Robertsa,

Charlene

S. Dezzuttib

Dioision of Viral and Rickettsial Diseases, Centers For Disease Control, Atlanta,

of Veterinary

Pathobiology,

The Ohio State University,

Columbus,

Ohio, 43210

Georgia,

(USA)

(Received 27 April 1992) (Revision received 8 June 1992) (Accepted

9 June 1992)

ShUIWh~ The immune response elicited by a synthetic peptide derioed from an immunodominant external envelope region (Env-5, amino acids 242 - 257) of human T-lymphotropic virus type 1 [HTLV-1) was tested in a rabbit model of HTLV-1 infection. The synthetic peptide elicited a strong antibody response to the HTLV-1 envelope protein gp46; however, these antibodies failed to inhibit HTLV-Imediated cell fusion. Immunized rabbits were not protected from HTLV-1 infection as determined by seroconuersion to uiral core proteins by immunoblot, HTLV-1 p24 antigen detection

in lymphocyte cultures and polymerase chain reaction for the HTLV-1 provirus in lymphocyte DNA. Env-5 peptide immunization failed to induce T-cell lymphocyte proliferative responses in rabbits, but induced antibody responses in T-cell deficient Balb c nu/nu mice

suggesting

that

represented

by the Env-5 peptide

the

antigenic

determinant is primarily a B-cell epitope. These results further define an immunodominant epitope of the HTLV-1 Correspondence to: Michael D. Lairmore, The Ohio University, Department of Veterinary Pathobiology, Coffey Road, Columbus, Ohio, 43210, USA.

0304-3835/92/$05.00 Printed and Published

State 1925

0 1992 Elsevier Scientific Publishers in Ireland

envelope protein and suggest that potential synthetic peptide vaccines against HTLV-1 infection must contain multiple antigens that induce both reactivity.

humoral

and

cellular

immune

Keywords: human T-lymphotropic virus type 1; synthetic peptide; animal model; vaccine; human

Introduction The immunopathogenesis of human Tlymphotropic virus type 1 (HTLV-I) infection and disease is poorly understood. The virus has been etiologically linked with adult T-cell leukemia/lymphoma (ATLL) and a chronic, degenerative myelopathy, HTLV-I-associated myelopathy, also referred to as tropical spastic paraparesis (HAM/TSP) [ 11. Synthetic peptide immunization is one approach to defining the immunoprotective epitopes necessary to abate HTLV-I infections. As vaccines, synthetic peptides have the theoretical advantage of providing selective humoral and cellular immune reactivity against natural viral epitopes without eliciting harmful responses against host Ireland Ltd

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tissues or necessitating the need to include the infectious agent in vaccine preparations. Several structural motifs of the HTLV-I envelope have been defined by the use of either recombinant proteins or synthetic peptides. These studies have demonstrated several major immunoreactive domains. Two domains located at the N-terminus of gp46 (amino acids 86- 107, [19] and 191- 196, [26]) have been demonstrated to elicit antibodies which block HTLV-I-mediated cell fusion. The central region of gp46 contains human B-cell epitopes defined by both a recombinant protein (MTA-4) [16] and a synthetic peptide (SP-4A amino acids 190 - 209) [18], a human cytotoxic T-cell epitope which spans envelope gp46 amino acids 196 -209 [9] and a murine T helper cell epitope (VlE8 amino acids 191- 209) [lo]. Plasmids which express sequences from the carboxyl terminal region of HTLV-I gp46 (amino acids 200 - 306 and 229 - 308) are also recognized by serum from HTLV-I-infected persons [25]. We have recently reported a unique region from the C-terminus of HTLV-I gp46 (Env-5; amino acids 242-257) which is immunodominant and discriminates antibodies against HTLV-I from those of HTLV-II-infected persons [13]. This peptide elicited antibodies in rabbits which specifically recognized HTLV-I gp68 envelope antigens on the surface of the HTLV-I-infected cell line MT-2 [14]. In addition, Env-5 shares significant amino acid sequences with the epidermal growth factor receptor and could stimulate signal transduction of lymphocytes [15]. In addition, we have reported the comparative ability of HTLV-I and HTLV-II isolates to infect and elicit immune responses in a rabbit model of the human infection [3,12]. To further investigate the ability of Env-5 to elicit a protective immune response, we challenged rabbits with HTLV-I following vaccination with the immunodominant peptide. We report the ability of Env-5 to elicit a strong antibody response to HTLV-I gp46. However, these antibodies failed to inhibit HTLV-I-mediated cell fusion and did not protect rabbits from HTLV-I infection.

The peptide did not induce cellular proliferative responses in immunized rabbits and elicited antibody responses in both T-cell deficient Balb c nu/nu and Balb c nu/ + mice suggesting that Env-5 represents a B-cell epitope of the HTLV-I external envelope. These data also suggest that potential synthetic peptide vaccines against HTLV-I infection may be required to contain multiple epitopes which induce both humoral and cellular-based antiHTLV-I activity. Materials and Methods Peptide origin and synthesis The methods of selection and synthesis of HTLV-I synthetic peptides have been described [13]. Briefly, the synthetic peptide was derived from the external envelope of HTLV-I (Env 5; amino acids 242 - 257) [22]. Synthetic peptides were made on the MilliGen 9050 Pepsynthesizer (Millipore, Bedford, MA) with 9-fluorenylmethyloxycarbonyl chemistry, using the manufacturer’s reagents and recommended chemistry cycles. Final purification was carried out by preparative high-performance liquid chromatography on a Waters (Milford, MA) Cl8 Delta-Fax (19 mm x 30 cm, 15-pm particle, 300~pm pore size), using 0.1% trifluoroacetic acid (TFA) in water as a solvent followed by a O-50% acetonitrile gradient in 0.1% TFA. Amino acid composition, amino acid sequence analysis and analytic reverse-phase HPLC were performed to confirm peptide sequence and purity. Rabbit inoculation procedures New Zealand white rabbits (12 weeks of age) were obtained from a specific pathogen free colony (CDC, Atlanta, GA) and maintained in biosafety level 3 facilities. Rabbits were vaccinated with either synthetic peptide Env-5 coupled with bovine serum albumin (BSA) or with BSA only. Immunized animals were inoculated four times subcutaneously with 100 mg of either Env-5-BSA or BSA only emulsified in complete Freund’s adjuvant (day 0) or incomplete Freund’s adjuvant

13

(weeks 2, 4 and 6). Rabbits were challenged with infectious HTLV-I cell lines and monitored virologically and clinically as described [3,12]. Each animal was inoculated intravenously with either of 2 strains of 1 x 10’ lethally irradiated (5000 rads) HTLV-I-infected cells. The HTLV-I cell lines used for virus challenge included two infectious strains derived from either an ATLL patient (HTLV-I-P3) or from a HAM/TSP patient (HTLV-I-Pl) [12]. Animal groups and inoculum are summarized in Table I. HTLV-1 antibody detection Antibodies reactive to HTLV-I were detected from rabbit serum specimens by peptidebased ELISA (detection of antibodies to Env-5 inoculations) and immunoblot assay (IB) (to monitor seroconversion to HTLV-I infection) as described [6,13]. Briefly, to perform the peptide-based ELISA, polyvinyl plates (Immunlon II, Dynatech Laboratories, Alexandria, VA) were coated with 50 ~1 of synthetic peptide (100 pg/m!) in 0.01 M carbonate buffer (pH 9.6) and incubated overnight prior to washing with phosphate buffer saline (PBS) containing 0.05% Tween 20 (PBS-T). Wells were blocked with 3% bovine serum albumin to prevent non-specificity reactivity. A 1:20 dilution of each test serum was added to duplicate wells and the plates incubated for 90 min at 37OC and rinsed in PBS-T. Alkaline phosphatase-conjugated goat anti-human IgG (Sigma, St. Louis, MO) and p-nitrophenylphosphate (Sigma) substrate was used to demonstrate antibody reactivity. The plates were read with an ELISA reader (SLT Labinstruments, Ronkonkome, NY) at 405 nm. In IB assays, strips were made using purified HTLV-I antigen from MT-2 cells Biologicals , (Hillcrest Cypress, CA). Biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) and an avidinbiotin-horseradish peroxidase conjugate was used to detect bound antibodies. Diaminobenzidine and nickel chloride-hydrogen peroxide was used as a substrate to visualize antibodyantigen bands.

Antibody reactivity to cell surface and intracellular antigens were measured by standard immunofluorescence assay. Briefly, MT-2 cells (HTLV-I-infected) were plated on adherent slides (MM Developments), fixed with acetone and stained with affinity-purified anti-Env-5 or anti-HTLV-I antibodies. HUT 78 cells (HTLVI-negative) were used as negative controls. Detection of HTLV-I in PBMC cultures HTLV-I was detected by co-culture of fico!l/diatrizoate-separated rabbit PBMC with normal mitogen-stimulated human PBMC as described [ 111. Culture supernatants were tested at 21 days of culture by an antigen capture assay for HTLV-I p24 capsid antigen (Coulter Immunology, Hialeah, FL). Polymerase chain reaction (PCR) PCR analysis for HTLV-I provira! DNA from rabbit PBMC was performed as previously described [3]. Briefly, oligonucleotide primer pairs from the gag and po! genes of HTLV-I [4] were used to amplify 1 pg of DNA (equivalent to approximately 150 000 cells) for each PCR amplification. The amplification conditions and probing methods for amplified products have been described [ll, 121. The amplified products were analyzed after electrophoresis in 1.5% agarose gels by Southern blot hybridization using 32P-!abe!ed oligonucleotide probes specific for the amplified products [4,13]. Syncytia inhibition assay HTLV-I and HTLV-II-mediated cell fusioninhibition assay was performed as described previously [2]. Briefly, 40 ~1 of C91/PL (HTLV-I infected cells at 1.25 x lo6 cells per ml) were mixed with 10 ~1 of serum and added to 50 ~1 of C8166 indicator cells at lo6 cells per ml. After an 18 hour incubation the number of syncytia observed were recorded; titers are the reciprocal of the serum dilution which caused inhibition of HTLV-I-mediated syncytia in the indicator cells. T-cell proliferation assay Heparinized blood from immunized

rabbits

14

was collected and peripheral blood mononuclear ceils (PBMC) were separated on ficolldiatriazoate gradients. PBMC were washed twice and then resuspended at 1 x lo6 cells/ml in RPM1 1640 media with 10% fetal bovine serum. PBMC were plated in 96-well round bottom microtiter plates (200 PI/well) containing Env-5 peptide or BSA (each tested with 50 and 100 pg/well); phytohemagglutinin (PHA-P, Difco Laboratory, Detroit, MI) was utilized as a positive control. Cultures

Table 1.

Post HTLV-I challenge: immunoblot,

were incubated for 5 days at 37OC, pulsed with 1 PCi of [3H]thymidine for 16 h prior to harvest. Glass fiber filters were counted with a liquid scintillation counter and triplicate values were averaged. Responses were expressed as a stimulation index (test-cell sample result divided by cell sample with medium alone). An index of 2 or more was considered a positive reaction. To further assess the T-cell dependency of Env-5 antibody responses Balb/c nu/nu (T-cell deficient) (n = 5) and Balb/c

PCR and virus culture data. HTLV-I p24 PBMC culture +

Group no. (immunogen)

Rabbit no.

Immunoblot’

HTLV-I PCR

No. 1 (Env-5)

51 57 218 220

+ + + +

+ + +

45 (Died wk 7) 49 208 210

ND

ND

ND

+ +

+ +

+ +

47 41 202 204

+

+ + ND ND

+ + + + + + + +

No. 2 (Env-5)

No. 3 (BSA)

No. 4 (BSA)

N’D +

l l

+ + -I+

+

39 33 206 212

N’D ND

+ ND ND

No. 5 (none)

43 216

+ +

ND ND

+ +

No. 6 (none)

53 214

ND ND

ND ND

-I+

lImmunoblot analysis on serum samples 4 weeks or greater following HTLV-I challenge. + , indicates positive for HTLVI gag antibodies (i.e., p19, ~24, ~55). ‘Polymerase chain reaction for HTLV-I gag and pol sequences in PBMC samples. +PBMC co-culture supematants monitored for HTLV-I p24 by antigen capture ELISA (Coulter Immunology). ND, not determined. l

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nu/ + (T-cell immunocompetent) (n = 5) mice (littermates) were injected intraperitoneally with 100 pg of Env-5-BSA and BSA emulsified in incomplete Freund’s adjuvant. Serum samples were collected at 7, 14 and 21 days after immunization and Env-5 specific antibodies were tested by ELISA (as above). Results

A total of 20 rabbits (6 groups) were inoculated with Env-5-BSA (n = 8), BSA only (n = 8), or remained uninoculated (n = 4) (Table I). S erum specimens [1:30 dilutions of specimens collected prior to immunization (day 0) and at 6 weeks (at time of 4th immunization)] were tested for immunoreactivity to Env-5-BSA or BSA only by ELISA. All Env-5-BSA immunized rabbits (groups 1 and 2) seroconverted to Env-5 while rabbits immunized to BSA only or uninocul-

EN’/5

ENV5

P3) n=4

W) n=4

ated rabbits (groups 3 - 6) remained negative (Fig. 1). Affinity purified rabbit anti-Env-5 antibodies reacted strongly to HTLV-I antigens from MT-2 cells in immunofluorescence assay similar to human anti-HTLV-I antibodies (data not shown). These results confirmed our previously published data [14] and indicated that Env-5-elicited antibodies recognize the surface of infected cells. The immunoblot assay was less sensitive for detecting Env-5 antibodies to gp46, which is most likely due to the denaturation of the confirmationally dependent Env-5 epitope. We further characterized the immune response to Env-5 immunization in rabbits and T-cell deficient mice. In immunized rabbits syncytia-inhibition antibody titers were low to insignificant ( < 1: 10) in both Env-5-BSA or BSA only groups (data not shown). Following immunization rabbit PBMC cultures did not proliferate in response to Env-5 peptide

Uninoculated controls

n=4

n=4

n=2

n=2

Fig. 1. Peptide ELISA data (week 6, average * S.D.) from immunized rabbits. Animal groups included Env-5 immunized rabbits that were challenged with either of 2 isolates of HTLV-I [Env5 (P3) and Env5(pl)], rabbits immunized with BSA carrier only and challenged with the same HTLV-I isolates (BS(P3) and BS(Pl] and uninoculated (nonimmunized/challenged) controls.

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1 2

3 4 5 6 7

8 0

10 11 12 13 14

Fis. 2. HTLV-I immunoblot assay of Env-5 immunized rabbits: (11, positive control serum from HTLV-I disease patient; (2), negative control normal human serum; (3 -5), serum from rabbit no. 41 (group 3, BSA only) at week 0, 6 weeks after BSA immunization and 6 weeks after HTLV-I challenge; (6 - 8), serum from rabbit no. 210 (group 2, Env-5) at week 0, 6 weeks after Env-5 immunization and 6 weeks after HTLV-I challenge; (9 - 11)) serum from rabbit no. 218 (group 1, Env 5) at week 0, 6 weeks after Env-5 immunization and 6 weeks after HTLV-I challenge; (12- 14), serum from rabbit no. 39 (group 4, BSA only) at week 0, 6 weeks after BSA immunization and 6 weeks after HTLV-I challenge. Note seroconversion to HTLV-I gag proteins in all rabbit groups. Band at 69 kDa indicates reactivity to BSA following immunizations. Immunoblot assay was less sensitive to detecting antibodies elicited by Env-5 immunization to gp46 due to limited glycoprotein transfer in the assay and denaturation of Env-5 antibody reactive epitopes.

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(Fig. 2). Only rabbit no. 49 (group 2) remained seronegative for antibodies to HTLV-I gag proteins by immunoblot assay. Seven of seven rabbit serum samples tested from groups 3 through 6 (BSA only or non-vaccinated groups) had immunoblot reactivity to HTLV-I gag proteins after virus challenge. Clinical or hematologic parameters remained within normal limits in all rabbits throughout the course of study, except 1 rabbit (no. 45, group 2) which died prior to virus challenge of causes unrelated to the immunization study. Since antibody reactivity to HTLV-I proteins could simply reflect an immune responses to viral proteins only, we next determined the presence of HTLV-I in rabbit PBMC cocultures. All rabbits were tested at 6 weeks and at 24 weeks after virus challenge for HTLV-I antigen from PBMC co-cultured samples. Six of seven Env-5-BSA rabbit PBMC co-cultures were positive by antigen capture assay (> 20 pg/ml HTLV-I p24 antigen). One rabbit (no. 49, group 2) was negative for HTLV-I antigen in both PBMC co-cultures. All rabbits which were inoculated with BSA only or remained unimmunized (groups 3 - 6) became infected by HTLV-I as detected by the presence of HTLV-I antigens from PBMC cocultures (Table I). Normal human PBMC (used

(stimulation indices all < 2.0) suggesting that Env-5 does not contain T-cell epitope(s) or elicited a low frequency of Env-5 reactive lymphocytes. To further investigate whether the Env-5 peptide represented a B-cell epitope, homozygous nude (nu/nu) mice and their heterozygous (nu/ + ) littermates were immunized with the Env-5 peptide. Both nu/ + immunocompetent (n = 5) and nu/nu T-cell deficient (n = 5) mice produced high levels of Env-5 specific antibodies in ELISA assays (nu/nu group O.D. = 1.015 * 0.16; nu/ + group O.D. = 0.505 l 0.12). Concurrently tested sera from unimmunized mice did not contain reactivity to Env-5 in our ELISA. Collectively, these data suggest that the Env-5 peptide represents a B-cell epitope. At 8 weeks (following 4 vaccinations) all animals were challenged with either HTLV-IP3 cells (groups 1,3 and 5) or HTLV-I-PI cells (groups 2,4 and 6) (Fig. 1 and Table I). Serum specimens were tested by immunoblot assay at day 0, 6 weeks (at 4th immunization) and 4- 6 weeks after virus challenge. Serum specimens from 6 of 7 rabbits tested from Env5-BSA immunized groups had reactivity to gag gene products (e.g., p19, ~24) after virus challenge. These data suggested that the rabbits were infected by the HTLV-I challenge

12

3456

7

8

9

10

11

12

130 bp

Flp, 3. Polymerase chain reaction to detect HTLV-I gag proviral sequences in rabbit PBMC samples 6 weeks after HTLV-I challenge: (l), MT-Z (HTLV-I) cells diluted 1: 1000 with negative control DNA (IIuT 78 ceils); (2), MT-2 (HTLVI) cells diluted 1: 10 000 with negative control HUT 78 cells; (3), negative control HUT 78 cells; (4), negative control normal human PBMC; (51, rabbit no. 33 (group 4, BSA); (61, rabbit no. 39 (group 4, BSA); (7), rabbit no. 41 (group 3, BSA); (8), rabbit no. 47 (group 3, BSA); (9)) rabbit no. 49 (group 2, Env-5); (lo), rabbit no. 208 (group 2, Env-5); (ll), rabbit no. 210 (group 2, Env-5); (12), rabbit no. 218 (group 1, Env-5). 130 bp = 130 basepair product amplified by HTLV-I gag primers, probed in by Southern blot assay.

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for co-culture with rabbit PBMC) were maintained in parallel cultures and remained negative for HTLV-I antigens. To further verify the presence of HTLV-I from rabbit PBMC samples, PCR was performed directly on PBMC DNA at 6 weeks post viral challenge. Five of seven Env-5-BSA rabbit PBMC samples were positive by PCR (Fig. 3). Two rabbit PBMC samples were negative by PCR (no. 49, group 2 and no. 51, group 1). Because all rabbits in groups 3 -6 were positive in PBMC co-cultures for HTLV-I antigens only selected PBMC samples were tested by PCR. Three of four PBMC samples tested from groups 3 and 4 were PCR positive. PCR analysis was not performed on PBMC samples from groups 5 and 6. Collectively, these data demonstrate that Env-5 immunization offered minimal protection from HTLV-I infection (6 of 7 rabbis were infected after challenge). Discussion Most of the major immunodominant epitopes of the HTLV-I envelope have not been evaluated to delineate which will elicit a protective immune response. We have previously reported that our Env-5 peptide is immunodominant and reactive to HTLV-I antibodies from infected persons [13]. We choose to test the immunoprotective capacity of Env-5, in part, because the peptide is derived from an immunogenic beta turn region of HTLV-I gp46 and elicited antibodies in rabbits which recognized surface antigens from HTLV-I-infected cells [14]. In addition, the Env-5 peptide shares sequence homology with the epidermal growth factor receptor and has the potential to serve as a signal transducer for HTLV-I-induced lymphocyte proliferation II51. Our results indicate that Env-5 is immunogenic in rabbits eliciting antibodies which recognize the Env-5 peptide in ELISA and HTLV-I cell-associated antigens in immunofluorescence assays. However, these antibodies did not inhibit the fusion of HTLV-I-

induced syncytia or protect challenged rabbits from HTLV-I infection. Antibodies against Env-5 apparently fail to bind gp46 epitopes critical for cell fusion, although these antibodies recognize gp46 epitopes on the surface of infected cells. Other linear peptides derived from gp46 (amino acids 86- 107, [19]; 191- 196, [26]) elicit antibodies which block HTLV-I-mediated cell fusion; however, the ability of these peptides to elicit protective immunity has not been reported. Takehara et al., [27] has reported that hyperimmune IgG, but neither heat-inactivated HTLV-I from infected MT-2 cells nor an envelope (env) peptide (amino acids 175 - 196) protected rabbits from HTLV-I infection. Their data also demonstrated that the synthetic peptide env 175 - 196 did not elicit antibodies capable of inhibiting HTLV-I cell fusion. Shida et al., [23] reported the successful vaccination against HTLV-I in rabbits by inclusion of multiple proteins of HTLV-I envelope in a vaccinia-based vaccine. However, these studies did not examine T-cell responses of vaccinated rabbits or examine challenged rabbits for HTLV-I provirus by PCR. In a limited study, Cynomolgus monkeys (Macaca fascicularis) have been reported to be protected from HTLV-I infection by immunization with env hybrid gene products produced in Escherichia co/i 1171. Unfortunately, this study did not examine ‘protected’ monkeys for HTLV-I provirus by PCR and reported inconsistent infections among control monkeys (non-vaccinated animals were negative at 16 and 20 weeks after challenge). Our data indicates that the Env-5 peptide represents a B-cell epitope; Balb c nu/nu mice produced similar levels of antibody responses compared to immunocompetent littermate controls. In addition, Env-5 did not appear to elicit T-cell proliferative responses in our immunized rabbits. Appropriately selected synthetic peptides have been reported to protect from experimental viral infections. A synthetic peptide derived from the nucleoprotein of lymphocytic choriomeningitis virus (LCMV) has been shown to induced anti-viral protection;

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however the LCMV peptide elicited a specific cytotoxic T-cell mediated immune response [Zl]. Synthetic peptides which elicit high affinity antibody responses have been demonstrated to protect cattle from foot-and-mouth disease virus infection [24]. Peptides derived from the E2 glycoprotein of Venezuelan equine encephalititis (VEE) virus have protected mice from experimental VEE infections [8]. Recently, immunization with simian immunodeficiency virus (SW) envelope derived peptides, produced as beta-galactosidase fusion proteins, have been reported to protect monkeys from SW challenge [ZO]. Successful vaccination against HTLV-I infection may require immunogens which elicit cytotoxic Tcell responses against HTLV-l-infected cells perhaps by the inclusion of mixtures of peptides representing multiple T-cell epitopes or inclusion of preparations which maintain conformational dependent epitopes [7]. Optimization of adjuvant preparations or the use of liposomes [5] may improve selective isotypic responses against HTLV-I B-cell epitopes; human sera which recognize our Env-5 peptide contain predominantly IgGl and IgG3 isotypes (R. Lal, unpublished data). Collectively, our results further define an immunodominant B-cell epitope of the HTLV-I envelope and suggest that vaccination against HTLV-I with synthetic peptides will likely require the inclusion of multiple immunodominant antigenic determinants of this human retrovirus. Acknowledgements The authors thank Dr. B. Brown and Mr. E. Jackson, Centers For Disease Control, for technical services and Dr. Gary Toedter, Coulter Immunology, for HTLV-I antigen capture assays. The research was supported, in part, by grants from the American Cancer Society (IRG- 16 - 30)) National Cancer Institute (CA-40714) and The Ohio Cancer Research Associates (OSURF-724954). Dr. C. Deuutti is supported by a Fellowship from The Cancer Research Foundation of America.

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