Identification of a novel human leucocyte antigen-A*01-restricted cytotoxic T-lymphocyte epitope in the respiratory syncytial virus fusion protein

Abstract

Virus-specific cytotoxic T lymphocytes (CTL) play a major role in the clearance of respiratory syncytial virus (RSV) infection. To begin monitoring the immunological response to infection, especially in infants, it is important to identify human leucocyte antigen (HLA)-restricted CTL epitopes. Herein, we used a novel, comprehensive peptide panel containing all possible 8-, 9- and 10-mer peptides spanning the RSV fusion protein to screen for novel HLA-restricted T-cell epitopes. These peptide sets were synthesized as 10-mer peptides overlapping by nine amino acids and contained corresponding 8- and 9-mer peptides generated by C-terminal truncation. Unselected and uncultured peripheral blood mononuclear cells from healthy adult subjects were screened by interferon-γ (IFN-γ) Elispot assays against the peptide panel. Seven of 19 subjects displayed positive responses against 10 of the 565 peptides analysed. An HLA-A*01-restricted CTL epitope detected in three healthy adult subjects is characterized. This is the first RSV-specific memory CTL response identified in the fusion protein of RSV.

Introduction

Respiratory syncytial virus (RSV) commonly initiates upper respiratory tract infections that result in relatively mild cold symptoms in immunocompetent adults. However, RSV is the single most important cause of severe lower respiratory tract infection during infancy and early childhood worldwide, and of hospitalization of infants in developed countries.^1,2 Nearly all children have had at least one RSV infection by 2 years of age and nearly one-third of infants who have primary RSV infections develop lower respiratory tract infections. Furthermore, RSV lower respiratory tract illness in early childhood may be an independent risk factor for the subsequent development of wheezing in children up to 11 years of age.³

Cytotoxic T lymphocytes (CTL) play a major role in host defences in most viral infections. CTL directly control acute infection in vivo by destroying virus-producing cells⁴ and possibly by releasing cytokines and chemokines with antiviral activity.⁵ There is substantial evidence that cell-mediated immunity plays a primary role in the clearance of established respiratory virus infection in humans. The best evidence to support this concept is the fact that patients with defects in cellular immunity, such as recipients of bone marrow transplantation or patients with cancer undergoing immunosuppressive chemotherapy, suffer more prolonged virus shedding and more frequent illness with RSV,^6,7 influenza virus^8,9 or parainfluenza virus type 3 (PIV-3),¹⁰ of greater severity. In addition to being an important immunological mechanism for clearing virus-infected cells, CTL are also capable of causing immunopathology.¹¹ This has been demonstrated in persistently infected nu/nu BALB/c mice in which RSV replication was terminated in lung by infusion of low numbers (<10⁶) of RSV-specific T cells without inducing illness,¹² yet transfer of a larger number of RSV-specific T cells resulted in haemorrhagic pneumonitis.¹³ Others have found CTL-mediated RSV clearance in mice without enhanced illness.¹⁴ Importantly, the natural occurrence of CTL responses in experimental infections of mice correlated with virus clearance and recovery.^15,16 This phenomenon has also been demonstrated in a calf model of bovine RSV.¹⁷

All of the RSV proteins examined to date have been shown to be targets for RSV-specific CTL,^18,19 and RSV-specific T cells can be detected in the blood of previously infected adults in whom CTL responses are associated with reduced clinical symptoms.²⁰ In addition, RSV-specific human leucocyte antigen (HLA) class I-restricted CTL have been derived from adult peripheral blood mononuclear cells (PBMC),^18,21 or from children following acute RSV infection.^22,23 While HLA class I-restricted CTL responses are considered to play a critical role in the clearance of RSV infections, the role of cell-mediated immune effectors in the protection against disease upon reinfection or during virus challenge following immunization is not clear. Therefore, characterization of human RSV-specific CTL epitopes may enhance our ability to define the role of CTL in the pathogenesis and protection from RSV infection, and to monitor and evaluate cellular immune responses, not only in wild-type infections but also in clinical vaccine trials.

Evaluation of human CTL responses is a challenging and complex undertaking. Requirements for autologous target cells and a low frequency of CTL effectors in the peripheral blood make human CTL analysis expensive and labour intensive. However, recent advances in the field of human immunodeficiency virus (HIV)-related immunology and vaccine evaluation have made a significant impact on the process of acquired immune-deficiency syndrome (AIDS) vaccine development, and provide a strong precedent for applying these approaches in RSV vaccine development. Herein we characterize a novel HLA-A*01-restricted RSV-specific CTL epitope identified as a memory response in healthy adult subjects. This is the first RSV-specific memory CTL response identified in the fusion protein.

Materials and methods

Subjects

The 19 HLA-A*02-positive healthy adult subjects were selected from eligible volunteers (18–60 years of age) participating in the Vanderbilt HIV Vaccine Trials Network. Seven of the 19 subjects used in this study were HLA-A*01 positive.

HLA typing

HLA typing was performed by DCI Laboratories (Nashville, TN) using standard serological and molecular techniques.

Synthetic peptides

The peptide sets used in Elispot screening experiments were 10 amino acids in length (overlapping adjacent peptides by nine amino acids) and synthesized to contain C-terminal truncations of the corresponding 9-mer and 8-mer peptides (Table 1). The overlapping peptide sets correspond to the fusion protein of subgroup A RSV, and were purchased from Mimotopes Pty. Ltd. (Clayton Victoria, Australia). Peptides used in confirmatory experiments were synthesized as free acids and purchased from SynPep Corporation (Dublin, CA). These peptides were >90% pure, as determined by analytical high-performance liquid chromatography (HPLC).

Table 1.

Peptide-synthesis strategy

	Amino acids
Sequence	Position	Length	Synthesis
MELLILKA	1st	8	Separate synthesis
MELLILKAN	1st	9	Synthesized together
″ELLILKAN	2nd	8
MELLILKANA	1st	10	Synthesized together (peptide pool 1)
″ELLILKANA	2nd	9
″″LLILKANA	3rd	8
″ELLILKANAI	2nd	10	Synthesized together (peptide pool 2)
″″LLILKANAI	3rd	9
″″″LILKANAI	4th	8
etc.			Peptide pools 3–565

Elispot assay

Prior to the addition of cells, 96-well polyvinylidene difluoride-backed plates (MAIP S4510; Millipore, Bedford, MA) were coated with the anti-interferon-γ (IFN-γ) monoclonal antibody (mAb), 1-D1K (5 µg/ml; Mabtech, Stockholm, Sweden), in phosphate-buffered saline (PBS) at 4° overnight. After washing the plates three times with PBS, each plate was blocked by the addition of 200 µl/well R10 medium [RPMI-1640 (Sigma-Aldrich, St Louis, MO), 10% fetal calf serum (FCS) (Atlanta Biologicals, Norcross, GA), and 10 mm HEPES buffer containing 2 mm glutamine and 0·5 mg/ml gentamicin (Gibco BRL, Grand Island, NY)] for at least 2 hr at room temperature. Peptides were added directly to wells in a volume of 50 µl and then freshly isolated PBMC were added at 100 000–200 000 cells/well in 50 µl of R10 media. The final concentration of the peptides in the screening assay was 10 µm. The plates were incubated for 18–20 hr overnight at 37° in 5% CO₂. The plates were then washed, labelled with biotinylated anti-IFN-γ mAb 7-B6-1 (2 µg/ml; Mabtech) in PBS containing 0·5% fetal bovine serum (FBS), and incubated at room temperature for 3 hr. After additional washes, Avidin-Peroxidase-Complex (APC; Vector Laboratories, Burlingame, CA) was added to each well in PBS containing 0·1% Tween-20 for 1 hr at room temperature. The plates were washed, and IFN-γ-producing cells detected as dark spots after a 4-min colour reaction using 100 µl of AEC substrate [20 mg of 3-amino-9-ethylcarbazol (Sigma-Aldrich) dissolved in 2·5 ml of dimethylformamide diluted 1 : 20 in 47·5 ml of sodium-acetate buffer + 25 µl 30% H₂O₂]. IFN-γ-producing cells were counted in an Automated Elispot Reader System using KS 4.3 software (Carl Zeiss, Thornwood, NY). Results were expressed as the number of spot-forming cells (SFC) per 10⁶ input cells. The number of peptide-specific IFN-γ-secreting T cells was calculated by subtracting the negative control value from the established SFC count.

Intracellular cytokine staining

Intracellular cytokine staining (ICS) was performed by incubating freshly isolated PBMC with 2 µm peptide and anti-CD28 and anti-CD49d mAbs (1 µg/ml each; Becton-Dickinson, San Jose, CA) at 37° in 5% CO₂ for 1 hr before the addition of brefeldin-A (10 µg/ml; Sigma-Aldrich). The cells were incubated for an additional 5 hr at a 5° slant, and then placed at 4° overnight prior to staining. The next day, EDTA (2 mm final concentration) was added to each sample for 15 min at room temperature and vortexed before transfer to polystyrene tubes containing 2 ml of cold wash buffer [1% bovine serum albumin (BSA), 0·1% sodium azide in PBS]. After an additional wash, the cells were lysed/permeabilized using BD-FACS Lysing and BD-FACS Permeabilization 2 Solutions (Becton-Dickinson) prior to staining. Cells were stained using fluorescein isothiocyanate (FITC)-conjugated anti-IFN-γ, Per-CP-conjugated anti-CD8, allophycocyanin-conjugated anti-CD3 and phycoerythrin-conjugated anti-CD69 (Becton-Dickinson). After a final wash, the cells were resuspended in 250 µl of PBS containing 1% paraformaldehyde. Six-parameter flow cytometry analysis was performed within 6 hr using a FACS™calibur flow cytometer (Becton-Dickinson). Gating was performed on CD3⁺ and CD8⁺ small viable lymphocytes and 50 000–100 000 events were collected. List mode data files were analysed using WinList software (Verity Software House, Inc., Topsham, ME). Responses were rated as positive when a population of IFN-γ⁺ and CD69⁺ events ≥ 0·03% (above background) of CD3⁺ CD8⁺ lymphocytes was observed. With very few exceptions the background expression observed in these experiments did not exceed 0·01%.

Peptide-specific cell lines

Peptide-stimulated lymphocyte cultures were established from healthy adult subjects, as previously described.²⁴ Briefly, freshly isolated PBMC (at a high cell density) in 100 µl of R10 were pulsed for 1 hr at a peptide concentration of 100 µm and then diluted to 2 × 10⁶ cells/ml in R10 containing 25 ng/ml recombinant human IL-7 (rIL-7; Endogen, Woburn, MA). Cells were plated out into 2-ml wells of 24-well tissue culture plates. After 3 days of culture (at 37° in an atmosphere of 5% CO₂), the culture medium was changed every 4 days, being replaced with R10 containing 10 U/ml rIL-2, obtained via the NCI BRB Preclinical Repository (Fredrick, MD) from Hoffman-La Roche, Inc. (Nutley, NJ). Peptide-specific CTL lines were tested in ⁵¹Cr-release cytotoxicity assays after 12–14 days of stimulation.

Cytotoxicity assay

Autologous Epstein–Barr virus (EBV)-transformed B-cell lines (BCL), or BCL presenting appropriate HLA-class I molecules, were pulsed for 1 hr at 37° in 5% CO₂ with 10 µm of peptide (unless indicated otherwise) and Na2 5l/hfill /hbox {CxsrO₄ (ICN Biomedicals, Costa Mesa, CA). Target cells were washed three times with cold R10 medium and then incubated with peptide-specific CTL at indicated effector to target (E : T) ratios at 37° and 5% CO₂ for 4 hr in triplicate wells. Cellular release of ⁵¹Cr into the supernatant was measured by using a Top Count Microplate scintillation counter (Packard Instrument Company, Meridien, CT), and the percentage specific lysis was calculated according to the following formula:

Results are reported as the means of triplicate values.

Vaccinia virus

In some experiments, BCL target cells (1 × 10⁶) were infected at a multiplicity of infection of 10 with vaccinia viruses and incubated for 16–20 hr (overnight) at 37° in 5% CO₂. All the vaccinia viruses used in this study were kindly provided by Peter Collins (NIAID, Bethesda, MD). The parental virus was the WR strain of wild-type vaccinia virus (Vac). Foreign genes were inserted under the control of the early late P7·5-kb vaccinia virus promoter, inside the thymidine kinase gene of the WR strain. Recombinant vaccinia virus Vac-F encodes the corresponding fusion protein of the RSV wild-type Long strain (a member of the A subgroup of RSV). The production and characterization of these recombinant vaccinia viruses was as described previously.²⁵ The virus stocks were grown in HEp-2 cell culture monolayers and titrated for infectivity by plaque assay in HEp-2 cell monolayers, as described previously.²⁶

Results and discussion

Observations from previously defined HLA class I-restricted CTL epitopes

Initial studies were carried out, using previously defined HLA-restricted virus-specific epitopes, to establish experimental protocols for the detection and characterization of memory CTL responses in healthy adult subjects. An HLA-A*02-restricted epitope from influenza matrix protein, designated MP.58 [GILGFVFTL],²⁷ and an HLA-B*07-restricted RSV-nucleoprotein epitope, designated N.306 [NPKASLLSL],²⁸ were tested. Multiple subjects expressing the appropriate HLA-haplotype, HLA-A*02 and/or HLA-B*07, were tested for epitope-specific CTL responses by Elispot, ICS and/or ⁵¹Cr-release assays. The highly characterized influenza MP.58 epitope elicited strong CTL responses in 60% (12 of 20) of the HLA-A*02 subjects tested, while the recently described RSV N.306 epitope induced comparatively weaker responses in three of five of the HLA-B*07 subjects tested.

Two healthy adult subjects with representative responses in each of the three assays are described. Significant responses against the MP.58 epitope (Subject 2, 110 SFC/10⁶ PBMC) and against the N.306 epitope (Subject 1, 80 SFC/10⁶ PBMC) were observed by IFN-γ Elispot assays (data not shown). These responses were confirmed by IFN-γ/ICS assays, in which positive responses were observed in both subject 1 and subject 2 (Fig. 1a and data not shown, respectively). Negative control samples incubated in the presence of the HLA-A*02-restricted HIV reverse transcriptase epitope, RT.476 [ILKEPVHGV],²⁹ or in the absence of peptide did not induce significant IFN-γ production. These data indicate that adult memory CTL responses to respiratory viruses can be measured in the absence of long-term in vitro cell culture.

Figure 1.

(a) Analysis of peptide-reactive T cells in peripheral blood mononuclear cells (PBMC) from subject 1 by interferon-γ (IFN-γ)/intracellular cytokine staining (ICS). The data are presented as the percentage CD3⁺ CD8⁺ lymphocytes that were CD69⁺ and scored positive for IFN-γ production. (b) Recognition of viral epitopes by peptide-specific effector cells in standard ⁵¹Cr-release assays. A respiratory syncytial virus (RSV) N.306-specific cytotoxic T-lymphocyte (CTL) line generated from subject 1 was used as effector cells. Targets were peptide-pulsed autologous (square), HLA-B*07 matched (circle), or mismatched (triangle) B-cell lines (BCLs). The data are presented as the percentage specific lysis at the indicated effector to target (E : T) ratios.

We confirmed the previously defined HLA restrictions of the MP.58 and N.306 epitopes using peptide-specific cell lines (MP.58, RT.476, or N.306) as effector cells against peptide-pulsed EBV-transformed BCL target cells in standard ⁵¹Cr-release assays. Partially HLA-matched or HLA-mismatched BCL were used to define the HLA restriction of cytolytic responses. The MP.58 peptide-specific cell line generated from subject 2 displayed significant lysis of MP.58 peptide-pulsed autologous and HLA-A*02-matched BCL targets. However, cytolytic activity was not detected for the RT.476 peptide-specific effector cells against either autologous or HLA-A*02-matched target cells, nor was cytolysis observed by any effector cells against peptide-pulsed target cells that did not present the HLA-A*02 allele (data not shown). Similar results were observed for subject 1 using N.306 peptide-specific cell lines against autologous or HLA-B*07-matched targets (Fig. 1b). These data confirmed the HLA restriction for the MP.58 and N.306 epitopes and showed that inadvertent primary CTL responses are not generated in vitro under our culture conditions. Furthermore, these data demonstrate that low-frequency CTL effector cells can be measured functionally and provide the basis for identification of memory CTL responses to RSV in healthy adult subjects.

Characterization of an HLA-A*01-restricted RSV-F-specific CTL epitope

These studies focused (based on earlier work) on the RSV fusion (F) protein as a potential CTL target. The F protein is composed of two disulphide-linked fragments of about 50 000 and 20 000 molecular weight and is responsible for virus-to-cell and cell-to-cell fusion activity, leading to viral entry and syncytia formation. RSV F is the protein to which most RSV neutralizing mAbs are directed³⁰ and is an important target for cytotoxic T lymphocytes.^19,31 Both HLA- and H-2-restricted epitopes have been described for the RSV F protein.^23,32,33 The previously described HLA-restricted CTL epitopes were characterized using T-cell clones isolated from two infants following severe RSV infection.²³

The approach we adopted for identification of novel RSV-specific CTL epitopes included the use of a panel of overlapping peptide sets containing all possible 8-, 9- and 10-mer peptides spanning the RSV F protein. These peptide sets were synthesized as 10-mer peptides overlapping by nine amino acids and contained corresponding 8- and 9-mer peptides generated by C-terminal truncation (Table 1). These peptide sets were used in Elispot assays to screen 19 healthy adult subjects, who expressed the HLA-A*02 allele, for IFN-γ production. PBMC were tested against the 565 peptide sets in duplicate and the number of SFC/10⁶ input cells was determined. Seven of 19 subjects displayed an IFN-γ Elispot response of > 100 SFC (Table 2). Of those subjects exhibiting a positive IFN-γ response, five of seven responded to more than one peptide, identifying a total of 10 potential CTL epitopes. The predominant response was observed with peptides F.519 and F.521, against which four donors reacted to one or both peptides.

Table 2.

Interferon-γ (IFN-γ) Elispot responses to respiratory syncytial virus (RSV) F peptides^*

	HLA
Subject	A		B		Cw		Peptide pool†	SFC	Sequence
							8	113	ANAITTILTA
G	2	–	14	44	05	08	519	118	GKSTINIMIT
							93	113	LQLLMQSTPA
H	2	3	7	8	6	7	521	105	STINIMITTI
A	1	2	8	39	7	–	109	403	RELPRFMNYT
							260	172	LINDMPITND
I	1	2	7	8	6	7	273	107	LMSNNVQIVR
							285	142	SYSIMSIIKE
							273	210	LMSNNVQIVR
J	2	28	51	60	3	–	519	131	GKSTINIMIT
							521	324	STINIMITTI
K	2	29	39	44	–	–	374	113	TLPSEVNLCN
							388	128	NPKYDCKIMT
L	2	31	27	37	–	–	519	178	GKSTINIMIT
							521	135	STINIMITTI

^*Only Elispot results where > 100 spot-forming cells (SFC)/10⁶ input cells were obtained, are shown.

^†Contains a 10-mer peptide at the corresponding amino acid position and the C-terminal 8- and 9-mer peptides (Table 1)

HLA, human leucocyte antigen.

The highest magnitude of response was observed against peptide RELPRFMNYT at amino acid position 109 (designated F10.109) in subject A (Table 2), and this peptide was chosen for further characterization. Interestingly, this peptide did not induce a positive response in any of the other subjects tested, suggesting that it was not an HLA-A*02-restricted response. Furthermore, as the peptides were synthesized as 10-mer peptides overlapping by nine amino acids, the results indicate that this response is to a 10-mer peptide. Otherwise, positive responses should have been detected in adjacent peptide sets. These data suggest that the F10.109 peptide, RELPRFMNYT, elicits an RSV-specific memory CTL response from this subject.

Analysis of the RSV F protein using computer algorithm programs that predict epitope HLA restriction³⁴ indicated that peptide F10.109 is HLA*01-restricted, which corresponded to the HLA type of subject A. Therefore, we further tested peptide F10.109 by standard chromium release assays in three additional subjects who also present the HLA-A*01 allele. Peptide-specific effector cells from two of these three HLA-A*01 subjects displayed significant cytolytic activity against autologous BCL pulsed with peptide F10.109 (Fig. 2a, 2b, 2c). This response was shown to be HLA-A*01 restricted in that only target cells presenting HLA-A*01 and pulsed with the F10.109 peptide were recognized by the F10.109-specific CTL lines (Fig. 2d, 2e, 2f, and data not shown).

Figure 2.

Recognition of peptide RELPRFMNYT (F10·109)-pulsed target cells by peptide-specific effector cells generated from subjects B (panel a), C (panel b) and D (panel c) [human leucocyte antigen (HLA) types are indicated]. Effector cells were peptide specific cytotoxic T-lymphocyte (CTL) lines and targets were autologous B-cell lines (BCLs) pulsed with either no peptide (white symbols) or with peptide F10.109 (black symbols). The data are presented as the percentage specific lysis at the indicated effector to target (E : T) ratios. Peptide F10.109-specific effector cells were generated and assayed for lytic function against autologous (d), A*0101-matched (e), or HLA-mismatched (f) BCL pulsed with F10.109, or BCL infected with a recombinant vaccinia virus encoding the respiratory syncytial virus (RSV) F gene (Vac-F), or with a control vaccinia virus (Vac). The data are presented as percentage specific lysis at an E : T ratio of 25 : 1.

To determine whether target cells that process the full-length RSV F protein within the cell can present this epitope, BCL were infected with a recombinant vaccinia virus encoding the RSV F gene (Vac-F), or with a control vaccinia virus (Vac). Vac-F-infected target cells were recognized in the same HLA-restricted manner as peptide-pulsed targets (Fig. 2d, 2e, 2f). Notably, only effector cells established from subjects B and C exhibited measurable cytolytic activity in these assays. We reasoned that this might have been a result of expression of different HLA alleles within the HLA-A*01 family. However, HLA molecular typing revealed that each of the subjects tested in these experiments expressed the HLA-A*0101 allele. Overall, three of six HLA-A*01 subjects tested had measurable responses against peptide F10.109 (Table 3). Importantly, there was a general correlation between the detection of F10.109-specific CTL responses for subjects B and C by each assay employed (Elispot, ICS, or ⁵¹Cr release), while those unresponsive subjects (D, E and F) did not display peptide-specific responses in any of the assays. Therefore, RSV-specific memory CTL responses can be identified by multiple methods of CTL analysis. Not all individuals expressing a particular allele, however, recognize individual epitopes within the virus.

Table 3.

Summary of responses from human leucocyte antigen (HLA)-A^*0101 subjects against peptide F10.109

Subject	Elispot (SFC/106 input cells)	ICS (% positive)	51Cr release (E : T, 25 : 1)
A	403	NA*	NA*
B	76	0·05%	24·8%
C	147	0·03%	29·1%
D	15	0·00%	1·2%
E	0	0·00%	0·8%
F	5	0·00%	0·0%

^*The subject was not available for additional analysis following the initial screen.

E : T, effector to target ratio; ICS, intracellular cytokine staining; SFC, spot-forming cells.

The analysis of IFN-γ-producing cells by Elispot from subject A suggested that the optimal HLA-A*01-restricted epitope is F10.109. Experiments were performed to identify the optimal epitope capable of inducing a CTL response. Peptide-specific CTL lines from subject C were tested in cytotoxicity assays against HLA-A*01 targets pulsed with different concentrations of peptides RELPRFMNY, RELPRFMNYT and ELPRFMNYT (F09.109, F10.109 and F09.110, respectively). The optimal epitope, defined as the ability to sensitize targets for 50% of maximal lysis at the lowest peptide concentration, was peptide RELPRFMNYT (Fig. 3). These results were confirmed in IFN-γ/ICS assays, in which the largest magnitude of IFN-γ-producing cells were those incubated in the presence of F10.109 (Fig. 4). Therefore, these data were consistent with the findings of the initial Elispot screening assay which suggested that the epitope was a 10-mer peptide. Interestingly, peptide F10.109 contains a recently described cleavage site for furin-like proteases at amino acid position 110.³⁵ The exact mechanism that proteolytic modification may play in the processing/presentation of F-protein epitopes remains to be determined.

Figure 3.

Identification of the optimal epitope within the 10-mer peptide F10.109. Effector cells were peptide-specific cytotoxic T-lymphocyte (CTL) lines generated from subject C [human leucocyte antigen (HLA) type A0101/11, B52/55, Cw3/12], and targets were HLA-A*0101-matched B-cell lines (BCLs) (HLA type A0101/3, B8/35, Cw7/–) pulsed at the indicated concentrations of peptide F10.109 (square), peptide F09.109 (circle), or peptide F09.110 (triangle). The data are expressed as percentage specific lysis at an effector to target (E : T) ratio of 25 : 1.

Figure 4.

Analysis of peptide-reactive CD8⁺ T cells from subject C. Peripheral blood mononuclear cells (PBMC) were incubated in the presence or absence of the indicated peptides (2 µm) for 6 hr in an interferon-γ (IFN-γ)/intracellular cytokine staining (ICS) assay. The data presented represent the percentage of CD3⁺ CD8⁺ lymphocytes that were CD69⁺ and scored positive for IFN-γ production.

In summary, we have utilized a comprehensive peptide-synthesis strategy for screening all possible 8-, 9- and 10-mer peptides spanning the RSV F protein to identify RSV-specific CTL epitopes from unselected and uncultured T cells in healthy adults. Therefore, HLA-restricted virus-specific CTL epitopes may be identified from PBMCs, despite a low frequency of HLA-restricted epitope-specific T cells present. We characterized a novel HLA-A*01-restricted CTL epitope that is presented by RSV F protein-infected cells. The definition of single epitopes is but a start towards the characterization of CTL responses to RSV in infants. However, as additional epitopes are characterized, and as the immunodominant RSV-specific CTL epitopes are identified, our ability to address the role of RSV-specific CTL in the resolution of disease and protection from rechallenge will expand rapidly. Evaluation of infant cellular immune responses following infections with wild-type RSV or during clinical vaccine trials will be greatly enhanced by the identification of RSV-specific CTL epitopes. Characterization of novel epitopes will begin to provide a panel of reagents allowing detailed analysis of infant CTL responses to RSV using individual peptides or pools of peptides in a variety of assay systems. The use of major histocompatibility complex (MHC)–peptide tetramers to evaluate the infant CTL repertoire in conjunction with functional analysis, such as ICS, will enable study of the role of CTL in RSV immunity. The growing number of HLA-restricted CTL epitopes in RSV, which we and others are identifying, will provide the necessary tools to examine the timing of the appearance of RSV-specific CTL with virus clearance and correlation of the magnitude of the CTL response with measures of disease severity and viral load in age-matched children.