Hemagglutinin Protein Is a Primary Target of the Measles Virus—Specific HLA-A2—Restricted CD8⁺ T Cell Response during Measles and after Vaccination

Abstract

To characterize the measles virus (MV)—specific T cell responses important for evaluation of measles vaccines, human leukocyte antigen (HLA)—A2—positive and —negative adults immunized with measles-mumps-rubella vaccine were studied. Both groups developed increases in antibody and in interferon (IFN)-γ—producing cells in response to pooled hemagglutinin (H) and fusion peptides. HLA-A2—binding peptides were predicted for all MV-encoded proteins and confirmed by T2 cell stabilization. Twenty-nine peptides were tested, and 19 (6 from H) stimulated increased IFN-γ secretion in a majority of vaccinees. Peptide-loaded HLA-A2 tetramers or immunoglobulin dimers documented MV-specific CD8⁺ T cell responses after vaccination and during measles and confirmed new A2 epitopes in H (250–259 and 516–525 aa) and matrix (M; 50–58 aa) protein and previously described epitopes in H (30–38 aa), M (211–219 aa), and nonstructural protein C (84–92 aa). No single peptide dominated the response. We conclude that H is an important stimulus for CD8⁺ T cell as well as for antibody responses in HLA-A2—positive individuals.

Measles remains a major cause of child morbidity and mortality. The live attenuated measles vaccine is safe and effective, and implementation of a 2-dose schedule has led to the elimination of endemic measles virus (MV) transmission in many countries. However, in 2002 there were 20–30 million cases of measles and 540,000 deaths, representing 21% of vaccine-preventable childhood deaths [1]. One of the challenges to measles control is the inability to immunize young infants. The live attenuated vaccine is poorly immunogenic in infants under the age of 9 months, both because of the immaturity of the immune response to MV and the presence of maternal antibody [2, 3]. Because maternal antibody decay rates differ, there is a variable window of susceptibility to infection before vaccination [4]. Thus, a new measles vaccine that could be used during early infancy would facilitate measles control. The development of such a vaccine requires a better understanding of the immune responses to MV and the correlates of measles protective immunity.

The level of MV neutralizing antibodies is generally accepted as the best correlate of protection from measles [5], but CD8⁺ T cells are important for viral clearance [6, 7], and both CD4⁺ and CD8⁺ T cells are likely to play a role with antibody in protective immunity. Thus, vaccines that elicit both cellular and humoral responses are likely to be optimal. Strategies for the development of new measles vaccines involve use or expression of individual MV proteins [8, 9]. Identification of MV epitopes recognized by CD8⁺ T cells is an important first step for the evaluation of cellular responses to natural measles and to MV vaccines.

The MV genome encodes 8 proteins [10]. V and C are nonstructural proteins that regulate innate responses. Phosphoprotein (P), large polymerase protein (L), and nucleoprotein (N) are complexed with viral RNA to form the ribonucleocapsid. The hemagglutinin (H), fusion (F), and matrix (M) proteins, together with cellular lipids, form the viral envelope. Neutralizing antibodies are primarily directed against H, with some contribution from F [11, 12]. Therefore, most new MV vaccines have focused on inducing immune responses to H and F [9, 12–14]. However, these proteins may not include T cell epitopes important for optimal protective immune responses [6, 15, 16]. CD8⁺ T cells recognize short peptides presented by highly polymorphic HLA class I molecules. HLA-A2 is one of the most common class I molecules [17], and the identification of MV-specific HLA-A2 epitopes will aid in understanding the pathogenesis of measles, evaluating the immune responses to vaccines, and choosing proteins for inclusion in a new vaccine.

Previous studies identified 1 probable HLA-A2 epitope each in H (29–37/30–38 aa), C (84–92 aa), M (211–219 aa), and L (519–527 aa) and 3 epitopes in N (210–218, 226–234, and 340–348 aa), using prediction algorithms and peptide elution from infected cells [18–20]. To identify HLA-A2 epitopes in F and additional epitopes in H, we previously predicted peptide sequences common to multiple MV strains. Four new A2-specific peptides were identified—H250–259, H516–525, H576–584, and F57–65—that stabilized A2 molecules on T2 cells and protected A2 transgenic mice from vaccinia virus expressing H or F [21].

Of the probable HLA-A2⁺ epitopes identified to date, only responses to H30, C84, and M211 have been confirmed in humans [22, 23]. To expand our knowledge, we have identified potential epitopes in the other MV proteins and extended the results of previous studies to humans. Peptide-specific responses were assessed in health care workers being revaccinated against measles and in children with measles. These studies showed a broad response to MV epitopes present in N, P, C, M, F, H, and L, with the largest number in H. Tetramer or immunoglobulin dimer staining showed that responses to reactive peptides were relatively balanced, with no single epitope dominating the response.

Subjects, Materials, and Methods

Study populations. Forty-five adult hospital workers receiving measles vaccine as part of routine immunization at Johns Hopkins Hospital were studied. New employees identified by the hospital laboratory as having nonprotective levels of antibody to MV by EIA were invited to enroll. Data were collected on age, current medications, and medical history, including the frequency and timing of measles vaccination. Those receiving immunosuppressive therapy, with a chronic disease, or with known HIV-1 infection were excluded. Each individual received the measles-mumps-rubella vaccine (MMR-II; Merck) subcutaneously. Blood was drawn before vaccination and 1–4 weeks later. We also studied cryopreserved peripheral blood mononuclear cells (PBMCs) from 3 Zambian children with acute measles (2, 4, and 7 days after the onset of rash) that had been collected as part of a separate prospective study of measles [24]. Written, informed consent was obtained from adult hospital employees and from the parents or guardians of the children in accordance with protocols approved by the Johns Hopkins Bloomberg School of Public Health Committee on Human Research and, for the Zambian children, the Ethics Committee of the University Teaching Hospital in Lusaka, Zambia.

Cell preparation, culture, and HLA typing. PBMCs were isolated from heparinized blood by Ficoll-Paque Plus density-gradient centrifugation (Amersham Pharmacia), washed, and suspended in RPMI 1640 that contained 20 U/mL penicillin, 20 μg/mL streptomycin, 2 mmol/L L-glutamine (GIBCO), and 10% human AB serum (Sigma-Aldrich). PBMCs not assayed immediately were frozen in fetal bovine serum that contained 10% dimethyl sulfoxide (DMSO), thawed, and washed before use. HLA-A2—positive individuals were identified by staining PBMCs with a fluorescein isothiocyanate (FITC)—conjugated mouse anti—HLA-A2 polyclonal antibody (Becton Dickinson).

For CD8⁺ T cell depletion or enrichment, PBMCs were incubated with antibodies to CD8 or to CD4 and CD56 conjugated to magnetic beads and then negatively selected (Miltenyi Biotec). To confirm depletions, cells were stained with FITC-labeled anti-CD56, phycoerythrin (PE)—labeled anti-CD4, or allophycocyanin (APC)—labeled anti-CD8 (Miltenyi Biotec); examined in a FACSCalibur flow cytometer (Becton Dickinson); and analyzed using FlowJo software (version 6.3; Tree Star). After depletion, the percentages of CD8⁺, CD4⁺, or CD56⁺ T cells were <5%.

Peptide prediction, synthesis, and analysis. Peptides, both 9-mers and 10-mers, from individual proteins were predicted, and binding scores were calculated using the algorithm developed by the Bioinformatics and Molecular Analysis Section at the National Institutes of Health [25]. The top scoring peptides were selected and synthesized to a minimum of 95% purity as measured by high-performance liquid chromatography. All peptides were dissolved in DMSO to produce 1-mmol/L stock solutions. Peptide binding to HLA-A2 was measured using the T2 cell line as described elsewhere [21, 26]. HLA-A2 expression was quantified by flow cytometry, and a fluorescence index (FI) was calculated as follows: FI = [(mean fluorescence with peptide — mean fluorescence without peptide)/mean fluorescence without peptide]. Background fluorescence was subtracted for each individual value. FI₅₀, the peptide concentration (in micromoles per liter) that increases HLA-A2.1 expression by 50% over the no-peptide control, was calculated from the titration curve for each peptide.

Interferon (IFN)—γ enzyme-linked immunospot (ELISpot) assay. Cells were assayed for IFN-γ—producing cells by ELISpot assay. Briefly, each well of a 96-well ELISpot plate (Millipore) was coated overnight at 4°C with 50 mL of monoclonal antibody to IFN-γ (2 μg/mL in sodium bicarbonate buffer [pH 9.5]; BD Pharmingen). Wells were washed and blocked with 10% human AB serum, and cells were added at 2 × 10⁵ cells/well in triplicate. MV peptides were added at a concentration of 1 μg/mL. Assessment of non—HLA-restricted T cell responses used pooled 20-mers overlapping by 11 aa derived from MV F (n = 60) or H (n = 68) proteins. Phytohemagglutinin (1 μg/mL) and appropriately diluted DMSO were used as positive and negative controls. After 40 h of culture at 37°C, plates were washed, secondary antibody was added, and plates were developed in accordance with the manufacturer's protocol (BD Pharmingen). Spots were counted with an automated reader (CTL Analyzers), and the data are expressed as spot-forming cells per 1 × 10⁶ cells after the subtraction of the negative control (0–5 sfc).

HLA-A2 tetramer and immunoglobulin dimer staining. HLA-A2 peptide PE tetrameric complexes for H250, H516, and M50 were synthesized by Beckman Coulter Immunomics Operation. The control tetramer folded with a proprietary 9-mer peptide that shows no response in human samples was supplied by the manufacturer. PBMCs were stained with tetramers at 2 μg/mL, followed by peridinin-chlorophyll-protein—labeledanti-CD3 and APC-labeled anti-CD8. After washing, stained cells were fixed with 0.5% paraformaldehyde and analyzed. HLA-A2 immunoglobulin dimers were synthesized in the laboratory of J. Schneck (Johns Hopkins School of Medicine, Baltimore, MD) [27]. PBMCs were stained with A2 immunoglobulin dimers loaded with H30, H250, H516, M211, and C84 peptides; washed; and stained with PE-conjugated goat anti—mouse immunoglobulin (Caltag Laboratories), followed by FITC-conjugated anti-CD8. Unloaded A2 immunoglobulin was the negative control. Data were acquired on a FACSCalibur flow cytometer and analyzed using FlowJo software. The frequency of tetramer-positive or dimer-positive CD8⁺ T cells was determined by gating on CD3⁺ cells. The 3 techniques identified similar numbers of cells.

Antibody assays. MV-specific neutralizing antibody was measured by reduction of plaque formation by the Chicago-1 strain of MV on Vero cells [28]. Data were normalized to a standard serum run with each assay.

Statistical analysis. Differences between 2 groups were assessed with Student's t test (2-tailed), whereas differences between 2 time points were evaluated using the Wilcoxon matched-pairs signed rank test. Analyses were done using Stata software (version 7; StataCorp).

Results

Study subjects and characteristics. We enrolled 45 adult health care workers. Of those who returned, 17 were HLA-A2 positive and 15 were HLA-A2 negative. Subjects' ages ranged from 25 to 39 years (mean, 30.3 years; median, 31 years). All had a history of measles immunization. No significant differences were identified between the HLA-A2—positive and HLA-A2—negative subjects in age, sex, or prior vaccine experience (data not shown), which suggests that the HLA-A2—positive group was a representative sample.

Antibody and T cell responses of HLA-A2—positive and HLA-A2—negative individuals to revaccination with MMR. The MV neutralizing antibody response was assessed using a plaque reduction assay (figure 1A). Subjects enrolled in the study had been determined by the hospital screening EIA to have levels of antibody below that considered protective, and they were called for revaccination. However, most (90%) had neutralizing antibody titers above the generally accepted protective level of 120 mIU/mL. Antibody titers increased within 1–2 weeks and were sustained over the course of the study period. Responses were similar in HLA-A2—positive and HLA-A2—negative individuals.

Figure 1.

Measles virus (MV)—specific immune responses of HLA-A2—positive (A2⁺) and HLA-A2—negative (A2⁻) adults to reimmunization with measles-mumps-rubella vaccine. The neutralizing antibody response was assessed using an MV plaque reduction assay (A). T cell responses were assessed by stimulating peripheral blood mononuclear cells with pooled MV hemagglutinin (B) or fusion (C) peptides and measuring the no. of interferon-γ—secreting cells by enzyme-linked immunospot assay. Points represent the mean of results from 15 A2⁺(squares) and 12 A2⁻(circles) subjects. Error bars indicate SEs. *P ⩽ .05 and **P ⩽ .01, for the comparison with prevaccination values (Wilcoxon signed rank test).

To assess CD4⁺ and CD8⁺ T cell responses, PBMCs from both HLA-A2—positive and HLA-A2—negative subjects were stimulated with pools of peptides derived from H and F. IFN-μ responses were measured by ELISpot assays (figure 1B and 1C). Most subjects had detectable IFN-μ spot-forming cells before vaccination. Responses increased at 2–3 weeks and then decreased to prevaccination levels. Responses were similar in HLA-A2—positive and HLA-A2—negative individuals. H peptides induced more IFN-γ spot-forming cells than F peptides.

Prediction of MV HLA-A2—specific peptides and induction of IFN-μ responses after MMR. By use of computer algorithms, peptides with HLA-A*0201—binding motifs and high binding scores were identified from the amino acid sequences of each of the 8 MV-encoded proteins (table 1) [25]. All proteins except V contained at least 1 predicted CD8 epitope. In general, binding scores provided by the algorithm correlated with FI₅₀ values. A majority of the peptides tested could bind HLA-A2, but C166, F205, F452, and H117 bound weakly (FI₅₀>50). Although H250 has arginine at position 4, an amino acid associated with poor binding to HLA-A2 [29, 30], the FI₅₀ was 12.5, which suggests that nonanchor residues contributed to the binding of this peptide. C84, M50, M164, and L1640 bound very strongly (FI₅₀< 1.0). Five peptides with high binding scores and low FI₅₀s were predicted from the L protein, probably because this is the largest protein. The H protein contained 9 (31%) of the predicted epitopes with 3 overlapping regions (H36, H38, and H41; H248 and H250; and H576⁹ and H576¹⁰).

Table 1.

HLA-A2 epitopes predicted for measles virus (MV) proteins.

To determine the peptides recognized, 5 HLA-A2—positive adults revaccinated with MMR were studied using IFN-γ ELISpot assays (table 2). Responders were identified by a 2-fold increase in the number of spot-forming cells over prevaccination values or by induction of >25 sfc/1 × 10⁶ PBMCs. Nineteen of 29 peptides were associated with responses in a majority of individuals. There were no responses to 3 of the peptides (H36, L942, and L1917), perhaps because they were not endogenously processed. All individuals responded to 5 of the peptides (M50, M164, H117, H250, and H516). Peak responses occurred 2–3 weeks after vaccination. The overlapping peptides from 2 of the H regions (H36/H38 and H248/250) showed very different responses, despite similar binding scores, which suggests that H36 and H248 may not be processed as efficiently as H38 and H250. The 9-mer H576⁹ and the 10-mer H576¹⁰ elicited similar responses. Ten peptides (1 each from F, C, and L; 2 from M; and 5 from H) that had not been previously confirmed in humans were chosen for further investigation.

Table 2.

Average (SE) no. of interferon-γ spot-forming cells/1 × 10⁶ peripheral blood mononuclear cells in response to stimulation with predicted HLA-A2—specific peptides before and after vaccination of 5 HLA-A2—positive individuals with measles-mumps-rubella vaccine.

Peptide stimulation of IFN-γ production by CD8⁺ T cells. To determine which cells were responding to peptide stimulation, PBMCs obtained from 5 HLA-A2—positive subjects 2 weeks after vaccination were depleted or enriched for CD8⁺ T cells and then stimulated (figure 2). For most of the peptides, the frequency of IFN-γ spot-forming cells was decreased by depletion of CD8⁺ T cells (figure 2A) and was increased by depletion of CD4⁺ and CD56⁺ T cells (figure 2B), compared with unfractionated PBMCs. H250, H516, and M50 peptides stimulated the highest frequencies of IFN-γ spot-forming cells in the bulk PBMC population, and responses to these peptides were decreased by CD8⁺ depletion.

Figure 2.

Cellular source of interferon (IFN)—γ in response to peptide stimulation in measles-mumps-rubella (MMR)—revaccinated adults. Comparison of IFN-γ secretion in response to phytohemagglutinin (PHA) and to individual measles virus peptides (1 μg/mL) of peripheral blood mononuclear cells (PBMCs), CD8⁺ T cell—depleted (CD8-depleted; A), and CD4⁺ T cell and CD56⁺ cell—depleted PBMCs (CD8 enriched; B) is shown. Results are expressed as the mean ± SE of spot-forming cells per 1 × 10⁶ cells from 5 subjects 2 weeks after MMR vaccination. The spot-forming cells for cells cultured in medium alone (7 ± 2) were subtracted. *P ⩽ .05 and **P ⩽ .01 for comparisons of responses of bulk PBMCs to responses of depleted PBMCs. C, nonstructural protein; F, fusion protein; H, hemagglutinin; L, large polymerase protein; N, nucleoprotein.

Tetramer staining to determine frequencies of MV peptide—specific CD8⁺ T cells. To further enumerate peptide-specific CD8⁺ T cells to the newly identified epitopes, H250, H516, and M50 tetramers were used to stain PBMCs obtained from 7 HLA-A2—positive subjects before and after MMR vaccination (figure 3A). The frequency of peptide-specific CD8⁺ T cells was increased for all 3 MV tetramers at 2 and 3 weeks, compared with prevaccination levels (P< .005) (figure 3B). At the fourth week, these numbers had decreased. By contrast, the frequency of CD8⁺ T cells binding to the control tetramer did not change.

Figure 3.

Time course of the measles virus (MV) tetramer—positive CD8⁺ T cell response after measles-mumps-rubella (MMR) reimmunization of adults. HLA-A2 tetramers made with hemagglutinin (H) 516 (T-H516), H250 (T-H250), matrix protein (M) 50 (T-M50), or negative control (Tc) were used. A representative example of percentages of CD8⁺ T cells stained with each MV tetramer in peripheral blood mononuclear cells from 1 HLA-A2—positive individual before (Prevac) and from 2 individuals 3 and 4 weeks after MMR immunization (A). The mean ± SE of percentages of tetramer-positive CD8⁺ cells from 7 HLA-A2—positive (B) and 2 HLA-A2—negative (C) subjects are shown. *P ⩽ .05, compared with prevaccination values (Wilcoxon signed rank test). APC, allophycocyanin; PE, phycoerythrin.

At the peak of the responses to MMR, the average proportion of CD8⁺ T cells reacting with the MV tetramers was 0.4%–0.55%, and that for the control tetramer was 0.18%–0.22%. The frequency of MV-specific cells measured by tetramer staining was ∼20-fold higher than that measured by IFN-γ ELISpot assay (figure 2; table 2). To confirm the HLA restriction of the response, PBMCs obtained from 2 HLA-A2—negative subjects were studied (figure 3C). There was no increase in the number of cells binding to the HLA-A2 tetramers, and this did not differ from the binding to the control tetramer.

Broad CD8⁺ T cell response to MV. To compare the frequencies of the CD8⁺ T cells recognizing the 2 newly identified A2 epitopes (H250 and H516) and the A2 epitopes previously confirmed in humans (H30, C84, and M211) [22, 23], PBMCs collected 1 and 2 weeks after vaccination from an individual with a particularly strong response to the vaccine were stained with A2 immunoglobulin dimers loaded with each peptide (figure 4). Similar percentages of CD8⁺ T cells were induced for each of the peptides after vaccination, which indicates that none of these epitopes exhibited strong immunodominance.

Figure 4.

Frequencies of CD8⁺ T cells to measles virus peptides after measles-mumps-rubella (MMR) immunization. A2 immunoglobulin dimers loaded with hemagglutinin (H) 30, H250, H516, matrix protein (M) 211, and nonstructural protein (C) 84 peptides were used to stain CD8⁺ T cells 1 (A) and 2 (B) weeks after immunization with MMR. FITC, fluorescein isothiocyanate.

High frequencies of MV peptide—specific CD8⁺ T cells during acute measles. To compare vaccine-induced recall responses to responses induced by wild-type MV infection, we used tetramers to measure the frequency of MV peptide—specific CD8⁺ T cells in cryopreserved PBMCs obtained from 3 HLA-A2—positive Zambian children during acute measles (2–7 days after the onset of rash; figure 5). The frequencies of CD8⁺ T cells specific to each of the peptides were higher in children with measles than in control children and were 2–3-fold higher than those after measles vaccination.

Figure 5.

Frequency of measles virus tetramer—positive CD8⁺ T cells in the peripheral blood of HLA-A2—positive Zambian children during acute measles. Peripheral blood mononuclear cells obtained from HLA-A2—positive Zambian children during acute natural measles (2, 4, and 7 days after rash) were stained with each tetramer. Means and SEs are shown. *P ⩽ .05, compared with the control tetramer. T-H250, hemagglutinin 250; T-H516, hemagglutinin 516; T-M50, matrix protein 50; Tc, negative control.

Discussion

Measles has been difficult to control in part because of the inability to immunize young infants. For this reason, new types of vaccines are being developed, and these typically focus on induction of immune responses to H and F [9, 12–14]. These proteins have been chosen for their importance in inducing neutralizing antibody, but little is known about their roles in inducing T cell responses. CD8⁺ T cells play an essential role in the clearance of MV and are an important component of the immune response to infection [6, 7, 31]. In the present study, we mapped HLA-A2—restricted MV epitopes and demonstrated that epitopes were present in most MV proteins but were most abundant in H. The CD8⁺ T cell response to vaccination and to natural infection involved a relatively balanced recognition of a number of epitopes. These studies provide tools for a better assessment of the cellular immune response to MV infection and vaccination and help to validate H as an appropriate antigen for the development of a new measles vaccine.

Because HLA-A2 has a high gene frequency in human populations [17], we identified T cell epitopes that could be presented by HLA-A2. Twenty-nine predicted MV-specific HLA-A2—restricted epitopes from 7 of 8 MV proteins were identified. Four peptides from H (H117, H250, H516, and H576) and 4 peptides from F (F57, F63, F205, and F452) were previously studied in A2 transgenic mice, and immunization with H250, H516, H576, and F57 induced protection against challenge with vaccinia virus expressing H or F [21]. Responses to the 3 H peptides occurred in 60%–100% of the vaccinees, and there was a response to F57 in 40% of them. T cells specific for 19 of the 29 peptides were induced by MV vaccination in a majority. Two previously reported epitopes from M and C are recognized by MV-infected humans [22, 23]. The addition of a previously reported H peptide (30–38) [18, 20] not predicted by our analysis but recognized by individuals with measles [22] results in a total of 20 known MV-specific A2 epitopes. Thus, we have greatly expanded the number and variety of validated MV-specific epitopes available for study of the CD8⁺ T cell response to vaccination and natural infection.

The CD8⁺ T cell response to viral infections may be characterized by an immunodominant response to a single epitope or by a more broadly reactive immunodemocratic response to many epitopes [32]. In the murine response, immunodominance hierarchies are common, but human responses tend to be more immunodemocratic [32, 33]. This is also the case for MV. After the vaccination of adults, responses were detected to many peptides, and tetramer or dimer staining indicated similar percentages of CD8⁺ T cells responding to at least 5 different peptides. The responses to 3 different epitopes (M50, H250, and H516) in children with measles had average percentages of tetramer-positive CD8⁺ T cells of 1.17%–1.85%, similar to a previous report of 1.28% for H30 and 0.73% for M211 [22].

We detected higher frequencies of MV-specific CD8⁺ T cells using tetramers, compared with IFN-γ ELISpot assays. Similar differences have previously been observed in response to other infections [34–36]. This may be due to an overestimation of tetramer-positive cells, as suggested by the 0.1%–0.25% staining in controls, or to cells binding to tetramers that may not be functional or producing IFN-γ.

Although our peptide prediction was based on HLA-A*0201, we used polyclonal antibody to detect all HLA-A2—positive subjects. The HLA-A2 allele is HLA-A*02011 in >95% of white persons and ∼60% of African Americans, in whom HLA-A*0202 occurs in 25.8% and A*0205 occurs in 12.9% [17]. HLA-A*0201 is closely related to subtypes HLA-A*0202 and A*0205, and cross-presentation of epitopes among allelic subtypes has been demonstrated [37, 38].

Subjects received measles revaccination because they were determined by EIA to have antibody levels below those required for protection. However, most had protective titers when determined by the plaque reduction neutralization test, which further confirms the poor correlation between EIA and neutralization at low levels of antibody [39]. The increase in MV neutralizing antibody after revaccination was prolonged, but the CD8⁺ T cell response was transient. It is likely that the higher frequency of MV-specific CD8⁺ cells generally observed in acute measles, compared with revaccination, results from higher antigenic challenge.

Ten (33%) of 30 (our 29 plus H30) studied peptides and 7 (35%) of 20 peptides with responses were from H, which suggests that H contributes significantly to the CD8⁺ T cell responses in HLA-A2—positive individuals. Furthermore, the frequency of IFN-γ—producing cells induced by the H peptide pool was higher than that induced by the F pool. H interacts with F to mediate fusion of the viral envelope with the host cell membrane, and its primary function is to bind to host cellular receptors CD46 and CD150 (SLAM) [40]. Antibodies produced against H and F protect against MV infection [10]. Comparison of the H epitopes with the H sequences from all reference MV genotypes [41], as well as Bilthoven and Chicago-1, indicated that they come from highly conserved regions of the protein [42]. H30, H36, H38, and H41 all include at least a portion of the transmembrane domain (H35–58) [43]; H117 is within the dimerization region of the H stem [44]. H248/250 is in a region of the globular head implicated in the interaction of H with F, whereas H516 and H576 are within propeller sheets 5 and 6, outside the predicted CD46 and SLAM receptor-binding regions [45, 46].

These studies show that H is important for the induction of MV-specific CD8⁺ T cells, as well as neutralizing antibody, and that epitopes are within conserved regions of the protein. Thus, H is appropriate for inclusion as a primary antigen in novel MV vaccines.

We are grateful to the study subjects and the mothers of the children who participated in the study; Laurie Sneed and the staff of the Johns Hopkins Hospital Occupational Health Services, for recruiting and obtaining blood samples from study subjects; Drs. Felicity Cutts, Judith Ryon, and Mwaka Monze, for playing key roles in setting up and managing the hospital-based measles study in Zambia; Dr. Jonathan Schneck, for preparing the measles virus peptide immunoglobulin dimers; and David Nyakiti and Jun Yang, for technical support.

Potential conflicts of interest: none reported.

Financial support: National Institutes of Health (NIH; grant AI-23047); Bill and Melinda Gates Foundation; Center for Cancer Research, National Cancer Institute, NIH (intramural funds).