Cytotoxic T-lymphocyte epitope vaccination protects against human metapneumovirus infection and disease in mice

Abstract

Human metapneumovirus (hMPV) has emerged as an important human respiratory pathogen causing upper and lower respiratory tract infections in young children and older adults. In addition, hMPV infection is associated with asthma exacerbation in young children. Recent epidemiological evidence indicates that hMPV may cocirculate with human respiratory syncytial virus (hRSV) and mediate clinical disease similar to that seen with hRSV. Therefore, a vaccine for hMPV is highly desirable. In the present study, we used predictive bioinformatics, peptide immunization, and functional T-cell assays to define hMPV cytotoxic T-lymphocyte (CTL) epitopes recognized by mouse T cells restricted through several major histocompatibility complex class I alleles, including HLA-A*0201. We demonstrate that peptide immunization with hMPV CTL epitopes reduces viral load and immunopathology in the lungs of hMPV-challenged mice and enhances the expression of Th1-type cytokines (gamma interferon and interleukin-12 [IL-12]) in lungs and regional lymph nodes. In addition, we show that levels of Th2-type cytokines (IL-10 and IL-4) are significantly lower in hMPV CTL epitope-vaccinated mice challenged with hMPV. These results demonstrate for the first time the efficacy of an hMPV CTL epitope vaccine in the control of hMPV infection in a murine model.

FIG. 1.

Peptide immunization of mice with predictopes of hMPV elicits CTL responses. C57BL/6J (H-2^b), BALB/c (H-2^d), or A2Kb (HLA-A*0201) mice were peptide immunized with predictopes from the N, M2-2, SH, or G proteins of hMPV as indicated. Control mice were immunized with well-defined epitopes from other viruses: either human papillomavirus (HPV E7/H-2^b) (18), respiratory syncytial virus (hRSV M2/H-2^d) (25), or influenza virus (FLU M/HLA-A*0201) (19). Immune status was evaluated 8 days later by T-cell assays (n = 3 mice per group). IFN-γ-secreting T cells were quantified by ELISPOT assay using spleen cells (A) ex vivo or (B) postrestimulation (post-restim) with peptide for 6 days. Spleen cells were incubated for 18 h with or without peptide as shown. Results are expressed as IFN-γ-positive cells/10⁶ spleen cells (ex vivo) or 10⁴ spleen cells (postrestimulation) (means ± standard errors). For controls, f and AI values, respectively, were 120/10⁶ and 45 for HPV, 33/10⁶ and 21 for hRSV, and 37/10⁶ and 10 for FLU (ex vivo) or 280/10⁴ and 43 for HPV, 28/10⁴ and 4 for hRSV, and 65/10⁴ and 17 for FLU (postrestimulation). Cytotoxic T cells were measured by a ⁵¹Cr release assay using spleen cells (C) postrestimulation with peptide for 6 days. Spleen cells were tested against target cells with or without peptide as shown. Results are expressed as percent cytotoxicities (means ± standard errors) (SEM was always <5%). Numbers represent peptide-specific cytotoxicities (psc) at an effector-to-target ratio of 50:1 (see Materials and Methods). For controls, psc values were 51% for HPV, 45% for hRSV, and 48% for FLU.

FIG. 2.

hMPV-directed CTL response is MHC-I-restricted and mediated by CD8⁺ cells. BALB/c (H-2^d), A2Kb (HLA-A*0201), or C57BL/6J (H-2^b) mice were immunized with peptide epitopes from the M2-2, SH, or N proteins of hMPV as indicated. Restimulated spleen cells were reacted (A) with H-2^b (EL4) and with H-2^d (P815) target cells, (B) with H-2^d and with HLA-A*0201 (EL4.A2) target cells, and (C) with H-2^d and with H-2^b target cells, with (closed symbols) or without (open symbols) peptide, as shown, in a ⁵¹Cr release assay. (C, third panel) Restimulated spleen cells were derived from mice which had been depleted of CD8⁺ cells. Results are expressed as percent cytotoxicities (means ± standard errors) (SEM was always <5%). Numbers represent peptide-specific cytotoxicities at an effector-to-target ratio of 50:1. (D) Flow cytometric analysis of CD8⁺ cells in peripheral blood of representative ¹⁶⁴VGALIFTKL¹⁷²-immunized mice depleted of CD8⁺ cells (right panel) or nondepleted (left panel).

FIG. 3.

Vaccination with hMPV CTL epitopes elicits CTL responses associated with a reduction of viral load in lungs of hMPV-challenged mice. A2bdF₁ (HLA-A*0201, H-2^b, H-2^d) mice were vaccinated with hMPV CTL epitopes (N/H-2^b and SH/HLA-A*0201). Control mice were vaccinated with irrelevant CTL epitopes from other viruses (human papillomavirus, HPV E7/H-2^b; influenza virus, FLU M/HLA-A*0201). Immune status was evaluated 8 days later by T-cell assays (n = 3 mice per group) or 16 days later by hMPV challenge. IFN-γ-secreting T cells were quantified by ELISPOT assay using spleen cells (A) ex vivo or (B) postrestimulation with peptide for 6 days. Spleen cells were incubated for 18 h with or without peptide as shown. Results are expressed as IFN-γ-positive cells/10⁶ spleen cells (ex vivo) or 10⁴ spleen cells (postrestimulation) (means ± standard errors). Significance was calculated using Student's t test (*, P < 0.05). (C) Cytotoxic T cells were measured by a ⁵¹Cr release assay using spleen cells postrestimulation with peptide for 6 days. Spleen cells were tested against target cells with or without peptide as shown. Results are expressed as percent cytotoxicities (means ± standard errors) (SEM was always <5%). Numbers represent peptide-specific cytotoxicities at an effector-to-target ratio of 50:1 (see Materials and Methods). (D) Viral load (n = 7 mice per group) was quantified by plaque assay using lungs at 4 days postchallenge with hMPV. Results are expressed as log₁₀ PFU/g lung (means ± standard errors). Significance was calculated using Student's t test (*, P < 0.05).

FIG. 4.

Vaccination with hMPV CTL epitopes upregulates expression of Th1-type cytokines in lungs and PBLNs of hMPV-challenged mice. A2bdF₁ mice were vaccinated with either hMPV CTL epitopes or irrelevant CTL epitopes and then evaluated by T-cell assays (day 8) or challenged with hMPV (day 16) (see Fig. 2). Chemokine/cytokine expression was evaluated at 4 days postchallenge (n = 4 mice). Expression in lungs was quantified at the mRNA level for (A) chemokines and (B) cytokines with (C) HPRT reference gene. Expression in PBLNs was quantified at the protein level for (D) cytokines. Data are presented as means of triplicate cultures ± standard errors. Significance was calculated using Student's t test (*, P < 0.05; comparison between hMPV and irrelevant vaccine groups).

FIG. 5.

Vaccination with hMPV CTL epitopes reduces histopathology in lungs of hMPV-challenged mice. A2bdF₁ mice were vaccinated with either hMPV CTL epitopes or irrelevant CTL epitopes and then evaluated by T-cell assays (day 8) or challenged with hMPV (day 16) (see Fig. 2). Histopathological changes in lungs at 4 days postchallenge were examined by hematoxylin and eosin staining. Representative sections are shown for mice that were (A) uninfected, (B) vaccinated with irrelevant CTL epitopes, or (C) vaccinated with hMPV CTL epitopes (magnification, ×10).

FIG. 6.

Identified CTL epitopes are present in type A (and some type B) hMPV strains according to protein sequence alignments. (A) N protein (amino acids 120 to 191 of 394) with similarity region (54) boxed. (B) M2-2 protein (amino acids 1 to 71 of 71). (C) G protein (amino acids 1 to 72 of 236) with transmembrane domain (54) boxed. (D) SH protein (amino acids 1 to 72 of 183) with transmembrane domain (54) boxed. Identified CTL epitope sequences are shaded. * indicates virus types used for epitope prediction; → indicates virus type used for challenge.