JOURNAL OF VIROLOGY, JUlY 1991, p. 3789-3796 0022-538X/91/073789-08$02.00/0 Copyright C) 1991, American Society for Microbiology

Vol. 65, No. 7

Immunodominant T-Cell Epitope on the F Protein of Respiratory Syncytial Virus Recognized by Human Lymphocytes MELISSA E. LEVELY,1 CAROL A. BANNOW,2 CLARK W. SMITH,2

AND

JUDITH A.

NICHOLAS'*

Departments of Infectious Diseases Research1 and Biochemistry,2 Upjohn Laboratories, Kalamazoo, Michigan 49007 Received 17 December 1990/Accepted 15 April 1991

The lymphocyte proliferative responses to respiratory syncytial virus (RSV) were evaluated for 10 healthy adult donors and compared with proliferative responses to a chimeric glycoprotein (FG glycoprotein) which consists of the extracellular domains of both the F and G proteins of RSV and which is produced from a recombinant baculovirus. The lymphocytes of all 10 donors responded to RSV, and the proliferative responses to the whole virus were highly correlated with the responses to the FG glycoprotein. These data suggested that one or both of these glycoproteins of RSV were major target structures for stimulation of the human lymphocyte proliferative response among virus-specific memory T cells. The lymphocytes of four donors were evaluated further for their proliferative responses to a nested set of overlapping peptides modeled on the extraceliular and cytoplasmic domains of the F protein of RSV. Strikingly, the lymphocytes of all 4 donors responded primarily to a region defined by a single peptide spanning residues 338 to 355, and the lymphocytes of 2 donors responded to an overlapping peptide spanning residues 328 to 342 also, thus defining a region of the Fl subunit within residues 328 to 355 that may circumscribe an immunodominant site for stimulation of human T cells from a variety of individuals. This region of the F protein is highly conserved among A and B subgroup viruses. As revealed by monoclonal antibody blocking studies, the lymphocytes responding to this antigenic site had characteristics consistent with T helper cells. Similar epitope mapping studies were performed with BALB/c mice immunized with the FG protein in which a relatively hydrophobic peptide spanning residues 51 to 65 within the F2 subunit appeared to be the major T cell recognition determinant. The data are discussed with respect to an antigenic map of the F protein and the potential construction of a synthetic vaccine for RSV. Human respiratory syncytial virus (RSV) is a member of the family Paramyxoviridae and has a single-stranded RNA genome which encodes at least 10 unique viral proteins within infected cells (7, 8, 13). RSV is the leading cause of severe lower respiratory tract infection in infants, and most, if not all, children become infected with RSV during the first few years of life. Intense research efforts have recently focused on characterizing both the virus structural proteins and the host immune responses which may contribute to protection from this important human pathogen. The two major surface glycoproteins of RSV, the G protein and the F protein, function in attachment to and fusion of susceptible host cells, respectively (17, 38). Polyclonal antibodies or monoclonal antibodies (MAbs) to the G or the F protein neutralize virus infectivity in vitro (2, 25, 37, 38, 40) and provide passive protection in experimental animal models (30, 39). The important contributions of the G and F glycoproteins in providing protection against RSV infection in experimental animals have been further demonstrated by immunization with recombinant vaccinia viruses expressing the glycoproteins of RSV (9, 15, 20, 22) or by immunization with purified glycoproteins derived either from infected cells (36) or from recombinant baculovirus (6, 41). RSV infection in humans and in experimental animals induces a vigorous cell-mediated immune response. The target proteins for RSV-specific cytotoxic T lymphocytes have been identified as the F protein, the nucleocapsid (N) protein, and the 22-kDa membrane-associated protein (4, 19, 23, 26). Although T-cell proliferative responses have been extensively documented after natural RSV infection or im-

*

munization of humans (10, 16, 28, 29), the viral proteins which elicit this response have not been precisely identified. In this study, we investigated the human lymphocyte proliferative response to whole RSV antigens in comparison to the proliferative response to a chimeric protein containing the extracellular domains of both the F and G glycoproteins as a method to determine the relevance of these two major glycoproteins to this cellular immune response. We then further defined a subset of T-cell-stimulating epitopes within the F protein of RSV by using a nested set of overlapping synthetic peptides. This topological identification of antigenic sites which stimulate human T-cell-mediated immune function may be important for the construction of a safe and effective vaccine against RSV. MATERIALS AND METHODS

Virus and the FG glycoprotein. Human RSV A2 was propagated in HEp-2 cells and titrated by plaque assay under 0.5% agarose. The cDNA clones containing the complete sequences for the F and G proteins of RSV A2 were received in pBR322 from G. W. Wertz (clones F-A25 and G-16) and manipulated for expression of the chimeric FG protein from the polyhedrin promoter of baculovirus, as previously described in detail (42). The chimeric FG glycoprotein contained the signal and extracellular regions of the F glycoprotein fused to the extracellular region of the G glycoprotein and contained a termination codon just upstream of the anchor region of the G sequence. The FG glycoprotein was secreted from Sf9 cells infected with the recombinant baculovirus and purified to apparent homogeneity by a combination of cation-exchange and immunoaffinity chromatographies as described previously (6).

Corresponding author. 3789

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Peptide synthesis. A series of 52 overlapping peptides was synthesized by solid-phase methodology on an Applied Biosystems, Inc. 430A peptide synthesizer using the smallscale rapid cycle on a 0.1-mmol scale. The small-scale rapid cycle utilizes abbreviated single couple cycles with standard t-Boc chemistry. The t-Boc L-amino acids were supplied by Applied Biosystems, Inc., with standard side-chain-protecting groups. The completed peptides were removed from the supporting phenylacetamidomethyl resin concurrently with the side-chain-protecting groups by a standard hydrogen fluoride cleavage procedure using appropriate cation scavengers (10% [vol/vol] anisole, p-cresol, and p-thiocresol; 1,4-butanedithiol and anisole; or dimethyl sulfide and anisole, depending on the amino acid sequence of the peptide). The crude peptides were purified by preparative reversephase chromatography on a Phenomenex C-18 column (250 by 22.5 mm) using water acetonitrile gradients; each phase contained 0.1% trifluoroacetic acid. The purity of fractions was determined by analytical high-pressure liquid chromatography, and pure fractions were pooled, the acetonitrile was evaporated, and the aqueous solution was lyophilized. All peptides were analyzed by fast atom bombardment mass spectroscopy and gave the anticipated (M + H)+. Lymphocyte proliferation assays. Peripheral blood mononuclear cells (PBMC) were obtained from healthy adult donors (age, >18 years) by leukapheresis and purified on Ficoll-Hypaque (Pharmacia). PBMC were frozen in aliquots in dimethyl sulfoxide (10% in RPMI 1640 medium [Whittaker Bioproducts] containing 20% fetal bovine serum [GIBCO]) and held in the vapor phase of liquid N2 until use. PBMC were rapidly thawed, diluted stepwise in ice-cold MLR medium (RPMI 1640, 10% fetal bovine serum, 2 mM L-glutamine, 10 mM HEPES buffer [N-2-hydroxyethylpiperazineN'-2-ethanesulfonic acid], 50 FM 3-mercaptoethanol, 50 p.g of gentamicin sulfate per ml), washed, and cultured in microtiter wells with antigen (RSV, FG glycoprotein, or peptide) at 4 x 105 cells per well in MLR medium substituting human AB serum (Hazleton Biologics, Inc.) for fetal bovine serum. After 5 days of exposure to antigen, PBMC were pulse-labeled with [3H]thymidine (ICN Pharmaceuticals, 1 ILCi per well) for 6 h, harvested onto filter disks (Brandell cell harvester), and evaluated for incorporation of radiolabeled precursor in a Beckman LS5801 liquid scintillation spectrometer. Murine popliteal lymph node cells (PLNC) were obtained at least 2 weeks after footpad immunization of BALB/c mice with 1 to 2 ,ug of FG glycoprotein emulsified in complete Freund's adjuvant. Freshly derived

PLNC were cultured in microtiter wells in MLR medium with antigen for 3 to 5 days and then pulse-labeled, harvested, and assayed as described for human PBMC. Data for human PBMC assays were expressed as amount of [3H]thymidine incorporated (in counts per minute) or as a stimulation index (counts per minute incorporated in the presence of antigen/counts per minute incorporated in the absence of antigen); data for murine PLNC assays were expressed as net counts per minute incorporated (counts per minute incorporated in the presence of antigen - counts per minute incorporated in the absence of antigen). Epstein-Barr virus (EBV) transformation of B lymphocytes and HLA typing. PBMC from some of the donors were transformed with EBV by standard procedures (14). Briefly, PBMC were cultured in the presence of EBV contained within the supernatant fluid of the B95-8 marmoset cell line (American Type Culture Collection) in the presence of cyclosporin A (Sandoz). The outgrowing EBV-transformed B lymphocyte cell lines were subcultured twice weekly in

J. VIROL.

MLR medium and were typed for human leukocyte antigen (HLA) class II determinants by the Research Immunohematology and Serology Laboratory, Department of Medicine, Michigan State University, Lansing, Mich., courtesy of Robert W. Bull. MAb blocking studies. Purified MAbs to monomorphic determinants of HLA-DR (L243), to CD4 (Leu3a), or to CD1 thymocyte antigen (Leu6; negative control) were from Becton Dickinson and were dialyzed against 1,000 volumes of phosphate-buffered saline to remove NaN3 before use. Purified MAbs (0.5 p.g/ml) were added to proliferation assays at the time of initiation of culture. Hybridoma cell culture supernatant fluid containing MAbs to murine L3T4 (CD4, GK1.5), to IAd (MKD6), to IE determinants (14.4.4S), or to determinants common to IAdIEd (M5/114) were added at 10% (vol/vol) at the time of initiation of culture. All hybridoma cell lines are available from the American Type Culture Collection. Results are expressed as the percent inhibition of proliferation calculated as follows: % inhibition = {1 - [(cpm with peptide plus MAb) - (cpm with MAb)]/[(cpm with peptide) - (cpm with medium)]} x 100. RESULTS Proliferative responses to RSV or to FG glycoprotein among healthy adult donors. PBMC were isolated from 10 healthy adult donors over the course of approximately 24 months. All donors were presumed to have been naturally infected with or exposed to RSV on multiple occasions during their lifetimes. One adult donor (donor 1 in our panel) had been hospitalized with confirmed RSV infection a few months prior to donation of PBMC; two donors (donors 10 and 6) provided a second leukapheresis donation (lOb and 6b) several months after the first donation, bringing the total sample size to 12. The lymphoproliferative responses of human PBMC upon in vitro restimulation with RSV or with decreasing doses of the FG glycoprotein are compared in Fig. 1 for the panel of 10 separate donors and 2 repeat donors. One donor (donor 2) had a relatively low lymphoproliferative response to RSV; all of the remaining donors showed moderate to vigorous lymphoproliferative responses to RSV. Increasing the RSV antigen load by 10-fold did not appreciably change the observed lymphoproliferative response of any of these donors, suggesting that these studies were performed under conditions of optimal RSV antigen concentration (data not shown). The single donor with low responsiveness to RSV (donor 2) was the only donor without a detectable lymphoproliferative response to restimulation with the FG glycoprotein (Fig. 1). Of the remaining donors with moderate to vigorous lymphoproliferative responses to RSV, all responded to restimulation with the FG glycoprotein and all showed corresponding moderate to vigorous lymphoproliferative responses to the FG glycoprotein. Donor 14 showed a vigorous response to the RSV antigen and a relatively minor response to the FG glycoprotein. Thus, the lymphoproliferative response to whole RSV was highly correlated with the response to the FG glycoprotein among the entire panel of adult donors (r = 0.73 overall and r = 0.80 excluding donor 14). Human lymphoproliferative responses to overlapping peptides of the F protein of RSV. It was of interest to use a series of overlapping synthetic peptides as a method to construct an antigenic map of the lymphoproliferative epitopes of the F glycoprotein of RSV. By using the published sequence of the F protein of the A2 strain of RSV, a series of 52 peptides of (typically) 15 residues each, which overlapped by 5 residues,

VOL. 65, 1991

T-CELL EPITOPES ON THE RSV F PROTEIN

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Donor # FIG. 1. Human lymphoproliferative responses to RSV and to FG glycoprotein. PBMC from 10 healthy adult donors and 2 repeat donors (lOb and 6b) were restimulated (from left to right) with RSV at a multiplicity of infection of 0.1 (X), with FG glycoprotein at 5 (O), 1 (ED), or 0.2 (ED) pug/ml, or with medium (-) for 5 days before being pulse-labeled with [3H]thymidine. Data represent the mean of triplicate samples and reveal trends representative of several independent experiments.

were synthesized, purified, and used to restimulate lymphoproliferation in PBMC from four individual donors selected as representative of the panel described above. The PBMC from two donors with moderate responses to RSV and the FG glycoprotein (donors 5 and 10b from Fig. 1) and from two donors with vigorous responses to RSV and the FG glycoprotein (donors 12 and 1 from Fig. 1) were cultured in the presence of each individual synthetic peptide, and proliferation was assayed 5 days after restimulation. Figure 2 reveals the lymphoproliferative responses of each of these four donors to the entire panel of overlapping synthetic peptides modeled on the F protein of RSV. As expected, when the panel of the four individual donors was evaluated, several peptides were found to stimulate lymphoproliferation. However, two donors (donors 1 and 12) showed a response, albeit modest, to a peptide spanning residues 131 to 145 or its overlapping peptide spanning residues 141 to 155 (peptides 12 and 13, respectively, in Fig. 2). Remarkably, all four donors responded predominately to a single synthetic peptide spanning residues 338 to 355 (peptide 33 in Fig. 2), and two of these four donors also responded to the overlapping peptide spanning residues 328 to 342 (peptide 32 in Fig. 2). These data suggested that the region spanning residues 328 to 355 contained an immunodominant epitope for all four

of our tested donors. HLA class II antigen expression in donors responding to the immunodominant peptide epitope (residues 338 to 355) or to whole RSV. The striking result obtained with all four donors

(i.e., responding predominantly to a single peptide) prompted us to look at the HLA class II phenotypes of these individuals. Furthermore, it was of interest to compare these phenotypes with the class II phenotype of donor 14, the single donor whose lymphoproliferative response to the FG

glycoprotein did not correlate with the response to whole RSV. As shown in Table 1, none of the four donors (donor 1, 5, 10, or 12) who responded to the immunodominant F peptide (residues 338 to 355) shared a single class II major histocompatibility complex (MHC) allele, suggesting that this peptide could be presented by more than one allele. Donors 1 and 12 shared alleles DR4 and DRw53 (Table 1), and PBMC from these donors recognized the overlapping F peptide (residues 328 to 342) (Fig. 2). Furthermore, it appeared that DR1, DR3, DRw52, DQwl, and DQw2 were not important in the proper processing and presentation of the FG glycoprotein since donor 14 expressed each of these alleles (Table 1) and responded very poorly to the FG glycoprotein despite a good lymphoproliferative response to whole RSV (Fig. 1). Only alleles DRw15(2) and DQw6(wl) could be detected for donor 2 (data not shown), the single donor showing a low response to RSV and no response to the FG glycoprotein (Fig. 1). Thus, these data suggested that the recognition of the FG glycoprotein as well as the recognition of the immunodominant peptidic epitope (residues 338 to 355) was not limited by the expression of a single class II MHC allele. MAbs to CD4 and HLA-DR block proliferation to F peptide (residues 338 to 355). Since the F peptide spanning residues 338 to 355 appeared to encompass at least part of an immunodominant epitope of the F protein, it was of interest to characterize the lymphoproliferative response to this peptide further. The results of the HLA class II typing indicated that donors 10 and 12 had determinants in DQ (DQwl) and in DR (DR2) but were otherwise heterogeneous at the class II locus (Table 1). Therefore, PBMC from these two donors were individually cultured with the immunodominant F peptide (residues 338 to 355) in the presence of MAb

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TABLE 1. HLA class II antigen expressiona

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a Class II antigen expression was determined on EBV-transformed B-lymphoblastoid cells (for donors 1, 5, 10, and 12) or on PBMC (for donor 14), as described in Materials and Methods. b Donors 1, 5, 10, and 12 showed a lymphoproliferative response to the FG glycoprotein which was highly correlated with that to whole RSV and also responded to the immunodominant epitope (residues 338 to 355) of the F glycoprotein of RSV. Donor 14 was the single donor who showed a good lymphoproliferative response to RSV but who responded poorly to the FG glycoprotein. c Donors 1 and 5 expressed the DRw11(5) antigen. d Donor 10 expressed the DRw6 antigen. I Donors 1, 5, and 12 expressed the DQw7(w3) antigen.

to monomorphic determinants of HLA-DR, MAb to CD4, or MAb to CD1 (a surface determinant expressed only on thymocytes), and the percent inhibition of proliferation by MAbs was determined (Fig. 3). For donor 12, lymphoproliferation induced by either 50 or 5 ,ug of F peptide (residues 338 to 355) per ml was strongly inhibited by MAb to DR determinants or by MAb to CD4, while inhibition by the control MAb to CD1 was negligible (Fig. 3, left). For donor 10b, proliferation induced by 50 ,ug of peptide per ml was specifically inhibited by MAb to DR determinants and proliferation induced by 5 ,ug of peptide per ml was specifically inhibited by MAb to either DR or CD4 determinants (Fig. 3, right panel). These data suggested that presentation of the F peptide (residues 338 to 355) was class II MHC restricted and that the responding lymphocytes were of the CD4+, T helper cell phenotype. Lymphoproliferative responses to overlapping peptides of the F protein in mice primed to the FG glycoprotein. Since mice of the H-2d haplotype have proven to be a valuable model for the study of human RSV pathogenesis (12, 31), it was of interest to immunize BALB/c mice with the FG glycoprotein and to determine the recognition of the F protein moiety by using our panel of overlapping synthetic peptides. The results of a representative experiment are shown in Fig. 4. The lymphocytes from FG-primed BALB/c mice responded to restimulation with either RSV or FG and responded significantly to a single synthetic peptide spanning residues 51 to 65 within the F2 subunit of the F protein (peptide 4 in Fig. 4). The proliferative response to this peptide was highly reproducible among several experiments with BALB/c mice (data not shown), and this response could be abrogated by MAb to CD4 determinants (L3T4) and

significantly reduced by MAb to IE determinants but not by MAb to IA determinants (Fig. 5). DISCUSSION These studies verified a role for the major glycoproteins of RSV in stimulation of the T lymphocyte proliferative response following natural infection of the human host. The high degree of correlation between the lymphoproliferative response to whole virus and the response to the chimeric FG glycoprotein further suggested that one or both of these glycoproteins were dominant target structures for human T-cell recognition, at least within the adult population, in which repeated exposure to or infection with RSV was likely to have occurred. Although these are the first studies to investigate the RSV protein specificity of the lymphoproliferative response in humans, our study design could not distinguish the contribution of each individual glycoprotein to this response. However, studies of mice (24) have previously identified a dominant role for the F glycoprotein versus that of the G glycoprotein in stimulation of the RSV-specific murine T helper cell response following immunization with recombinant vaccinia virus vectors, thus supporting the notion that the human response we observed in our studies may have been similarly dominated by recognition of the F moiety of the FG glycoprotein. Since the lymphoproliferative response to RSV has been reported to gradually rise during the 3 years following primary infection (10), it will be important to determine whether there is a corresponding correlation between the lymphoproliferative responses to whole virus and to the FG glycoprotein following primary

FIG. 2. Human lymphoproliferative response to synthetic peptides modeled on the F protein of RSV. PBMC from four healthy adult donors selected from the group described in the legend to Fig. 1 as representing moderate or vigorous responses to RSV and to the FG glycoprotein were restimulated in vitro with individual, overlapping synthetic peptides modeled on the F protein of RSV. Peptide was added to a final concentration of 50 ,ug/ml except where shown as closed bars, which represent the response to peptide at a final concentration of 100 ,ug/ml. Data represent the mean ± standard deviation of the stimulation index, calculated as described in Materials and Methods. A mean stimulation index of 2 or greater was considered significant and is marked with *. Error bars represent mean variation among at least two separate experiments; values without error bars represent the mean of duplicate or triplicate samples from a single experiment. Peptide number corresponds to amino acid residues as follows: 1, 21 to 35; 2, 31 to 45; 3, 41 to 55; 4, 51 to 65; 5, 61 to 75; 6, 71 to 85; 7, 81 to 95; 8, 91 to 105; 9, 101 to 115; 10, 111 to 125; 11, 121 to 135; 12, 131 to 145; 13, 141 to 155; 14, 151 to 165; 15, 161 to 175; 16, 171 to 185; 17, 181 to 195; 18, 191 to 205; 19, 201 to 215; 20, 211 to 225; 21, 221 to 235; 22, 231 to 245; 23, 241 to 255; 24, 251 to 265; 25, 261 to 275; 26, 271 to 285; 27, 281 to 295; 28, 291 to 305; 29, 301 to 315; 30, 311 to 321; 31, 317 to 332; 32, 328 to 342; 33, 338 to 355; 34, 351 to 365; 35, 361 to 375; 36, 371 to 385; 37, 381 to 392; 38, 388 to 405; 39, 401 to 415; 40, 411 to 421; 41, 417 to 435; 42, 431 to 445; 43, 441 to 455; 44, 451 to 465; 45, 461 to 475; 46, 471 to 485; 47, 481 to 495; 48, 491 to 505; 49, 501 to 515; 50, 511 to 525; 51, 551 to 565; and 52, 561 to 574.

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RSV or whether the cellular recognition of the viral glycoproteins develops upon repeated exposure. We used a nested set of overlapping synthetic peptides to identify antigenic sites on the F glycoprotein capable of stimulating T cells from either humans or mice. Strikingly, our data revealed an immunodominant site for human T cells from several individuals in a region of the F glycoprotein within residues 338 to 355 of the Fl subunit. The response appeared to be MHC class II restricted and mediated by the exposure to

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VOL. 65, 1991

T-CELL EPITOPES ON THE RSV F PROTEIN

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may be important in the presentation of this peptide. We are currently working to test the hypothesis that a composite peptide spanning residues 328 to 355 or alternately truncated peptides within this region would have the potential for maximal stimulation of the human T helper cell response from several individuals within our panel. This region of the F glycoprotein (spanning residues 328 to 355) is highly conserved among several isolates representing both the A and B subgroups of RSV (27), including the Edinburgh,

Long, A2, and RSS-2 subtype A strains and the 18537 subtype B strain, thereby increasing the potential utility for incorporation of this peptidic region into a vaccine for RSV. By using similar criteria, the murine lymphoproliferative response following immunization with the FG glycoprotein mapped to a conserved region spanning residues 51 to 65 within the F2 subunit, and the response appeared to be MHC class II (IEd) restricted. It is possible that recognition of different regions of the F protein by T cells from humans and BALB/c mice resulted from differences in the nature of to antigen, namely, natural infection of humans administration of purified FG glycoprotein with adjuvant in mice. However, there is no a priori reason to expect an immunodominant T-cell site for humans and BALB/c mice to be within the same region of the F glycoprotein, regardless of the method of antigen exposure. A more precise antigenic map of the F and G glycoproteins of RSV has begun to emerge, with the focus being thus far on the identification of antibody-binding sites. Competitive binding assays with MAbs (3, 5, 25, 34, 35, 37), selection of neutralization-escape mutants (5, 11, 18), Western immunoblotting (33, 34), and analysis of the binding of MAbs or polyclonal antibodies to proteolytic digestion fragments or synthetic peptides (1, 18, 21, 27, 32) have defined four to five antigenic sites on the F glycoprotein and two to three antigenic sites on the G glycoprotein. At least one immunodominant site for binding of human postinfection serum with high neutralization titer has been localized to a disulfidebonded loop of the G protein which has been identified for both the A and B subgroups of RSV (1, 21). At least one neutralization determinant maps to the Fl subunit of the exposure versus

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fusion protein (5, 18, 32-35), and a site for binding of fusion-inhibiting MAb maps proximal to a neutralization determinant on the Fl subunit (35). At least some of these fusion-inhibiting and neutralizing antibody-binding sites appear to be continuous, linear determinants (18, 27, 32, 34, 35) which are highly conserved among group A and B isolates of RSV (2, 3, 18, 32). The highly conserved nature of neutralization determinants on the F protein of RSV coupled with the purported linear, sequential nature of these sites and of the highly antigenic loop site on the G protein (1, 21) suggests that incorporation of these sites into synthetic vaccines or their expression from prokaryotic vectors might require minimal protein refolding and reconfiguration. Thus, the identification of an immunodominant human T-cellstimulating site on the F glycoprotein, as reported herein, would be an important, additional component for such a proposed synthetic or subunit vaccine for RSV. ACKNOWLEDGMENTS We thank Ed Hausknecht and Mike Wathen for providing purified FG glycoprotein for these studies.

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Immunodominant T-cell epitope on the F protein of respiratory syncytial virus recognized by human lymphocytes.

The lymphocyte proliferative responses to respiratory syncytial virus (RSV) were evaluated for 10 healthy adult donors and compared with proliferative...
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