Human T-Lymphotropic Virus Type 1 Peptides in ... - Journal of Virology

JOURNAL OF VIROLOGY, Oct. 1995, p. 6077–6089 0022-538X/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 69, No. 10

Human T-Lymphotropic Virus Type 1 Peptides in Chimeric and Multivalent Constructs with Promiscuous T-Cell Epitopes Enhance Immunogenicity and Overcome Genetic Restriction MICHAEL D. LAIRMORE,1,2* ANN M. DIGEORGE,3 SUSAN F. CONRAD,2 ALEX V. TREVINO,1† RENU B. LAL,4 AND PRAVIN T. P. KAUMAYA2,3 Center For Retrovirus Research and Department of Veterinary Biosciences, College of Veterinary Medicine,1 Department of Obstetrics and Gynecology, College of Medicine,3 and Comprehensive Cancer Center,2 The Ohio State University, Columbus, Ohio 43210, and Retroviruses Diseases Branch, Centers for Disease Control and Prevention, Atlanta, Georgia 303334 Received 24 February 1995/Accepted 22 June 1995

Conventional strategies of viral peptide immunizations often elicit low-affinity antibody responses and have limited ability to elicit immune responses in outbred animals of diverse major histocompatibility (MHC) haplotypes. This genetically restricted T-cell-stimulatory activity of peptides is a serious obstacle to vaccine design. However, the use of promiscuous T-cell epitopes may circumvent this problem. Promiscuous T-cell epitopes from tetanus toxin (amino acids [aa] 580 to 599) and the measles virus F protein (aa 288 to 302) that bind to several isotypic and allotypic forms of human MHC class II molecules have been identified and have been used in highly immunogenic constructs to overcome haplotype-restricted immune responses. Chimeric and b-template peptide constructs incorporating known human T-lymphotropic virus type 1 (HTLV-1) B- and T-cell epitopes from the surface envelope protein gp46 (SP2 [aa 86 to 107] and SP4a [aa 190 to 209]) and promiscuous T-cell peptides were synthesized, and their immunogenicities were evaluated in both rabbits and mouse strains of divergent haplotypes (C3H/HeJ [H-2k], C57BL/6 [H-2b], and BALB/c [H-2d]). In addition, peptide preparations were structurally characterized by analytical high-performance liquid chromatography, mass spectrometry, and circular dichroism. In contrast to their linear forms, the chimeric constructs of both the SP2 and SP4a epitopes displayed a-helical secondary structures. Immunogenicity of the peptide constructs was evaluated by direct and competitive enzyme-linked immunosorbant assay (ELISA), as well as by radioimmunoprecipitation, syncytium inhibition, and antigen-induced lymphocyte proliferation assays. Immunization with the SP4a peptide without conjugation to a carrier protein produced antibodies specific for SP4a in two mouse strains (C3H/HeJ and C57BL/6). However, BALB/c mice failed to respond to the peptide, indicating that the T-cell epitope of the SP4a sequence is MHC restricted. In contrast, the chimeric constructs MVF-SP2 and SP4a-measles virus F protein were highly immunogenic, producing elevated ELISA titers after only two immunizations. Elicited antibodies recognized native forms of gp46 in ELISAs and radioimmunoprecipitation assays, as well as inhibited HTLV-1-mediated syncytium formation. In addition, chimeric constructs were effective at induction of lymphocyte proliferation to the T-cell epitope, SP4a, in each strain of immunized mice. Our data demonstrate that the antibody response to retroviral peptides is enhanced by promiscuous peptide constructs, in part because of the ability of such constructs to promote appropriate secondary structural forms of viral epitopes. In addition, these constructs promote virus-specific helper T-cell responses, thereby overcoming genetically restricted immune responses to the synthetic peptides. have not been fully elucidated. Recently, immunodominant epitopes within the HTLV-1 protein that are recognized by antibodies and cytotoxic T lymphocytes have been identified. Palker et al. (44, 45), using synthetic peptides, have identified at least two functional domains in the amino terminus and central region of gp46 (SP2, amino acids [aa] 86 to 107, and SP4a, aa 190 to 209). The SP4a region overlaps with neutralizing determinants mapped with both human (4) and rat (56) monoclonal antibodies. This central gp46 region also contains human B-cell epitopes defined by recombinant proteins (37) and a human cytotoxic T-cell epitope which spans aa 196 to 209 (21), as well as a murine defined T-helper cell epitope (V1E8, aa 191 to 209) (30). Recently, data regarding neutralizing Bcell epitopes within HTLV-1 gp46 were used to design effective peptide-based vaccines against HTLV-1 infection in initial animal studies (3, 55). Using synthetic peptides, we have identified several immunodominant regions in the gag, pol, tax, rex,

Knowledge regarding the immunopathogenesis of human T-lymphotropic virus type 1 (HTLV-1) infection and disease is incomplete. The virus is causally linked with adult T-cell leukemia/lymphoma, as well as a variety of lymphoproliferative disorders (reviewed in reference 19). The most important of these nonneoplastic disorders is tropical spastic paraparesis, which is clinically and pathologically identical to HTLV-1associated myelopathy (16, 41). The viral determinants which determine a protective immune response against HTLV-1

* Corresponding author. Mailing address: Center for Retrovirus Research, Department of Veterinary Biosciences, The Ohio State University, 1925 Coffey Rd., Columbus, OH 43210-1092. Phone: (614) 292-4819. Fax: (614) 292-6473. Electronic mail address: mlairmor@ magnus.acs.ohio-state.edu. † Present address: Coulston Foundation, Holloman Air Force Base, Holloman Air Force Base, NM 88330. 6077

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FIG. 1. (a) Schematic representation and amino acid sequences of SP4a-SP2-TT-MVF-template. The amino terminus of each peptide epitope and the N-terminal residue (Gly) of the template are shown in their acetylated forms (Ac). The template consists of alternating Leu-Lys connected by a 6-residue loop sequence consisting of Gly-Leu-Pro-Ser-Gly-Gly. (b) Schematic representation and amino acid sequences of chimeric HTLV-1 SP4a (aa 190 to 209) and SP2 (aa 86 to 107) constructs. The measles virus promiscuous T-cell epitope (MVF) was colinearly synthesized via a 4-residue b-turn sequence (Leu-Ser-Pro-Gly) at the N terminus of SP4a and at the C terminus of SP2.

and env regions of the virus (31–34). While several epitopes have been identified for HTLV-1, the characterization of these regions is not complete, as exemplified by the significant seroreactivity of infected persons to recombinant proteins from the C-terminal regions of gp46 (aa 200 to 306 and 229 to 308) (54) and synthetic peptides from the amino-terminal regions of the transmembrane protein (45). Although synthetic peptides can be used to delineate immunoprotective regions of a viral protein, critical parameters which regulate epitope selection and presentation of an antigen are highly complex. Theoretically, synthetic peptide immunogens are capable of eliciting selective immune responses against viral infection without triggering harmful responses to host tissues. Knowledge of the cellular and molecular basis of antigen processing and antigen recognition by both T cells and B cells provides the opportunity to rationally design effective immunogens (1, 2, 5, 39, 42, 53). Thus, strategies to design protective vaccines must be able activate the relevant arm(s) of the immune system (reviewed in reference 24). Antibodies elicited by conventional strategies of peptide immunization (peptide-carrier conjugates), in general, bind with less affinity to their corresponding native protein partly because native epitope conformations have been significantly modified during the process of conjugation (10, 38). Alternatively, antigen processing of the peptide-carrier conjugate in vivo presents a different conformational resemblance. Another major disadvantage is that this process results in carrier-induced epitopic suppression (35). Several proposed alternatives to improve peptide immunogens such as polymerized or chimeric constructs containing Band T-cell epitopes have been used successfully to generate immune responses (10, 13, 15, 35, 38, 43). The introduction of the concept of multiple antigenic peptides resolved many of the problems associated with conjugation of carrier proteins.

In this strategy, peptides are synthesized in multiple branches from a lysine core (48). However, a disadvantage that remains for peptide immunization is the limited ability of peptides to elicit effective immune responses in an outbred population due to genetic major histocompatibility complex (MHC) polymorphism (49). Thus, vaccination based on chimeric B- and Thelper cell epitopes will result in a proportion of nonresponders or poor responders in outbred animals and variable responses in different inbred mouse strains. This genetically restricted T-cell-stimulatory activity of peptides presents a serious obstacle to vaccine design using synthetic peptides. However, the use of promiscuous T-cell epitopes may circumvent this problem and allow the design of vaccines that bind divergent histocompatibility types (23, 25, 50). A number of promiscuous T-cell epitopes for tetanus toxin (TT) and the measles virus F protein (MVF) have been identified (18, 46, 47). A key feature of these T-cell epitopes is their capacity to bind several isotypic and allotypic forms of human MHC class II molecules. We have demonstrated that chimeric peptides comprising these promiscuous T-cell epitopes and a B-cell epitope from the testis-specific isozyme of lactate dehydrogenase C4 are highly immunogenic in several inbred strains of mice (23, 25, 50). In parallel studies, we have also developed a multiepitope b-template approach to present structurally defined constructs that preserve appropriate molecular mimicry of B-cell epitopes and also incorporate a number of different B- and T-cell epitopes to bypass MHC restriction (24, 28). Therefore, in the present study, in an attempt to develop a universal HTLV-1 vaccine, chimeric and b-template vaccine constructs incorporating known HTLV-1 B-cell, helper T-cell, and cytotoxic T-lymphocyte epitopes with promiscuous T-cell peptides were synthesized, and their immunogenicities were evaluated in inbred mouse strains and outbred rabbits and

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FIG. 2. Haplotype restriction of the SP4a epitope. Antibody responses (titers were determined as the serum dilution that gave an absorbance of 0.2 U above the background) in three inbred strains of mice (BALB/c, H-2d; C3H/HeJ, H-2k; C57BL/6, H-2b). The figure shows antibody titers (2y 1 1) (1 week after the second injection) against plated antigens SP4a, chimeric SP4a-MVF, and recombinant protein MTA-1 (left to right in each group of columns). Individual titers were determined for each mouse (the number of responders versus the number immunized), and pooled serum titers were determined.

mice. The constructs were highly immunogenic in both animal species. Furthermore, the linkage of our retroviral peptides in these immunogenic constructs extended our ability to elicit antibody responses in mice of divergent MHC haplotypes. We provide structural data of peptide constructs and demonstrate that the constructs mimic native viral envelope proteins. These findings demonstrate that retroviral peptides linked with promiscuous peptides promote appropriate secondary structural forms of viral epitopes and enhance the ability of such constructs to promote helper T-cell responses and overcome genetic restriction associated with synthetic peptide immunization. MATERIALS AND METHODS Peptide synthesis and purification. All synthetic peptide constructs were assembled with a Milligen/Biosearch 9600 synthesizer. The HTLV-1 viral epitope peptides (SP2 [aa 86 to 107] and SP4a [aa 190 to 209] [44, 45]) and the chimeras (MVF-SP2 and SP4a-MVF) were synthesized with 4-methylbenzyhydrylamine resin (0.54-mmol/g substitution) as the solid support. The acid-labile linker 4-(hydroxymethyl)phenoxyacetic acid was attached to the resin by double coupling of the preformed pentafluorophenyl ester. The carboxy-terminal amino acid was attached to this support by double coupling the preformed pentafluorophenyl ester in the presence of 0.1 equivalent of dimethylaminopyridine (60 min for each coupling), and 1 equivalent of hydroxybenzotriazole was added during the final 15 min of each coupling. Amino acids were sequentially added by benzotriazole-1-yl-oxy-tris(dimethylamine)phophonium hexafluorophosphate–hydroxybenzotriazole activation and coupling protocols. When the synthesis was complete, the peptides were cleaved from the resin, and side chain protecting groups were removed, by treatment with trifluoroacetic acid in the presence of various scavengers (to prevent irreversible side chain modification reactions). The template constructs were synthesized with phenylacetamidemethyl resin (0.31-mmol/g substitution with butyloxycarboxy-glycine, the carboxy-terminal residue) as the support. Segments of the b-strand template moiety were synthesized by the Fmoc (9-fluoroenylmethoxycarbonyl)-benzyl strategy, with temporary a-NH2 protection by the nitropyridine sulfenyl group. Butyloxycarboxy groups were removed by treatment (two times for 20 min) of the resin with 40% trifluoroacetic acid in dichloromethane, followed by neutralization with 10% diisopropylethylamine in dichloromethane (three times for 5 min). The TT and MVF promiscuous T-cell epitopes were assembled sequentially (butyloxycar-

boxy-benzyl chemistry) on εNH2 of Lys in the b-strand template. The SP2 sequence was synthesized similarly by an Fmoc-benzyl protocol, and the SP4a epitope was constructed by an Fmoc–t-butyl strategy. Upon completion of the synthesis, peptide was cleaved from the support (and side chain protecting groups were removed) by the low-high hydroxyfluoride cleavage protocol. Crude peptides were gel filtered (Sephadex G-25; 0.1 M acetic acid as the eluant) to remove low-molecular-weight contaminants prior to purification by high-performance liquid chromatography (HPLC). The segmented epitopes and the chimeras were purified by semipreparative reverse-phase HPLC (Vydac C4 column; 10 mm by 25 cm) at 32.58C with a flow rate of 5 ml/min. Peptides (15 to 30 mg per run) were loaded in 0.1 M acetic acid and chromatographed for 20 to 30 min with linear gradients (0 to 60% or 10 to 90%) of acetonitrile in water containing 0.1% trifluoroacetic acid. The recombinant protein MTA-1 (containing HTLV-1 aa 162 to 209) and vector control protein SJ26 (37) were provided by Steven Foung, Stanford University Blood Bank, Palo Alta, Calif. Peptide characterization. Analytical HPLC was run using a Vydac C4 column (4.6 mm by 25 cm) at 1 ml/min using the gradients listed above. Eluants were monitored at 214, 254, and 280 nm. Purified peptides were obtained in .95% purity as assessed by reverse-phase HPLC. Samples also were analyzed by capillary electrophoresis using a Beckman PACE 2100 instrument. Samples were injected under pressure, and separations were performed in 100 mM sodium borate, pH 8.2, at 258C in a 10-kV applied field; detection was at 214 nm. Amino acid analysis was performed at the Ohio State University Biochemical Instrument Center. Peptides were hydrolyzed in 6 N HCl for 24 h at 1108C; this was followed by derivatization and analysis of the amino acids as their phenylthiohydantoin derivatives. The analysis was consistent with the expected composition for each peptide. Circular dichroism spectra were obtained with a Jasco J500 spectropolarimeter interfaced with an IBM computer. The instrument was calibrated with a 0.06% (wt/vol) solution of ammonium-D-10-camphorsulfonate. Peptides were dissolved (100 mM) in water or 50% trifluoroethanol in buffer, and spectra were obtained from 200 to 260 nm with a 0.1-cm cylindrical quartz cuvette at 258C under a constant flow of nitrogen. Molar ellipticity values ([Q]l) were calculated according to the following equation: [Q]l 5 (100 3 Q)/(n 3 c 3 l), where Q is the ellipticity (in millidegrees), n is the number of residues in the peptide, c is the peptide concentration (in millimolars), and l is the cell path length (in centimeters). Helicity of peptides was calculated with the equation of Chen et al. (11) with Q222 5 233,000 for 100% helix of the polylysine reference. [Q]222 (percent helix) in buffer: SP2, 21,770 (5.4%); SP4a, 22,275 (6.9%); MVF-SP2, 21,940 (5.9%); SP4-MVF, 22,426 (7.4%). [Q]222 (percent helix) in 50% trifluoroethanol–buffer: SP2, 22,891 (8.8%); SP4a, 22,795 (8.5%); MVF-SP2, 27,949 (24.1%); SP4-MVF, 27,738 (23.4%).

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FIG. 3. Antibody responses of chimeric MVF-SP2 and SP4a-MVF. Individual rabbit sera (2y 1 1) were reacted against chimeric immunogens as well as the individual epitopes. Titers were determined as described in the legend to Fig. 2. (Top) Columns (left to right in each group): SP4a-MVF, SP4a, and MVF-turn; (bottom) columns (left to right in each group): MVF-SP2, SP2, and MVF-turn. Animals and immunization. Female inbred strains of mice (BALB/c, C3H/ HeJ, and C57BL/6) were obtained from Jackson Laboratories (Bar Harbor, Maine), and outbred ICR mice were obtained from Harlan Industries (Indianapolis, Ind.). All mice were housed in the Ohio State University vivarium in accordance with an approved animal care and use protocol and in compliance with National Institutes of Health guidelines for the care and use of laboratory animals in research. Mice were immunized subcutaneously at the base of the tail and on the back with 100 mg of peptide emulsified in 4:1 squalene-arlacel supplemented with 0.1 mg of muramyl dipeptide (36). A subsequent booster was given at 3 weeks following the primary immunization by the same protocol. Sera were collected weekly, aliquoted, and stored frozen prior to testing by direct enzyme-linked immunosorbent assay (ELISA). New Zealand White rabbits (12 weeks of age) were obtained from Hazelton Laboratories (Kalamazoo, Mich.)

and maintained under conditions similar to those used for the mice (see above). Rabbits (equal numbers of male and female) were immunized subcutaneously at multiple sites with 500 mg of peptide emulsified in 4:1 squalene-arlacel supplemented with 0.5 mg of muramyl dipeptide. Subsequent booster immunizations were administered at 2-week intervals under conditions identical to those used for the primary immunization. Animals were bled weekly, and their sera were collected (as described above). For comparative studies, serum was obtained from rabbits hyperimmunized with SP2, SP3 (aa 176 to 189), and SP4a coupled to TT (obtained from Thomas Palker, Department of Medicine, Duke University Medical Center, Durham, N.C.) (45) and from rabbits immunized with a recombinant protein, MTA-1, containing HTLV-1 aa 162 to 209 (37) (obtained from Steven Foung, Stanford Blood Bank). Direct ELISA. For peptide and recombinant protein ELISA, U-bottom poly-

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FIG. 4. Comparative immunogenicities of SP2/3/4a and SP4a-MVF. Antibodies generated to a mixture of peptides (SP2/3/4a) conjugated to TT (hyperimmunization with a series of six inoculations [44]) (■) were compared with those in a serum sample obtained at 1 week post-secondary injection from animals immunized with the chimeric construct SP4a-MVF (h). Titers are expressed as described in the legend to Fig. 2 and were screened by direct ELISA with the plated antigens SP4a, SP4a-MVF, recombinant protein MTA-1, and SP4a-SP2-TT-MVF-template.

vinyl plates from Dynatech were coated with 100 ml of antigen at a concentration of 2 mg/ml in phosphate-buffered saline (PBS) overnight at 48C. Nonspecific sites were blocked with 1% bovine serum albumin for 1 h at room temperature. Plates were washed repetitively with PBS containing 0.05% Tween 20 and 1% horse serum (PBT). Rabbit antiserum (1/500 initial dilution) or mouse antiserum (1/100 initial dilution) was added to antigen-coated plates, further diluted (serially twofold), and incubated at room temperature for 2 h. Plates were washed with PBT and incubated at room temperature for 1 h with, respectively, goat anti-rabbit immunoglobulin G or goat anti-mouse immunoglobulin G conjugated to horseradish peroxidase diluted 1/500 in PBT. Plates were washed repeatedly in PBT and then repeatedly washed with cold tap water. Bound antibody was detected with 50 ml of 0.15% H2O2 in 24 mM citric acid–5 mM sodium phosphate buffer, with 0.5 mg of 2,29-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) per ml. The color reaction was allowed to proceed for 10 min in the dark before being stopped with 25 ml of 1% sodium dodecyl sulfate solution. The A410s of plates were read with a Dynatech MR700 ELISA reader. Results are recorded as the means from duplicate wells minus background absorbance. To test for mouse and rabbit antibodies against complete disrupted HTLV-1, a commercially available ELISA for human antibodies (Cambridge Biotech Corp., Worcester, Mass.) was modified. Serial twofold dilutions of sera were evaluated to determine titers. HTLV-1 (Hut-102, B2)-coated microtiter plates and ELISA reagents were used according to the manufacturer’s instructions with the exception of a substitution of anti-human antibody conjugates with either alkaline phosphatase-conjugated anti-mouse or anti-rabbit whole immunoglobulin G conjugates (Sigma Chemical Co., St. Louis, Mo.). Competitive ELISA. U-bottom polyvinyl plates were coated and blocked for nonspecific binding as in the direct ELISA protocol. Primary antibody was added to plates as 50 ml of a constant antiserum dilution along with 50 ml of an inhibitor, using concentrations of each ranging from 0 to 20 mM. Inhibitor and antiserum were allowed to incubate at room temperature for 2 h, and then the secondary antibody-peroxidase conjugate was added. Readings and results were obtained and processed in same manner as direct ELISA protocol. RIPA. The radioimmunoprecipitation assay (RIPA) was performed as previously described (17). Briefly, HTLV-1-infected MT-2 cells were metabolically labeled (200 mCi) with [35S]cysteine and [35S]methionine (Translabel; ICN Biomedical Inc., Irvine, Calif.). Labeled cells were washed and disrupted in RIPA lysing buffer, and lysates were prepared by centrifugation. Labeled lysates were incubated with 20 ml of test serum for 16 h at 48C. Immune complexes were precipitated with protein A–Sepharose CL-4B (Sigma), and bound immune complexes were washed with RIPA lysing buffer and then eluted by boiling. Samples were electrophoretically analyzed in sodium dodecyl sulfate–10% polyacrylamide gels and visualized by autoradiography. SIA. A human osteosarcoma cell-based assay was used to test the ability of serum samples to prevent HTLV-1-mediated syncytium formation (40). Briefly, human osteosarcoma cells maintained in complete Dulbecco’s minimal essential medium with 10% fetal bovine serum were trypsinized and resuspended at 2.5 3 105 cells per ml. Aliquots (45 ml) of human osteosarcoma cells were placed in each well of a 96-well, flat-bottom microtiter plate and allowed to adhere for 2 h at 378C in a 7% CO2 incubator prior to the addition of appropriate dilutions (10 ml) of heat-inactivated test serum in duplicate wells. Plates were then incubated for an additional 30 min prior to the addition of HTLV-1-positive Hut102 cells (2.5 3 104 cells per well) to induce syncytium formation. Following an additional 18-h incubation period, plates were washed gently with PBS, fixed with

cold methanol, and stained with Giemsa stain prior to air drying. The number of syncytia was recorded for each of four fields observed with a 103 objective of an inverted microscope. The total number of syncytia from the four fields was added to calculate a syncytium inhibition assay (SIA) titer: the reciprocal dilution which inhibits syncytium formation .80% compared with that in the negative control (medium alone). Other controls included prebleed serum samples and normal mouse or rabbit serum. In vitro stimulation of lymph node cells with antigen. To measure antigeninduced proliferation, periaortic and inguinal lymph nodes were collected from immunized mice and prepared as single-cell suspensions for in vitro stimulation as described elsewhere (14). Lymph nodes were collected 10 days following the second immunization with MVF-peptide constructs (see above). Cell suspensions were washed twice with Hanks’ balanced salt solution and adjusted to a concentration of 4 3 106 cells per ml in complete RPMI 1640 supplemented with 2 mM L-glutamine, 5 3 1025 M 2-mercaptoethanol, and 10% fetal bovine serum. One hundred microliters of the cell suspension and 100 ml of peptides (6.25, 12.5, and 25 mg/ml) in medium were added to 96-well plates. Peptides tested included SP2, SP4a, SP4a-MVF, MVF-SP2, MVF, and TT3 (TT peptide as a nonspecific control, aa 580 to 599, configured as described in reference 24). All test samples were assayed in triplicate. Cells were incubated with peptides for 4 days at 378C in a 7% CO2 incubator. All wells were pulsed with 1.0 mCi of [3H]thymidine (NEN Research Products) and incubated for the last 18 h of the assay. Cells were harvested onto glass fiber filter strips (Cambridge Technology, Inc., Watertown, Mass.), dried, and counted by b-emission counting. Results expressed as counts per minute of [3H]thymidine incorporation were used to calculate stimulation indices (mean counts per minute of cells in presence of medium containing peptide/mean counts per minute of cells in medium alone).

RESULTS Epitope design, synthesis, and characterization. Two regions of HTLV-1 env gp46, one encoding a neutralizing B-cell epitope (SP2, aa 86 to 107) and the other encoding an overlapping B-cell and cytotoxic T-cell epitope (SP4a, aa 190 to 209), were selected for peptide synthesis. The choice of these epitopes was governed by the fact that both humoral and cellular immune responses to SP2 and SP4a have been extensively characterized by others (21, 44, 45). Although antibodies raised to these epitopes conjugated to carrier proteins have been shown to block HTLV-1-mediated cell fusion, they failed to elicit a protective immune response. Several reasons may account for these results: (i) poor presentation of the epitopes when coupled to the carrier protein results in low-affinity interaction and (ii) boundaries and structure of the epitopes are not mimicked accurately. Thus, to further enhance the immunogenicity, antigenicity, and immunoprotective capacity of SP2 and SP4a, we redesigned the linear sequences as chimeric constructs incorporating a promiscuous T-cell epitope from

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FIG. 5. (a) Immunogenicity of MVF-SP2 in various inbred strains of mice. The figure shows the serum samples reacted against the immunogen MVF-SP2, SP2, and MVF-turn (left to right in each group of columns) 2 weeks post-secondary injection. Individual titers for each strain (number of responders versus number immunized) and pooled serum specimen titers were determined. Titers are expressed as described in legend to Fig. 2. (b) Immunogenicity of SP4a-MVF in various strains of mice. The figure shows the serum samples of mice immunized with SP4a-MVF 2 weeks post-secondary injection. Individual titers for each strain, pooled serum specimen titers, and ELISA antigens are as described for panel a. Columns (left to right in each group): SP4a-MVF, SP4a, MTA-1, and MVF-turn.

measles virus (MVF) (Fig. 1a) and as template constructs incorporating multiple epitopes (Fig. 1b). The chimeric constructs were designed with MVF (measles virus aa 288 to 302) at the N terminus (SP4a-MVF) or at the C terminus (MVFSP2). The HTLV-1 epitopes SP2 and SP4a were synthesized (Fmoc–t-butyl strategy) colinearly with MVF via a 4-residue linker sequence (Leu-Ser-Pro-Gly) essentially as previously described (25). All constructs were HPLC purified and gave correct amino acid and mass spectral analyses.

The template peptides were designed with two promiscuous T-cell epitopes, MVF and a TT motif (aa 580 to 599) to maximize coverage of different MHC alleles. In one template construct (SP4a-SP2-TT-MVF-template) both regions of HTLV-1 were incorporated to maximize the benefits of multiple immunodominant epitopes. The other template construct was synthesized with only the neutralizing epitope SP2 (SP2TT-MVF-template) on the b-sheet template to examine MHC coverage as compared with that of the chimeric construct. The

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FIG. 6. Comparative immunogenicities of recombinant MTA-1 and HTLV(SP4a, SP2)-TT-MVF-X (template). Rabbit antisera raised to both the recombinant HTLV-1 protein MTA-1 and the template construct were reacted against MTA-1, SP4a-MVF, SP4a, HTLV(SP4a, SP2)-TT-MVF-X (template), and SP2 (left to right in each group of columns; SP2 appears only in the right panel) by direct ELISA. Titers are expressed as described in the legend to Fig. 2.

template peptides were synthesized by a combination of butyloxycarboxy-benzyl, Fmoc–t-butyl, and Fmoc-benzyl strategies. Additionally, to allow selective growth of individual epitopes from the S side chains of lysine, each N-terminal Leu was temporarily protected with the 3-nitro-2-pyridylsulfenyl protecting group. The folding properties of these peptides were evaluated by circular dichroism spectroscopy. The SP2 sequence is predicted (44) to encode a turn-loop motif, whereas SP4a is predicted to be a-helical (12). Both the linear sequences SP2 and SP4a as well as the chimeric constructs MVF-SP2 and SP4aMVF showed little (5.4 to 7.4%) a-helix content. On addition of trifluoroethanol (50%), only the chimeric constructs showed significant a-helicity (;24%). Thus, only the SP4a epitope when assembled into the chimeric construct displayed the predicted a-helical secondary structure, whereas MVF-SP2 displayed a relative high a-helical content contrary to its predicted secondary structure. SP4a (aa 190 to 209) as a peptide immunogen is MHC restricted. In order to establish whether the SP4a epitope was MHC restricted, three inbred strains of mice, C3H/HeJ (H-2k), C57BL/6 (H-2b), and BALB/c (H-2d), were immunized (five per group) with 100 mg of peptide emulsified in adjuvant. Three weeks later, the mice were boosted in a similar fashion. Serum specimens were screened from individual animals from samples taken 1 week after the second immunization (1 week post-secondary immunization) to obtain the number of re-

sponders, and pooled sera were examined to obtain titers in each mouse strain (Fig. 2). Serum specimens were tested for reactivity against the immunogen SP4a, chimeric construct SP4a-MVF, and the recombinant protein MTA-1 (aa 162 to 209). Our data indicated that two mice strains (C3H/HeJ and C57BL/6) produced antibodies specific for SP4a, but BALB/c mice failed to respond to the peptide. High titers (12,800) were elicited when the relatively large chimeric peptide SP4a-MVF and recombinant MTA-1 protein were used to coat ELISA plates, as compared with those obtained when the relatively small SP4a epitope was used as the coating antigen. This result is not entirely unexpected, as it is well-known that some small peptides can denature during the immobilization procedure (27). These data also show that all (five of five) mice in the two responder strains exhibited reactivity. We conclude that SP4a sequence contains an overlapping T-helper epitope which is under MHC restriction. HTLV-1 peptides in constructs with promiscuous T-cell peptides are highly immunogenic and bypass MHC restriction. We tested the ability of a promiscuous T-cell epitope (MVF) to (i) enhance the immunogenicity of the HTLV-1 epitopes (SP4a and SP2) and (ii) demonstrate whether immune responses by chimeric constructs can be broadened in nonresponder strains of inbred mice. The chimeric constructs MVF-SP2 and SP4aMVF were used to immunize outbred New Zealand White rabbits (n 5 2) and three inbred strains of mice (five per strain) of divergent haplotypes (C3H/HeJ [H-2k], C57BL/6 [H-2b], and

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FIG. 7. Immunogenicity of HTLV(SP4a, SP2)-TT-MVF-X (template) in three inbred mouse strains and outbred mice. Mice were immunized as described in Materials and Methods. Serum specimens 2 weeks post-secondary injection were reacted against the various constructs, HTLV(SP4a, SP2)-TT-MVF-X (template), SP4a, SP2, and MTA-1 (left to right in each group of columns). Individual titers were determined for each mice strain (number responding versus number immunized) and pooled sera.

BALB/c [H-2d]). The chimeric construct SP4a-MVF was highly immunogenic in rabbits, with titers of 5,000 to 16,000 within 1 week post-secondary immunization (Fig. 3). Sera obtained from rabbits hyperimmunized with SP2, SP3 (aa 176 to 189), and SP4a coupled to TT (44) show titers comparable to those from rabbits hyperimmunized with SP4a-MVF (Fig. 4). Thus, two immunizations of MVF promiscuous T-cell epitope was equal or superior at enhancing the immunogenicity of SP4a epitope compared to the peptide coupled to TT, which required multiple immunizations (four to six) to obtain high titers. Although the SP2-MVF construct was poorly immunogenic in rabbits, our studies with mice (Fig. 5a) show that all individual mice in three strains responded with medium titers (2,500). High titers against the immunogen were obtained in C57BL/6 mice. On the other hand, the SP4a-MVF construct was highly immunogenic in all three strains of mice, showing consistently high titers (12,800) against both the immunogen and the recombinant protein (Fig. 5b). These studies provide additional evidence that the MVF promiscuous T-cell epitope was able to provide the necessary help to SP4a in BALB/c mice, thus overcoming the genetic restriction associated with SP4a. In addition, the mean titer to SP4a was greater in both C3H/HeJ and C57BL/6 strains when SP4a was coupled to MVF. Multivalent b-template constructs containing HTLV-1 peptides elicit responses predominantly to SP4a. In order to develop a universal HTLV-1 vaccine able to elicit optimal B-cell, helper T-cell, and cytotoxic responses, we constructed a multivalent immunogen incorporating two promiscuous T-cell epitopes (MVF and TT) with both SP2 and SP4a on a b-sheet template (SP4a-SP2-TT-MVF-template) or with SP2 epitope alone on the same b-sheet template (SP2-TT-MVF-template). Outbred rabbits (five or six per group) and the three inbred strains of mice (five per group), as well as outbred mice, were immunized with these constructs. Our results in rabbits (1 week post-secondary immunization) indicated that both template constructs were highly immuno-

genic (titers versus immunogen, .30,000). For comparison, we tested rabbit antisera (1 week post-secondary immunization) raised against the recombinant protein MTA-1 and the SP4aSP2-TT-MVF-template construct (Fig. 6). High-titer antibodies were elicited by both constructs. Among rabbits immunized with SP4a-SP2-TT-MVF-template, four of six had responses against the recombinant protein MTA-1. Our studies indicated that the template constructs were highly immunogenic in all strains of mice (Fig. 7). Titers against the immunogen and the recombinant protein MTA-1 were consistently high (12,800) in all three mouse strains and in outbred mice. The exception was in the C57BL/6 strain, in which titers against the recombinant protein were relatively low (,2,000). These same serum samples failed to recognize the corresponding vector protein (SJ26) lacking the SP4a sequence (data not shown). HTLV-1 peptide constructs elicit antibodies which recognize native HTLV-1 gp46. Competitive ELISAs were performed to further test the ability of antibodies generated against the SP4a motif to recognize the recombinant protein MTA-1 (includes the amino acid sequence of SP4a). Antisera from rabbits receiving two immunizations of SP4a-MVF were compared with antisera from rabbits hyperimmunized with SP2, SP3 (aa 176 to 189), and SP4a coupled to TT (44). By using MTA-1-coated ELISA plates, each antiserum was effectively competed against by preincubation with the soluble MTA-1 protein, indicating that each antiserum specimen recognized this form of the protein (data not shown). These data indicate that after only two immunizations rabbits immunized with SP4a-MVF produced antibodies that recognized a native form of the motif and had titers similar to those in rabbits hyperimmunized against SP2, SP3, and SP4a. Preincubation with the SP4a peptide was effective at competition only against antiserum generated to SP4a-MVF and did not reduce the titer of antiserum generated against SP2, SP3 (aa 176 to 189), and SP4a coupled to TT. These results are not unexpected because of the greater number of motifs used to generate the hyperimmunized anti-

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TABLE 1. Summary of rabbit serum reactivity against HTLV-1 in ELISA, SIA, and RIPA Immunogen

TT-MVF-template SP4a-SP2-TT-MVF-template SP2-TT-MVF-template None

No. of No. Optical density Syncytium rabbits/ positive inhibitionb 6 SDa group by RIPA

5 6 6 5

0.2996 6 0.08 0.8192 6 0.56c 0.3765 6 0.14 0.2858 6 0.10

2 1 2 2

0 4 0 0

a Rabbit serum specimens (1:20 dilution) were tested 1 week post-secondary immunization in a disrupted whole-virus ELISA (see Materials and Methods). b See Materials and Methods. The titer is defined as 80% inhibition of HTLV1-mediated syncytium formation: 2, titers of 1:10 or less in all rabbits; 1, titers of 1:20 in five of six rabbits. c P , 0.05 by analysis of variance with Student-Newman-Keuls multiple comparison post-ELISA; other results were not significant.

serum (three peptides versus one for SP4a-MVF). Similarly, by using SP4a-MVF-coated ELISA plates, soluble MTA-1 protein effectively competed against SP4a-MVF antiserum and prevented binding to the peptide-coated plate (data not shown). To further test the ability of template constructs to elicit antibody responses against native forms of HTLV-1, rabbits inoculated with template constructs were tested by ELISA against disrupted whole virus. Rabbits inoculated with the b-template constructs containing only the SP2 peptide failed to recognize the whole virus, while the mean titer of antiserum from rabbits (n 5 6) inoculated with b-templates containing both SP2 and SP4a had significantly elevated titers to whole virus (Table 1). This more limited response to whole virus in the ELISA was likely due to the limited concentration of SP2 and SP4a epitopes in the whole-virus preparation. Selected serum samples were tested by RIPA to confirm that antibody responses were directed to native HTLV-1 gp46. Pooled sera from each inbred mouse strain inoculated with either chimeric or b-template constructs, as well as individual serum specimens from four of six rabbits inoculated with b-templates containing each peptide, react positively against the HTLV-1 envelope gp68 in RIPA (Fig. 8). These data together with the results from competitive and direct ELISA indicate that our peptide constructs elicited antibodies capable of recognizing native HTLV-1 envelope proteins. Antibodies produced in response to HTLV-1 peptide constructs inhibited virus-mediated cell fusion. The SIA was utilized to test serum from both the inbred mouse strains and outbred rabbits. This assay is an indirect measure of the ability of antibodies to neutralize HTLV-1-mediated syncytium formation and cell-to-cell transmission of the virus. Pooled serum samples from each of the three inbred mouse strains inoculated with chimeric or template constructs containing either SP4a or SP2 contained syncytium inhibition activity (1:20 to 1:40) (Fig. 9). Lower titers (1:10 to 1:20) were detected in serum specimens from five of six rabbits inoculated with b-templates with both SP4a and SP2. As previously reported (44), our peptide constructs containing SP2 only (without SP4a) did not elicit detectable syncytium-inhibitory antibodies in rabbits. Prebleed serum samples and sera from control animals inoculated with our b-template only or b-template with MVF and TT did not have detectable syncytium-inhibitory antibodies. Lymph node cells from immunized mice proliferate in response to HTLV-1 construct immunization. HTLV-1 peptide SP4a has been previously shown to contain a T-cell epitope that elicited helper T-cell responses in inoculated animals (30).

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To test whether our constructs containing promiscuous T-cell peptides would stimulate the proliferation of regional lymph node cells, we tested periaortic and inguinal lymph nodes from inbred mice inoculated with the chimeric construct MVF-SP2 or SP4a-MVF. Inbred mice of each MHC haplotype immunized with the colinear construct SP4a-MVF exhibited lymph node cell proliferation responses to the chimeric inoculum and the SP4a peptide alone (Table 2). In contrast, significant proliferation was detected only in wells pulsed with the inoculum MVF-SP2, and responses to SP2 antigen alone failed to elicit lymphocytes. These results are consistent with previous reports that have suggested that the SP2 epitope is a B-cell determinant (44). Further, these results suggest that SP4a-MVF constructs resulted in demonstrable T-cell stimulation in vivo. DISCUSSION Peptide-based vaccines have significant theoretical advantages in terms of safety and efficacy over conventional vaccines that utilize attenuated or killed strains of the pathogenic organism and recombinant protein products. However, the presentation of synthetic B- and T-cell epitopes to specific immune cells is critical to the progression of an effective immune response (6, 7, 9). Thus, approaches to the development of peptide vaccines require the rational manipulation of the in vivo immune response so that optimal B-cell and T-cell specificities are obtained (24). The design of peptide vaccines with improved binding affinities, titers, and immunity must rely on the engineering of structured peptides that mimic antibody recognition sites (22, 26, 27). Also, an ideal peptide vaccine would be universally immunogenic in a genetically diverse out-

1

2

3

4

5

68 kD

FIG. 8. RIPA of rabbit serum specimens. Lane 1, serum specimen from HTLV-1-infected rabbit; lanes 2, 3, and 4, serum specimens from rabbits inoculated with peptide constructs (MVF, TT, or template only, respectively); lane 5, rabbit 110 inoculated with HTLV(SP4a, SP2)-TT-MVF-X (template). The molecular size of the HTLV-1 envelope precursor gp68 from MT-2 cells persistently infected with HTLV-1 is indicated on the left.

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J. VIROL.

FIG. 9. SIA of pooled serum specimens from mice immunized with chimeric or template constructs with HTLV-1 peptides. The horizontial axis indicates the dilution of serum specimen. } and h, C3H/HeJ mice immunized with SP4a-MVF (chimeric construct) and HTLV(SP4a, SP2)-TT-MVF-X (template construct), respectively; E and ■, C57BL/6 mice immunized with SP4a-MVF and HTLV(SP4a, SP2)-TT-MVF-X, respectively; 3 and å, BALB/c mice immunized with SP4a-MVF and HTLV(SP4a, SP2)-TT-MVF-X, respectively. Each datum point represents data for pooled serum specimens (n 5 5 for each mouse strain). The horizontal line with the arrow indicates mean number of syncytia when human osteosarcoma indicator cells are incubated with prebleed sera. Note: chimeric constructs containing MVF-SP2 elicited SIA titers similar to those elicited by SP4a-MVF constructs (data not shown).

bred human population and overcome MHC restriction of the T-cell response (23, 24, 50). In this study, we are the first to report the use of promiscuous peptide constructs containing retroviral peptides. These constructs retained secondary structural characteristics of native proteins and were designed to overcome genetically restricted immune responses to peptide immunogens. Two immunodominant HTLV-1 peptides, SP2 (aa 86 to 107, a neutralizing epitope) and SP4a (aa 190 to 209, containing overlapping B-cell and T-cell epitopes), were designed as chimeric and b-template peptides constructs. These engineered peptides were synthesized as previously described (24, 28, 29), and their structures were biochemically characterized by using analytical HPLC, mass spectrometry, and circular dichroism. Data for the SP4a epitope were consistent with the predicted a-helical conformation of the native protein, whereas the SP2 epitope displayed a conformation opposite to the predicted turn motif. SP4a was highly immunogenic as the chimeric construct after only two vaccinations in both mice and rabbits. Importantly, we have demonstrated that the response to our peptide constructs resulted in the production of antibodies that recognize native forms of the HTLV-1 surface envelope protein in direct and competitive ELISA, as well as in RIPAs. These same antibodies functionally blocked HTLV-1-mediated syncytium induction, as demonstrated by the SIA. In the absence of crystal structure data for HTLV-1, both the circular dichroism spectra and the cross-reactivity observed suggest that the helical propensity of this peptide is the conformation adopted in context of the whole viral protein. On the other hand, structural data for the SP2 epitope are indicative that the conformation has not been adequately mimicked and correlates with the relatively low cross-reactivity observed in both mice and rabbits. For this epitope to display high immunogenicity, further engineering of this construct into a turn-loop mimic will be required. The SP4a peptide constructs elicited antibody responses in three strains of mice of divergent MHC haplotypes (H-2k, H-2d, and H-2b). Our results indicate that the response among responder mice strains was enhanced and that the colinear

presentation of SP4a overcame the genetically restricted response in BALB/c mice. In this strain, no response to the peptide alone was observed, whereas SP4a presented as a colinear peptide with MVF resulted in a high-titer response to the immunization. A small number of T-cell promiscuous peptides have been recently described (18, 46, 47) to be universally immunogenic. These epitopes, which bind several human MHC molecules in a permissive way, are able to be recognized by a single T-cell clone, implying that some peptide sequences are not restricted. T-cell epitopes capable of binding to the majority of immune response gene product motifs are preferable in strategies to overcome genetic restriction in the context of vaccine design. Most helper T-cell epitopes defined thus far have limited activity across divergent MHC class II haplotypes; that is, different mouse strains appear to preserve and present different regions of proteins for stimulation of T cells, and no single site has been stimulatory for all MHC types (51, 52). This genetic restriction results in a T-cell epitope that binds an MHC molecule of only one individual but not the MHC molecule of another individual of the same species and elicits immune responses that are in most cases restricted to only one or a few alleles of the MHC with limited activity across divergent class II haplotypes. We have previously used the MVF and other promiscuous T-cell epitopes to overcome the genetic restriction of immune response to a lactate dehydrogenase C4 determinant, thus ensuring immunogenicity of the chimeras in genetically diverse populations (23, 24, 50). Thus, we have been able to bypass certain haplotype-restricted immune responses and provide broad immunogenicity in a greater number of individuals typical for an outbred population. The data from our present study regarding the HTLV-1 SP4a motif confirm and extend our previous work on the immunogenicity of chimeric and template constructs incorporating promiscuous T-cell epitopes. Our chimeric MVF constructs elicited significant lymphocyte proliferative responses against either immunogen (SP4a-MVF and MVF-SP2), as well as SP4a peptide alone. These results further support our tenet that our constructs elicited T-helper lymphocyte responses and confirm the SP4a immunodominant T-cell motif (21, 44). This motif

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6087

TABLE 2. Lymph node cell proliferative response to HTLV-1 envelope peptidesa Resultd in: Immunogenb

MVF-SP2

MVF-SP4a

Concn (mg/ml) and antigenc

25 None MVF MVF-SP2 SP2 TT3 12.5 None MVF MVF-SP2 SP2 TT3 6.25 None MVF MVF-SP2 SP2 TT3 25 None MVF MVF-SP4a SP4a TT3 12.5 None MVF MVF-SP4a SP4a TT3 6.25 None MVF MVF-SP4a SP4a TT3

C3H/HeJ mice

C57BL/6 mice

BALB/c mice

cpm (SD)

SI

cpm (SD)

SI

cpm (SD)

SI

32,117 (4,236) 13,938 (6,897) 142,278 (6,777) 46,644 (6,511) 40,934 (8,501)

1.0 0.4 4.4* 1.5 1.3

15,134 (3,744) 20,176 (1,357) 78,921 (23,046) 23,960 (5,967) 15,310 (2,863)

1.0 1.3 5.2* 1.6 1.0

12,196 (3,092) 3,112 (1,893) 48,474 (15,005) 8,860 (506) 10,413 (1,087)

1.0 0.3 4.0* 0.7 0.8

40,285 (10,998) 13,070 (3,995) 134,488 (9,104) 43,188 (6,595) 33,942 (2,555)

1.0 0.3 3.3* 1.1 0.8

18,210 (1,631) 20,472 (6,123) 83,181 (6,885) 25,616 (11,724) 16,146 (597)

1.0 1.1 4.6* 1.4 0.9

10,177 (3,560) 3,041 (804) 48,000 (9,506) 9,101 (579) 8,668 (3,711)

1.0 0.3 4.7* 0.9 0.9

49,281 (3,515) 20,923 (1,694) 141,506 (13,362) 50,777 (8,603) 42,352 (10,614)

1.0 0.4 2.9* 1.0 0.9

16,840 (5,800) 27,284 (3,432) 73,454 (13,995) 21,620 (2,836) 22,974 (5,917)

1.0 1.6 4.4* 1.3 1.4

14,705 (5,905) 28,904 (16,037) 36,050 (5,168) 9,877 (693) 24,614 (22,489)

1.0 2.0 2.5 0.7 1.7

29,750 (6,045) 30,925 (3,957) 128,679 (39,043) 133,513 (38,549) 41,261 (11,426)

1.0 1.0 4.3* 4.4* 1.3

24,441 (7,358) 4,858 (2,172) 112,185 (13,264) 110,036 (12,060) 20,701 (859)

1.0 0.2 4.6* 4.5* 0.8

20,037 (4,920) 4,376 (998) 100,490 (4,800) 46,186 (6,196) 43,275 (13,148)

1.0 0.2 5.0* 2.3 2.1

36,679 (5,344) 18,640 (2,217) 114,050 (9,119) 100,533 (33,088) 34,692 (2,551)

1.0 0.5 3.1* 2.7* 0.9

42,988 (30,394) 7,290 (2,148) 100,983 (15,987) 110,872 (13,018) 15,662 (8,131)

1.0 0.2 2.3* 2.6* 0.4

18,826 (4,721) 14,667 (12,608) 105,300 (33,642) 32,561 (5,208) 13,590 (4,069)

1.0 0.8 5.6* 1.7 0.7

39,447 (9,815) 37,565 (10,186) 131,667 (6,665) 100,155 (4,620) 40,787 (8,124)

1.0 0.9 3.3* 2.5* 1.0

28,423 (6,299) 12,497 (4,757) 84,603 (2,218) 77,530 (6,744) 35,112 (12,602)

1.0 0.4 3.0* 2.7* 1.2

30,309 (9,022) 23,619 (11,636) 57,055 (19,700) 34,835 (10,489) 24,124 (3,355)

1.0 0.8 1.9** 1.1 0.8

a Lymph node cells harvested from periaortic and inguinal lymph nodes were prepared as single-cell suspensions for in vitro stimulation as described in Materials and Methods. b Immunogen was used for inoculation of mouse strains as described in Materials and Methods. c Antigen (peptide) was used to stimulate lymph node cells in microtiter assays as described in Materials and Methods. d SI, stimulation index; *, P , 0.001; **, P , 0.05 (compared by analysis of variance and Student-Neuman-Keuls posttest).

overlaps with recently identified neutralizing B-cell epitopes within HTLV-1 gp46 that have been demonstrated to prevent HTLV-1 infection in animal models of the viral infection (3, 55). Animal studies are in progress to determine the immunoprotective capacity of our peptide constructs. For an effective peptide vaccine, rationally designed constructs must incorporate enough antigenic determinants to elicit the appropriate B-cell, T-helper, and cytotoxic T-cell responses. Thus, minimally, three to four distinct peptide moieties need to be incorporated into a construct. For example, in the case of viruses, a strategy that allows incorporation of several variants of important sequences to cover a critical number of pathogenic strains would be highly desirable (8, 20). With these facts in mind, we have recently described a singlematrix, multicomponent combination strategy that will allow the construction of fundamentally different epitopes onto a single template (28). The core b-sheet template consists of two strands of alternating Leu-Lys. The ε-side chains of four Lysyl residues allow the growth of individual or multiple epitopes in various permutations. Our unique synthesis strategy incorpo-

rated four different epitopes using combinational Fmoc–t-butyl, Fmoc-benzyl, and butyloxycarboxy-benzyl methods, as well as a differential protection scheme employing 3-nitro-2-pyridylsulfenyl groups. We have previously used this strategy to produce model peptides incorporating various combinations of B- and T-cell epitopes and shown these to be highly immunogenic in several inbred strains of mice, outbred mice, and rabbits (25). In contrast to other approaches to presentation of peptide immunogens, our template method allows peptide epitopes to be constructed individually and therefore allows selectivity of incorporating fundamentally different epitopes in defined combinations with promiscuous T-cell epitopes. This strategy also allows incorporation of conformationally defined epitopes, an important criterion often overlooked in peptidebased vaccine design. In summary, our data indicate that promiscuous T-cell epitopes, which bind to several forms of human MHC class II molecules, can be used with immunodominant peptides derived from retroviruses to produce highly immunogenic preparations capable of overcoming haplotype-restricted immune

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responses. Further topographic manipulation of these constructs will provide important information for the development of synthetic peptide vaccines against retroviral infections. ACKNOWLEDGMENTS This work was supported, in part, by National Institutes of Health National Cancer Institute grants P30 CA16058-18 and CA55185. We thank Steven Foung, Stanford University, for the use of the recombinant protein MTA-1; Thomas Palker, Duke University, for peptide antiserum; and Tim Vogt, The Ohio State University, for his assistance in preparation of the manuscript. REFERENCES 1. Allen, P. M. 1987. Antigen processing at the molecular level. Immunol. Today 8:270–273. 2. Amit, A., R. Mariuzza, S. Phillips, and R. Poljak. 1986. Three-dimensional structure of an antigen-antibody complex at 2.8 Å resolution. Science 233: 747–753. 3. Baba, E., M. Nakamura, K. Ohkuma, J. Kira, Y. Tanaka, S. Nakano, and Y. Niho. 1995. A peptide-based human T cell leukemia virus type I vaccine containing T and B cell epitopes that induces high titers of neutralizing antibodies. J. Immunol. 154:399–412. 4. Baba, E., M. Nakamura, Y. Tanaka, M. Kuroki, Y. Itoyama, S. Nakano, and Y. Niho. 1993. Multiple neutralizing B-cell epitopes of human T-cell leukemia virus type 1 (HTLV-1) identified by human monoclonal antibodies: a basis for the design of an HTLV-1 peptide vaccine. J. Immunol. 151:1013– 1024. 5. Babbit, B., P. Allen, G. Matsueda, E. Haber, and E. Unanue. 1985. Binding of immunogenic peptides to Ia histocompatibility molecules. Nature (London) 317:359. 6. Berzofsky, J. 1991. Mechanisms of T cell recognition with application to vaccine design. Mol. Immunol. 28:217–223. 7. Berzofsky, J. 1991. Antigenic peptide interaction with MHC molecules: implication for the design of artificial vaccines. Semin. Immunol. 3:203–216. 8. Berzofsky, J., C. Pendelton, M. Clerici, J. Ahlers, D. Lucey, S. Putney, and G. Shearer. 1991. Construction of peptides encompassing multideterminant clusters of human immunodeficiency virus envelope to induce in vitro T cell responses in mice and humans of multiple MHC types. J. Clin. Invest. 88:876–882. 9. Bjorkman, P., M. Saper, B. Samraoui, W. Bennett, J. Strominger, and D. Wiley. 1987. Structure of the human class I histocompatibility antigen, HLAA2. Nature (London) 329:506–512. 10. Borras-Cuesta, F., A. Petit-Camurdan, and Y. Fedon. 1987. Engineering of immunogenic peptides by co-linear synthesis of determinants recognized by B and T cells. Eur. J. Immunol. 17:1213–1218. 11. Chen, Y., J. Yang, and K. Chan. 1974. Determination of the helix and b-form of proteins in aqueous solution by circular dichroism. Biochemistry 13:3350– 3362. 12. Chou, P., and G. Fasman. 1978. Prediction of secondary structure of proteins from amino acid sequence. Adv. Enzymol. (Biochem.) 47:45–69. 13. Clarke, B., S. Newton, A. Carroll, M. Francis, G. Appleyard, A. Syred, P. Highfield, D. Rowlands, and F. Brown. 1987. Improved immunogenicity of a peptide epitope after fusion to hepatitis B core protein. Nature (London) 330:381–384. 14. Corradin, G., H. Etlinger, and J. Chiller. 1977. Lymphocyte specificity to protein antigens: characterization of the antigen-induced in vitro T celldependent proliferative response with lymph node cells from primed mice. J. Immunol. 119:1048–1053. 15. Francis, M., G. Hastings, A. Syred, B. McGinn, F. Brown, and D. Rowlands. 1987. Non-responsiveness to a foot-and-mouth disease virus peptide overcome by addition of foreign helper T cell determinants. Nature (London) 330:168–170. 16. Gessain, A., F. Barin, J. Vernant, O. Gout, L. Maurs, A. Calender, and G. Dethe. 1985. Antibodies to human T lymphotropic virus type 1 in patients with tropical spastic paresis. Lancet ii:407–410. 17. Hartley, T. M., R. F. Khabbaz, R. O. Cannon, J. E. Kaplan, and M. D. Lairmore. 1990. Characterization of antibody reactivity to human T-cell lymphotropic virus types I and II using immunoblot and radioimmunoprecipitation assays. J. Clin. Microbiol. 28:646–650. 18. Ho, P. C., D. A. Mutch, K. D. Winkel, A. J. Saul, G. L. Jones, T. J. Doran, and C. M. Rzepczyk. 1990. Identification of two promiscuous T-cell epitopes from tetanus toxin. Eur. J. Immunol. 20:477–483. 19. Hollsberg, P., and D. Hafler. 1993. Pathogenesis of diseases induced by human lymphotropic virus type I infection. N. Engl. J. Med. 328:1173–1182. 20. Hosmalin, A., P. Nara, M. Zweig, N. Lerche, K. Cease, E. Gard, P. Markham, S. Putney, M. Daniel, R. C. Desrosiers, and J. Berzofsky. 1991. Priming with T helper cell epitope peptides enhances the antibody response to the envelope glycoprotein of HIV-1 in primates. J. Immunol. 146:1667– 1673.

J. VIROL. 21. Jacobson, S., J. Reuben, R. Streilen, and T. Palker. 1991. Induction of CD41 human T lymphotropic virus type 1 specific cytotoxic T lymphocytes from patients with HAM/TSP. J. Immunol. 146:1155–1162. 22. Kaumaya, P. T. P., K. D. Berndt, D. B. Heidorn, J. Trewhella, F. J. Kezdy, and E. Goldberg. 1990. Synthesis and biophysical characterization of engineered topographic immunogenic determinants with aa topology. Biochemistry 29:13–23. 23. Kaumaya, P. T. P., N. Feng, Y. Hoon Seo, S. F. Kobs-Conrad, A. M. VanBuskirk, and J. F. Sheridan. 1992. Immunogenicity and antigenicity of a promiscuous T-cell epitope and topographic B-cell determinant of the protein antigen LDH-C4, p. 883–885. In J. A. Smith, and J. Rivier (ed.), Peptides: chemistry and biology. ESCOM, Leiden, The Netherlands. 24. Kaumaya, P. T. P., S. Kobs-Conrad, S. DiGeorge, and V. Stevens. 1994. Denovo engineering of protein immunogenic and antigenic determinants, p. 133–164. In G. Anantharamaiah and C. Basava (ed.), Peptides: design, synthesis, and biology. Springer-Verlag, New York. 25. Kaumaya, P. T. P., Y. Seo, S. Kobs, I. Ngua, J. Sheridan, and V. Stevens. 1993. Peptide vaccines incorporating a ‘‘promiscuous’’ T cell epitope bypass certain haplotype restricted immune responses and provide broad spectrum immunogenicity. J. Mol. Recognit. 6:81–91. 26. Kaumaya, P. T. P., A. VanBuskirk, E. Goldberg, and S. Pierce. 1990. Predetermined supersecondary structural motifs in the de novo design of protein antigenic sites, p. 709–713. In J. Rivier and G. Marshall (ed.), Peptides: chemistry, structure, and biology. ESCOM Scientific Publishing, Leiden, The Netherlands. 27. Kaumaya, P. T. P., A. M. VanBuskirk, E. Goldberg, and S. K. Pierce. 1992. Design and immunological properties of topographic immunogenic determinants of a protein antigen (LDH-C4) as vaccines. J. Biol. Chem. 267:6338– 6346. 28. Kobs-Conrad, S. F., A. Gerdau, and P. T. P. Kaumaya. 1992. 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