Skip Navigation

Major Histocompatibility Complex Class I Cytotoxic T Lymphocyte Immunity to Human Metapneumovirus (hMPV) in Individuals with Previous hMPV Infection and Respiratory Disease

  1. Karen A. Herd1,
  2. Michael D. Nissen1,2,
  3. Peter M. Hopkins3,
  4. Theo P. Sloots1 and
  5. Robert W. Tindle1
  1. 1Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, and Clinical Medical Virology Centre, University of Queensland, Brisbane, Australia
  2. 2Department of Infectious Diseases, Royal Children's Hospital, Herston
  3. 3The Prince Charles Hospital, Brisbane, Australia
  1. Reprints or correspondence: Dr. Robert W. Tindle, Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, Herston Rd., Herston, Queensland 4029, Australia (r.tindle{at}uq.edu.au).

  1. Contribution no. 279 of the Sir Albert Sakzewski Virus Research Center.

 
Next Section

Abstract

Recently identified human metapneumovirus (hMPV) is an important respiratory pathogen in children and adults worldwide. Little is known about cytotoxic T lymphocyte (CTL) responses that may control hMPV infection in humans. To address this, we evaluated major histocompatibility complex (MHC) class I T cell immunity in 7 patients with previous hMPV respiratory disease. CTL responses were evident in most patients and to most proteins of hMPV. Individual patients had responses to at least 2 hMPV proteins (particularly theMprotein) and had multiallele responses. In addition, we identified 9 CTL epitopes that are presented by human leukocyte antigen alleles of the most common MHC “supertypes.” Many of these CT Lepitopes are conserved acrosshMPVtypes, and there is epitope similarity betweenhMPVand human respiratory syncytial virus. This study provides the first report of MHC class I T cell immunity to hMPV in humans. These findings have significance for understanding cellular immunity to hMPV infection and for future vaccine development.

Human metapneumovirus (hMPV) is a recently identified respiratory pathogen. It was first isolated in 2001 from young children with respiratory tract disease not attributable to other viruses [1] and has circulated in the human population for at least 50 years [1, 2]. Human metapneumovirus is a single-stranded, negative-sense RNA virus, classified with other mammalian respiratory pathogens, including human respiratory syncytial virus (hRSV). The clinical syndrome associated with hMPV infection is similar to that observed for hRSV infection, ranging from mild respiratory illness to bronchiolitis and pneumonia [3, 4]. Young children, the elderly, and immunocompromised individuals are particularly susceptible to hMPV-associated disease, underscoring the need to understand the role played by antiviral immunity in the control of infection.

The immunobiology of infection with hMPV is incompletely characterized, both in rodent [5–7] and nonhuman primate models [7, 8] and in humans. Although extensive literature describes sustained humoral immune responses in humans to both hMPV [1, 9] and hRSV [10, 11], clinical infection with these viruses occurs throughout life, suggesting that neutralizing antibody is not sufficient to control infection. Very little is known about the presence, nature, or control of cellular responses to hMPV, although some data are available for the hMPV F protein in African green monkeys [12]. CD8+ T cells play an important role in host defenses during most viral infections. They are probably necessary to resolve hMPV infection in humans, by analogy with hRSV, for which children with T cell immune dysfunction shed virus for months, in contrast to healthy children, who clear hRSV in 7–21 days [13].

In the present study, we describe for the first time cytotoxic T lymphocyte (CTL) immunity in patients with previous hMPV infection and respiratory disease. We demonstrate CTL immunity in most patients and to most proteins of hMPV. In addition, we describe the first hMPV human CTL epitopes. These findings contribute to an understanding of cellular immunity to hMPV in humans and offer encouragement for the prospect of a vaccine designed to induce protective T cell responses against hMPV infection. We have recently demonstrated that a murine CTL epitope vaccine is protective in a mouse model of hMPV infection [14].

Previous SectionNext Section

Materials and Methods

T cell epitope prediction and peptide synthesis. T cell (major histocompatibility complex [MHC] class I) epitopes of type A hMPV were predicted for MHC class I binding and proteosomal cleavage, by use of independent online algorithms [15–17] in 7 of 9 hMPV proteins (N, M, F, M2-1, M2-2, SH, and G). The protein encoded by the polymerase (L) gene is not readily amenable to epitope mapping because of its large size (∼2000 aa). The protein encoded by the phosphoprotein (P) gene was not included by analogy to related hRSV, because no CTL epitopes have been detected in that protein [18]. Predictions were made for HLA alleles representing 9 MHC “supertypes” (A1, A2, A3, A24, B7, B27, B44, B58, and B62) [19]. For initial screening, predicted epitopes (hereafter, “predictopes”) were synthesized as a PepSet (>1 µmol scale; Mimotopes). For further screening, purified individual syntheses were used. Peptides were dissolved in dimethyl sulfoxide or dimethylformamideat 10 mg/mL. Experiments were designed to preclude nonspecific toxicity and T cell stimulation by individual peptides. A total of 74 predictopes were selected (24 for F, 18 for N, 11 for M2-1, 9 for M, 6 for G, 4 for SH, and 2 for M2-2), across the range of hMPV proteins and MHC supertypes [19, 20]. This predictope set was used to evaluate CTL responses in 7 patients with previous hMPV-associated respiratory disease. In this study, we present results for T cell responses restricted by HLA alleles of the 3 most common MHC supertypes (A2, A3, and B7, each with mean frequencies of ∼40%–50% in the human population). PutativeMHC class I anchor residues are indicated in boldface for individual epitopes.

Patients and samples. Study approval was obtained from the Ethics Committee of the Royal Children's Hospital and Health Service District, Queensland, Australia. Informed consent of patients was obtained before commencement of the study. Patients with confirmed hMPV infection (diagnosed during 2003–2005) and symptoms of respiratory tract infection were included in this study. All 7 patients (age range, 37–66 years; mean age, 54 years) were former lung transplant recipients with recovered immunocompetencewhoexperienced hMPV-associated respiratory disease within 1–7 years of transplantation (within 2 months for patient 16). Nasopharyngeal aspirates and/or bronchoalveolar lavage fluid were tested for the presence of hMPV RNA by real-time reverse transcription polymerase chain reaction targeting the nucleoprotein gene [21]. These samples were negative for other common respiratory pathogens, including hRSV, adenovirus, parainfluenza virus, and influenza virus (except for patient 2, who had influenza A virus coinfection). Blood samples were collected with informed consent. Peripheral blood mononuclear cells (PBMCs) were isolated and either used immediately for generation of cell lines or stored frozen before T cell assay. Subsets of MHC-appropriate predictopes were used to evaluate T cell immunity. Patient HLA serotypes (supertypes) were as follows: patient 1, A1, A2, B8, and B61 (A1, A2, B7, and B44); patient 2, A25, A31, B7, and B51 (A1, A3, B7, and B7); patient 3, A2, A29, B44, and B44 (A2, Ax, B44, and B44); patient 9, A2, A2, B44, and B51 (A2, A2, B44, and B7); patient 13, A2, A2, B7, and B62 (A2, A2, B7, and B62); patient 15, A29, A31, B37, and B57 (Ax, A3, B44, and B58); patient 16, A2, A11, B7, and B55 (A2, A3, B7, and B7) (x denotes unassigned supertype).

Generation of cell lines. PBMCs were cultured, as described elsewhere [22], in T cell growth medium containing purified human recombinant interleukin-2 (10–20 U/mL; Sigma) and supernatant (30% vol/vol) from the T cell line MLA-144. Target and stimulator cell lines were generated as described elsewhere [22]. In brief, phytohemagglutinin (PHA) blasts were generated by stimulating PBMCs with PHA. Epstein-Barr virus-transformed lymphoblastoid cell lines (LCLs) were established by exogenous virus transformation of peripheral B cells using B95-8 virus isolates. Autologous LCLs (peptide pulsed) were used to stimulate T cell lines weekly. HLA-matched LCLs were used as target cells in cytotoxicity assays. For some assays, LCL target cells were infected with type A2 hMPV at an MOI of ∼1. Protein expression was demonstrated in ∼10% of cells by an immunofluorescence assay using monoclonal antibody to the N protein and anti-mouse immunoglobulin-fluorescein isothiocyanate (Chemicon).

T cell lines were established essentially as described elsewhere [22]. T cell lines were restimulated weekly with peptide-pulsed, γ-irradiated (8000 rad) autologous LCLs and were maintained in T cell growth medium.

T cell assays. Ex vivo T cell responses were evaluated by enzyme-linked immunospot (ELISpot) assay, essentially as described elsewhere [22]. In brief, ex vivo PBMCs were added (2.0 × 105-2.5 × 105/well) to plates coated with anti-human interferon (IFN)-γ capture antibody (clone 1-D1K; Mabtech) and incubated with MHC-appropriate peptide (10 µg/mL) or without, for 16–20 h at 37°C in 5% CO2. After washing, detection antibody (biotinylated anti-human IFN-γ, clone 7-B6 -1; Mabtech) was used at 2 µg/mL, and plates were developed using streptavidin-alkaline phosphatase (BD PharMingen) and BCIP/ NBT substrate (Sigma). Spots were counted with an automated ELISpot reader system. Results were expressed as IFN-γ-positive cells/1 × 106 PBMCs. Frequency was defined as (positive cells with peptide − positive cells without peptide)/1 × 106 PBMCs. The activation index (AI) was defined as (positive cells with peptide)/(positive cells without peptide). Results >3 SDs above the mean of the negative control (AI>3) were considered positive.

Memory T cell responses were evaluated by 51Cr release assays, using in vitro restimulated T cell lines and epitope peptides essentially as described elsewhere [22, 23]. In brief, 51Cr-labeled target cells were reacted with T cell lines at specified effector to target cell ratios for 4–6 h. Radioactivity in cell-free supernatant was determined using a Microbeta counter (Perkin Elmer). Results were calculated as the percentage of cytotoxicity using the formula [(E − S)/(T − S)]×100, where E represents counts per minute released in the presence of effector cells, S represents spontaneous counts per minute with medium only, and T represents total counts per minute in the presence of 5% sodium dodecyl sulfate (S values were always <25% of T values).

Protein sequence alignments. Protein sequences for various hMPV strains were obtained from GenBank, and alignments were performed with ClustalW software (version 1.8.3). Representative hMPV strains were NDL 00-1, CAN 97-83, CAN 97-82, and CAN 98-75 (for A1, A2, B1, and B2hMPVsubtypes, respectively). When available, protein sequences for other hMPV strains detected in Australia, Canada, and Japan were also included in sequence alignments.

Previous SectionNext Section

Results

Evidence of MHC class I T cell immunity to most hMPV proteins by most patients. Four of 5 patients with HLA alleles representing the B7 supertype displayed ex vivo and postrestimulation T cell responses to an M194 predictope (figure 1A). One of these patients also displayed low T cell responses to an M12 predictope (figure 1B).

Figure 1.
View larger version:
Figure 1.

T cell immunity to the M protein. T cell immunity was evaluated at various times (months) after diagnosis of human metapneumovirus (hMPV) infection, as indicated. A and B, Quantification of interferon (IFN)-γ-secreting T cells by enzyme-linked immunospot assay. Peripheral blood mononuclear cells (PBMCs; ex vivo) were incubated for 18 h with or without predictope peptide, as shown. Results are expressed as IFN-γ-positive cells/1 × 106 PBMCs. Cytotoxic T lymphocytes (CTLs) were measured by a 51Cr release assay. T cell lines were tested against autologous target cells with or without epitope peptide, as shown; values in the upper left corner denote the percentage of specific cytotoxicity after subtraction of the with-peptide value from the without-peptide value. Results are expressed as the percentage of cytotoxicity (mean ± SD; SDs were always <5%, and some are masked by the symbols); nos. represent peptide-specific cytotoxicity at an effector to target cell (E:T) ratio of 10:1. C and D, Sequence alignment for the M protein (254 aa). The alignment indicates that the identified CTL epitopes are present in types A and B hMPV strains. Amino acid positions are shown above the sequences. The conserved hexapeptide [24] is boxed, and the 2 CTL epitopes (M12, IPYTAAVQV/HLA-B7; M194, IAPYAGLIMI/HLA-B7, B51, B55) are shaded. AI, activation index; restim, restimulated.

Two of 3 patients with alleles representing the A3 supertype displayed T cell responses to an F429 predictope (figure 2A). One of 5 patients with alleles representing the A2 supertype displayed T cell responses to an F157 predictope (figure 2B).

Figure 2.
View larger version:
Figure 2.

T cell immunity to the F protein. T cell immunity was evaluated at various times (months) after diagnosis of human metapneumovirus (hMPV) infection, as indicated. A and B, Quantification of interferon (IFN)-γ-secreting T cells by enzyme-linked immunospot assay. Peripheral blood mononuclear cells (PBMCs; ex vivo) were incubated for 18 h with or without predictope peptide, as shown. Results are expressed as IFN-γ-positive cells/1 × 106 PBMCs. Cytotoxic T lymphocytes (CTLs) were measured by a 51Cr release assay. T cell lines were tested against target cells with or without peptide, as shown; values in the upper left corner denote the percentage of specific cytotoxicity after subtraction of the with-peptide value from the without-peptide value. Results are expressed as the percentage of cytotoxicity (mean ± SD; SD was always <5%); nos. represent peptide-specific cytotoxicity at an effector to target cell (E:T) ratio of 10:1. C and D, Sequence alignment for the F protein (539 aa). The alignment indicates that the identified CTL epitopes are present in types A and B hMPV strains. Amino acid positions are shown above the sequences. The heptad repeat 1 (HR-1) [25] is boxed. The 2 CTL epitopes (F157, VLATAVREL/HLA-A2; F429, KVEGEQHVIK/HLA-A11 and A31) are shaded. AI, activation index; restim, restimulated.

One of 3 patients with alleles representing the A3 supertype displayed T cell responses to the M2-1149 predictope (figure 3A). One of 5 patients with alleles representing the A2 supertype displayed T cell responses to the predictope M2-1157 (figure 3B).

Figure 3.
View larger version:
Figure 3.

T cell immunity to the M2-1 protein. T cell immunity was evaluated at various times (months) after diagnosis of human metapneumovirus (hMPV) infection, as indicated. A and B, Quantification of interferon (IFN)-γ-secreting T cells by enzyme-linked immunospot assay. Peripheral blood mononuclear cells (PBMCs; ex vivo) were incubated for 18 h with or without predictope peptide, as shown. Results are expressed as IFN-γ-positive cells/1 × 106 PBMCs. Cytotoxic T lymphocytes (CTLs) were measured by a 51Cr release assay. T cell lines were tested against target cells with or without peptide, as shown. Results are expressed as the percentage of cytotoxicity (mean ± SD; SD was always <5%); nos. represent peptide-specific cytotoxicity at an effector to target cell (E:T) ratio of 10:1. C, Sequence alignment for the M2-1 protein (187 aa). The alignment indicates that the identified CTL epitopes are present in types A and B hMPV strains. Amino acid positions are shown above the sequences. The 2 CTL epitopes (M2-1149, RLPREKLKK/HLA-A11; M2-1157, KLAKLIIDL/HLAA2) are shaded. AI, activation index; restim, restimulated.

Two of 5 patients with alleles representing the A2 supertype displayed T cell responses to the G32 predictope (figure 4A). A G32-specific T cell line also killed HLA-A2-matched LCL target cells infected with hMPV, for which infection was confirmed by indirect immunofluorescence (data not shown). One patient (patient 16) without an ex vivo response had a memory T cell response when tested further (data not shown). One of 5 patients with alleles representing the B7 supertype displayed ex vivo and memory T cell responses to the N307 predictope (figure 5A) as well as to the SH152 predictope (figure 5B).

Figure 4.
View larger version:
Figure 4.

T cell immunity to the G protein. T cell immunity was evaluated at various times (months) after diagnosis of human metapneumovirus (hMPV) infection, as indicated. A, Quantification of interferon (IFN)-γ-secreting T cells by enzyme-linked immunospot assay. Peripheral blood mononuclear cells (PBMCs; ex vivo) were incubated for 18 h with or without predictope peptide, as shown. Results are expressed as IFN-γ-positive cells/1 × 106 PBMCs. Cytotoxic T lymphocytes (CTLs) were measured by a 51Cr release assay. T cell lines were tested against autologous target cells with or without peptide, as shown. Results are expressed as the percentage of cytotoxicity (mean ± SD; SD was always <5%). Nos. represent peptide-specific cytotoxicity at an effector to target cell (E:T) ratio of 10:1. B, Sequence alignment for the G protein (236 aa). The alignment indicates that the identified CTL epitope is present in type A hMPV strains. Amino acid positions are shown above the sequences. The transmembrane domain [24] is boxed, and the CTL epitope (G32, SLILIGITTL/HLA-A2) is shaded. For patient 13, the autologous virus sequence was TLILIGLSAL. AI, activation index; restim, restimulated.

Figure 5.
View larger version:
Figure 5.

T cell immunity to the N and SH proteins. T cell immunity was evaluated at various times (months) after diagnosis of human metapneumovirus (hMPV) infection, as indicated. A and B, Quantification of interferon (IFN)-γ-secreting T cells by enzyme-linked immunospot assay. Peripheral blood mononuclear cells (PBMCs; ex vivo) were incubated for 18 h with or without predictope peptide, as shown. Results are expressed as IFN-γ-positive cells/1 × 106 PBMCs. Cytotoxic T lymphocytes (CTLs) were measured by a 51Cr release assay. T cell lines were tested against autologous target cells with or without peptide, as shown. Results are expressed as the percentage of cytotoxicity (mean ± SD; SD was always <5%); nos. represent peptide-specific cytotoxicity at an effector to target cell (E:T) ratio of 10:1. C, Sequence alignment for the N protein (394 aa). The alignment indicates that the identified CTL epitope is present in types A and B hMPV strains. The similarity region is boxed, and the CTL epitope (N307, SPKAGLLSL/HLA-B7) is shaded. D, Sequence alignment for the SH protein (183 aa). The alignment indicates that the identified CTL epitope is present in subtype A1 hMPV strains. The external/second hydrophobic region [24] is boxed, and the CTL epitope (SH152, KPAVGVYHIV/HLA-B7) is shaded. AI, activation index; restim, restimulated.

Overall, most patients (4 of 5 patients with HLA alleles of the B7 supertype, 2 of 3 patients with HLA alleles of the A3 supertype, and 3 of 5 patients with HLA alleles of the A2 supertype) showed T cell immunity to the M, F, and G proteins, respectively. One of 3 patients with HLA alleles of the A3 supertype and 1 of 5 patients with HLA alleles of the A2 supertypes showed T cell immunity to the M2-1 protein. Only 1 of 5 patients with HLA alleles of the B7 supertype showed T cell immunity to theN and SH proteins (summarized in table 1). Responses toMand G proteins were recorded significantly more frequently than were responses to the F, M2-1, N, and SH proteins (χ2 = 40.1 [5 df ]; P > .001; normalized for the number of predictopes per protein). However, the frequency of response was not significantly different among the B7, A3, and A2 supertypes (χ2 = 0.01 [2 df]; P > .05). In summary, CTL responses were evident in most patients and to most proteins of hMPV. We identified 9 human CTL epitopes in the M, F, and M2-1 proteins (2 each) and in the G, N, and SH proteins (1 each). These CTL epitopes are presented by HLA alleles of the most common MHC supertypes in the human population (4 by B7, 3 by A2, and 2 by A3) (table 1).

Conservation of identified CTL epitopes across hMPV types. We asked whether the identified CTL epitopes are present in both virus types (A and B). Protein sequence alignments were performed for hMPV strains representing subtypes A1, A2, B1, and B2.

Among the highly conserved proteins, epitopes M194, M12, F429, F157, and N307 were present in all strains analyzed (figures 1C, 1D, 2C, 2D, and 5C, respectively). The M2-1149 and M2-1157 CTL epitopes were present in most (8/11) strains analyzed (figure 3C); representative type B strains differed by 1 aa at a nonanchor position, which may not affect MHC binding.

Among the highly variable G and SH proteins, the G32 epitope was present in all (n = 24) type A strains analyzed (figure 4B). Representative type B strains differed by 3 aa at nonanchor positions, which may still allow MHC binding. The SH152 epitope was present in only 1 of the 2 representative subtype A1 strains analyzed (figure 5D). These strains differed by 1 aa at a nonanchor position. The remaining subtypes had important amino acid differences at anchor positions and did not fit the MHC-binding supermotif for B7 [16]. Furthermore, representative type strains had almost no sequence identity (KTMV EKHRKA; underscored/italicized sequences denote amino acid differences from the SH152 epitope).

Overall, 5 CTL epitopes in the M, F, and N proteins were conserved across hMPV types, and 2 CTL epitopes in the M2-1 protein were conserved across most hMPV subtypes (with some strain variation). The CTL epitope in the G protein was conserved across type A strains (but may also be recognized in type B strains), and the CTL epitope in the SH protein was probably subtype A1 specific.

T cell responses in individual patients. Individuals had T cell responses to at least 2 proteins (particularly M; also F, G, and M2-1). Patient 2 had responses to 2 proteins (M and F), as did patient 9 (M and G); patient 16 had responses to 4 proteins (M, F, G, and M2-1); and patient 13 had responses to 6 (M, F, G, M2-1, N, and SH).

Individuals recognized several (up to 7) of the 9 identified CTL epitopes (table 1). Patient 2 recognized 2 CTL epitopes (M194 and F429), as did patient 9 (M194 and G32); patient 16 recognized 4 (M194, F429, G32, and M2-1149); and patient 13 recognized 7 (M194, M12, F157, G32, M2-1157, N307, and SH152).

The M protein had 2 CTL epitopes (table 1). All 4 individuals with B7 supertype alleles recognized the M194 epitope. The F protein had 2 CTL epitopes. Both individuals with A3 supertype alleles recognized the F429 epitope. The G protein had 1 CTL epitope. All 3 individuals with A2 supertype alleles recognized the G32 epitope. Thus, M194, F429, and G32 are preferred CTL epitopes within these proteins.

Individuals had multiallele responses to hMPV. Patient 2 had 2 responses (restricted by B7 and A3), as did patient 9 (restricted by B7 and A2); patient 16 had 4 responses (restricted by A2 and A3, 2 responses each); and patient 13 had 7 responses (restricted by B7 and A2, 4 and 3 responses, respectively).

In summary, CTL responses (ex vivo and postrestimulation) were evident in most patients and to most hMPV proteins. We found no evidence of ex vivo T cell immunity in 3 patients. Responding individuals had multiallele responses to at least 2 proteins (particularly the M protein) and recognized several (up to 7) CTL epitopes, some preferred within the protein.

CTL epitope similarity between hMPV and hRSV. The identification of the first published human CTL epitopes of hMPV provided by this study allows comparison with known human CTL epitopes of related hRSV (9 in other studies) [26–30]. The hMPV-N307 (SPKAGLLSL) and hRSV-N306 (NPKASLLSL) [28] epitopes are very similar sequences at the same position in the respective viruses (figure 6A), presented by the same HLA allele (B7). They differ by only 2 aa (italicized and underscored), 1 of which is expected to be involved inTcell receptor binding [16]. The hMPV-M194 (IAPYAGLIMI) and the hRSV-M195 (IPYSGLLLV) epitopes [30] are at the same position in the respective viruses (figure 6B), presented by HLA alleles of the same MHC supertype (B7). Similarly, the hMPV-M2-1149 (RLPREKLKK) and hRSV-M2-1151 (RLPADVLKK) epitopes are at the same position in the respective viruses (figure 6C), presented by HLA alleles of the same MHC supertype (A3), even though the 3 aa at which they differ (underscored) are likely to be involved in T cell receptor binding. These intriguing similarities raise the possibility ofcommonCTLepitopes shared by hMPV and hRSV.

Figure 6.
View larger version:
Figure 6.

Cytotoxic T lymphocyte (CTL) epitopes in the N, M, and M2-1 proteins of human metapneumovirus (hMPV) and human respiratory syncytial virus (hRSV). Shown are sequence alignment for the N, M, and M2-1 proteins of hMPV and hRSV, with amino acid positions indicated above the sequences. For the N proteins (A), a similarity region for mononegaviruses [24] is boxed; the identified CTL epitopes hMPV-N307 and hRSV-N306 [27] are shaded. For the M proteins (B), the nuclear export signal (NES) [31] is boxed; the identified CTL epitopes hMPV-M194 and hRSV-M195 [30] are shaded. For the M2-1 proteins (C), the identified CTL epitopes hMPV-M2-1149, hMPV-M2-1157, and hRSV-M2-1151 are shaded.

Table 1.
View larger version:
Table 1.

Identified human cytotoxic T lymphocyte epitopes of human metapneumovirus (hMPV).

Previous SectionNext Section

Discussion

It is important to understand the immunobiology of hMPV because of the morbidity associated with infection in young children, the elderly, and immunocompromised individuals [3, 4]. As for other viruses displaying tropism for epithelium of the upper and lower respiratory tract (e.g., hRSV), proteins expressed during infection are likely to be targets of CTL responses, which may be associated with reduced virus titers and disease [32].

In this study, we evaluated MHC class I T cell immunity in patients with previous hMPV infection and respiratory disease. CTL responses were evident in most patients and to most proteins (M, F, M2-1, N, G, and SH) of hMPV. Seven of the 9 epitopes are in conserved proteins (M, F, M2-1, and N). The magnitude of the responses was in the range seen in some other viral infections (e.g., [33]). Responses targeting cells expressing these hMPV proteins could prevent virus assembly and/or spread and would be expected to reduce disease severity. In particular, we identified CTL epitopes that are presented by HLA alleles of the most commonMHCsupertypes (B7, A2, and A3). In this patient set, responses restricted through other supertypes were not detected. The epitopes we describe are clearly relevant to natural infection, because they are processed out of whole hMPV proteins during immune response induction in the patients and are presented by hMPV-infected cells for T cell-mediated killing. Further evidence for relevance to natural infection is likely to come from a mouse model; the G32 epitope shows complete homology to an hMPV A2-restricted epitope that we identified in HLA-A2 transgenic mice [14]. Immunization with a HLA A2 epitope was protective in a murine model of hMPV infection [14].

Many of the identified CTL epitopes we describe are conserved across hMPV types, suggesting the possibility of crossprotective T cell immunity. We are unaware of data from elsewhere describing CTL immunity to hMPV infection in humans.

Our results indicate that ex vivo T cell responses (IFN-γ release by PBMCs) can be detected up to 10 months after recovery from clinical disease. That these responses represent true “effector” T cell responses seems unlikely, because they were recorded so long after presumed induction during clinical infection. This is particularly so given the mature age (and assumed declining immunocompetence) of the patients. Alternative explanations are that hMPV persists after resolution of disease [34, 35] and that patients acquired subsequent subclinical reinfection(s) [2, 36]. We were able to recall memory T cell responses (cytotoxic activity of restimulated PBMCs) up to 21 months after disease resolution. The responses we report after 5 restimulations were also evident after 2 restimulations in most cases (data not shown). Note that in this study only patients who displayed ex vivo responses were subject to further investigation. Thus, we cannot preclude that memory responses existed in those individuals in whom ex vivo responses were not detected. Although most people are seropositive for hMPV by 5–10 years of age, we were unable to detect ex vivo hMPV-directed CTLs in adults without a history of hMPV-associated disease (data not shown).

The similarity between some hMPV and hRSV CTL epitopes in conserved proteins likely relates to epitope location in the functional domains [24, 25, 31] of these paramyxovirus proteins. This suggests that functional constraints may limit the capacity for variation within these epitopes. The degree of sequence identity (particularly at anchor positions for MHC binding) opens up the intriguing possibility of cross-protective T cell immunity betweenhMPVand hRSV, brought about either by natural infection or by vaccination.

Although immunity acquired by natural infection withhMPV is insufficient to prevent reinfection, it is sufficient to prevent serious lower respiratory tract disease, except in the elderly, the very young, and immunocompromised individuals. Thus, a vaccine to protect against hMPV infection and respiratory disease is required. The CTL epitopes identified in the present study are conserved across hMPV types, are from many (6/9) viral proteins, and are presented by HLA alleles of the most common MHC supertypes, with mean frequencies of ∼40%–50% in the overall human population [19]. Therefore, there are implications for the role these epitopes could play in the development of a multiallelic hMPV vaccine with broad population coverage.

In conclusion, we have evaluatedMHCclass I T cell immunity in patients with previous hMPV infection and respiratory disease. CTL responses were evident in most patients and to most proteins of hMPV. We identified 9 CTL epitopes that are presented by HLA alleles of the most common MHC supertypes. Many of these CTL epitopes are conserved across hMPV types, and there is epitope similarity between hMPV and hRSV. Individuals had multiallele responses to at least 2 hMPV proteins (particularly the M protein). This study provides the first detailed description of MHC class I T cell immunity to hMPV in humans and identifies protein targets of CTL responses at the epitope level. These findings have significance for our understanding of cellular immunity to hMPV and for future vaccine development.

Previous SectionNext Section

Acknowledgments

Wethank Jane Yunus and Emily McQueen for coordinating the collection of patient samples and Ian Mackay for viral gene sequencing. Maxine Preston provided the type A2 human metapneumovirus.

Previous SectionNext Section

Footnotes

  • Potential conflicts of interest: none reported.

  • Financial support: Royal Children's Hospital Foundation, Brisbane (grant 913-008).

  • Received July 5, 2007.
  • Accepted August 7, 2007.
Previous Section
 

References

  1. 1.↵
    1. van den Hoogen BG,
    2. de Jong JC,
    3. Groen J,
    4. et al
    . A newly discovered human pneumovirus isolated from young children with respiratory tract disease. Nat Med 2001;7:719-24.
    CrossRefMedlineWeb of Science
  2. 2.↵
    1. Williams JV,
    2. Wang CK,
    3. Yang CF,
    4. et al
    . The role of human metapneumovirus in upper respiratory tract infections in children: a 20-year experience. J Infect Dis 2006;193:387-95.
    Abstract/FREE Full Text
  3. 3.↵
    1. van den Hoogen BG,
    2. Osterhaus DM,
    3. Fouchier RA
    . Clinical impact and diagnosis of human metapneumovirus infection. Pediatr Infect Dis J 2004;23:S25-32.
    MedlineWeb of Science
  4. 4.↵
    1. van den Hoogen BG,
    2. van Doornum GJ,
    3. Fockens JC,
    4. et al
    . Prevalence and clinical symptoms of human metapneumovirus infection in hospitalized patients. J Infect Dis 2003;188:1571-7.
    Abstract/FREE Full Text
  5. 5.↵
    1. Alvarez R,
    2. Harrod KS,
    3. Shieh WJ,
    4. Zaki S,
    5. Tripp RA
    . Human metapneumovirus persists in BALB/c mice despite the presence of neutralizing antibodies. J Virol 2004;78:14003-11.
    Abstract/FREE Full Text
  6. 6.
    1. Hamelin ME,
    2. Yim K,
    3. Kuhn KH,
    4. et al
    . Pathogenesis of human metapneumovirus lung infection in BALB/c mice and cotton rats. J Virol 2005;79:8894-903.
    Abstract/FREE Full Text
  7. 7.↵
    1. Macphail M,
    2. Schickli JH,
    3. Tang RS,
    4. et al
    . Identification of small-animal and primate models for evaluation of vaccine candidates for human metapneumovirus (hMPV) and implications for hMPV vaccine design. J Gen Virol 2004;85:1655-63.
    Abstract/FREE Full Text
  8. 8.↵
    1. Kuiken T,
    2. van den Hoogen BG,
    3. van Riel DA,
    4. et al
    . Experimental human metapneumovirus infection of cynomolgus macaques (Macaca fascicularis) results in virus replication in ciliated epithelial cells and pneumocytes with associated lesions throughout the respiratory tract. Am J Pathol 2004;164:1893-900.
    MedlineWeb of Science
  9. 9.↵
    1. Ebihara T,
    2. Endo R,
    3. Kikuta H,
    4. Ishiguro N,
    5. Ishiko H,
    6. Kobayashi K
    . Comparison of the seroprevalence of human metapneumovirus and human respiratory syncytial virus. J Med Virol 2004;72:304-6.
    CrossRefMedlineWeb of Science
  10. 10.↵
    1. Baumeister EG,
    2. Hunicken DS,
    3. Savy VL
    . RSV molecular characterization and specific antibody response in young children with acute lower respiratory infection. J Clin Virol 2003;27:44-51.
    CrossRefMedlineWeb of Science
  11. 11.↵
    1. Ward KA,
    2. Lambden PR,
    3. Ogilvie MM,
    4. Watt PJ
    . Antibodies to respiratory syncytial virus polypeptides and their significance in human infection. J Gen Virol 1983;64:1867-76.
    Abstract/FREE Full Text
  12. 12.↵
    1. Tang RS,
    2. Mahmood K,
    3. Macphail M,
    4. et al
    . A host-range restricted parainfluenza virus type 3 (PIV3) expressing the human metapneumovirus (hMPV) fusion protein elicits protective immunity in African green monkeys. Vaccine 2005;23:1657-67.
    CrossRefMedlineWeb of Science
  13. 13.↵
    1. Hall CB,
    2. Powell KR,
    3. MacDonald NE,
    4. et al
    . Respiratory syncytial viral infection in children with compromised immune function. N Engl J Med 1986;315:77-81.
    MedlineWeb of Science
  14. 14.↵
    1. Herd KA,
    2. Mahalingam S,
    3. Mackay IM,
    4. Nissen M,
    5. Sloots TP,
    6. Tindle RW
    . Cytotoxic T-lymphocyte epitope vaccination protects against human metapneumovirus infection and disease in mice. J Virol 2006;80:2034-44.
    Abstract/FREE Full Text
  15. 15.↵
    1. Kuttler C,
    2. Nussbaum AK,
    3. Dick TP,
    4. Rammensee HG,
    5. Schild H,
    6. Hadeler KP
    . An algorithm for the prediction of proteasomal cleavages. J Mol Biol 2000;298:417-29.
    CrossRefMedlineWeb of Science
  16. 16.↵
    1. Rammensee H,
    2. Bachmann J,
    3. Emmerich NP,
    4. Bachor OA,
    5. Stevanovic S
    . SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics 1999;50:213-9.
    CrossRefMedlineWeb of Science
  17. 17.↵
    1. Singh H,
    2. Raghava GP
    . ProPred1: prediction of promiscuous MHC class-I binding sites. Bioinformatics 2003;19:1009-14.
    Abstract/FREE Full Text
  18. 18.↵
    1. Cherrie AH,
    2. Anderson K,
    3. Wertz GW,
    4. et al
    . Human cytotoxic T cells stimulated by antigen on dendritic cells recognize the N, SH, F, M, 22K, and 1b proteins of respiratory syncytial virus. J Virol 1992;66:2102-10.
    Abstract/FREE Full Text
  19. 19.↵
    1. Sette A,
    2. Sidney J
    . Nine major HLA class I supertypes account for the vast preponderance of HLA-A and -B polymorphism. Immunogenetics 1999;50:201-12.
    CrossRefMedline
  20. 20.↵
    1. Sette A,
    2. Sidney J
    . HLA supertypes and supermotifs: a functional perspective on HLA polymorphism. Curr Opin Immunol 1998;10:478-82.
    CrossRefMedlineWeb of Science
  21. 21.↵
    1. Mackay IM,
    2. Jacob KC,
    3. Woolhouse D,
    4. et al
    . Molecular assays for detection of human metapneumovirus. J Clin Microbiol 2003;41:100-5.
    Abstract/FREE Full Text
  22. 22.↵
    1. Elkington R,
    2. Walker S,
    3. Crough T,
    4. et al
    . Ex vivo profiling of CD8+ T-cell responses to human cytomegalovirus reveals broad and multispecific reactivities in healthy virus carriers. J Virol 2003;77:5226-40.
    Abstract/FREE Full Text
  23. 23.↵
    1. Rist M,
    2. Cooper L,
    3. Elkington R,
    4. et al
    . Ex vivo expansion of human cytomegalovirus-specific cytotoxicTcells by recombinant polyepitope: implications forHCMVimmunotherapy. Eur J Immunol 2005;35:996-1007.
    CrossRefMedlineWeb of Science
  24. 24.↵
    1. van den Hoogen BG,
    2. Bestebroer TM,
    3. Osterhaus AD,
    4. Fouchier RA
    . Analysis of the genomic sequence of a human metapneumovirus. Virology 2002;295:119-32.
    CrossRefMedlineWeb of Science
  25. 25.↵
    1. Miller SA,
    2. Tollefson S,
    3. Crowe JE Jr.,
    4. Williams JV,
    5. Wright DW
    . Examination of a fusogenic hexameric core from human metapneumovirus and identification of a potent synthetic peptide inhibitor from the heptad repeat 1 region. J Virol 2007;81:141-9.
    Abstract/FREE Full Text
  26. 26.↵
    1. Brandenburg AH,
    2. de Waal L,
    3. Timmerman HH,
    4. Hoogerhout P,
    5. de Swart RL,
    6. Osterhaus AD
    . HLA class I-restricted cytotoxic T-cell epitopes of the respiratory syncytial virus fusion protein. J Virol 2000;74:10240-4.
    Abstract/FREE Full Text
  27. 27.↵
    1. Goulder PJ,
    2. Lechner F,
    3. Klenerman P,
    4. McIntosh K,
    5. Walker BD
    . Characterization of a novel respiratory syncytial virus-specific human cytotoxic T-lymphocyte epitope. J Virol 2000;74:7694-7.
    Abstract/FREE Full Text
  28. 28.↵
    1. Rock MT,
    2. Crowe JE Jr.
    . Identification of a novel human leucocyte antigen-A*01-restricted cytotoxic T-lymphocyte epitope in the respiratory syncytial virus fusion protein. Immunology 2003;108:474-80.
    CrossRefMedlineWeb of Science
  29. 29.
    1. Venter M,
    2. Rock M,
    3. Puren AJ,
    4. Tiemessen CT,
    5. Crowe JE Jr.
    . Respiratory syncytial virus nucleoprotein-specific cytotoxic T-cell epitopes in a South African population of diverse HLA types are conserved in circulating field strains. J Virol 2003;77:7319-29.
    Abstract/FREE Full Text
  30. 30.↵
    1. Heidema J,
    2. de Bree GJ,
    3. de Graaff PM,
    4. et al
    . Human CD8+ T cell responses against five newly identified respiratory syncytial virus-derived epitopes. J Gen Virol 2004;85:2365-74.
    Abstract/FREE Full Text
  31. 31.↵
    1. Ghildyal R,
    2. Ho A,
    3. Jans DA
    . Central role of the respiratory syncytial virus matrix protein in infection. FEMS Microbiol Rev 2006;30:692-705.
    CrossRefMedlineWeb of Science
  32. 32.↵
    1. Chiba Y,
    2. Higashidate Y,
    3. Suga K,
    4. Honjo K,
    5. Tsutsumi H,
    6. Ogra PL
    . Development of cell-mediated cytotoxic immunity to respiratory syncytial virus in human infants following naturally acquired infection. J Med Virol 1989;28:133-9.
    MedlineWeb of Science
  33. 33.↵
    1. Duraiswamy J,
    2. Burrows JM,
    3. Bharadwaj M,
    4. et al
    . Ex vivo analysis of T-cell responses to Epstein-Barr virus-encoded oncogene latent membrane protein 1 reveals highly conserved epitope sequences in virus isolates from diverse geographic regions. J Virol 2003;77:7401-10.
    Abstract/FREE Full Text
  34. 34.↵
    1. Larcher C,
    2. Geltner C,
    3. Fischer H,
    4. Nachbaur D,
    5. Muller LC,
    6. Huemer HP
    . Human metapneumovirus infection in lung transplant recipients: clinical presentation and epidemiology. J Heart Lung Transplant 2005;24:1891-901.
    CrossRefMedlineWeb of Science
  35. 35.↵
    1. Debiaggi M,
    2. Canducci F,
    3. Sampaolo M,
    4. et al
    . Persistent symptomless human metapneumovirus infection in hematopoietic stem cell transplant recipients. J Infect Dis 2006;194:474-8.
    Abstract/FREE Full Text
  36. 36.↵
    1. Ebihara T,
    2. Endo R,
    3. Ishiguro N,
    4. Nakayama T,
    5. Sawada H,
    6. Kikuta H
    . Early reinfection with human metapneumovirus in an infant. J Clin Microbiol 2004;42:5944-6.
    Abstract/FREE Full Text
| Table of Contents

This Article

  1. J Infect Dis. (2008) 197 (4): 584-592. doi: 10.1086/526536
  1. AbstractFree
  2. » Full Text (HTML)Free
  3. Full Text (PDF)Free

Classifications

Share

  1. Email this article

Navigate This Article

Current Issue

  1. March 1, 2012 205 (5)
  1. Journal of Infectious Diseases
  1. Alert me to new issues

Most