In this paper, we describe dominant and subdominant H-2d-restricted CTL responses against hMPV, characterize the diversification of the anamnestic CTL response targeting multiple epitopes, and contextualize the role of CTLs against the previously described M2-256-64 region. Our data show that a dominant CTL response against hMPV is directed against amino acids 81 to 89 of the antitermination factor M2-1 and, in addition, identify a subdominant target in N307-315 during primary and secondary infection. Responses against both of these epitopes can decrease the hMPV load in the lungs. Other epitopes, including M2-256-64, are incorporated as targets during memory responses.
HMPV, like other respiratory viruses, causes repetitive infections throughout life (26
). Infection in infants and young children is often associated with bronchiolitis, while it causes upper respiratory illness or asthmatic exacerbations in older patients (14
). The diversification of the anamnestic CTL response provides a broad repertoire of virus-specific CD8+
T cells for viral clearance and protects the host against the emergence of escape mutants. A similar diversification has been described for RSV CTL epitopes in H-2b
), while the extent of the anamnestic H-2d
-restricted response after RSV infection, beyond dominant cytotoxicity against a region in RSV M2-1 and subdominant epitopes in RSV F (4
), has not been fully characterized. Other viruses, like influenza virus, present a shift in dominance in CTL epitopes between primary and secondary responses (7
). Differences in immunodominance during primary and secondary responses against influenza virus have been attributed to differential antigen presentation by dendritic and nondendritic cells (7
During infections with RSV, CTLs are important for virus clearance (3
) and appear to play a critical role in regulating the immune response through the secretion of factors, notably IFN-γ (11
). During hMPV infection, a role for T cells in the control of replication is supported by our findings and increased virus titers in mice after T-cell depletion (1
). Furthermore, immunization of H-2b
mice with hMPV peptides elicited IFN-γ production and cytotoxicity, and HLA A*0201 transgenic mice infected with hMPV had lower virus titers in the lungs when preimmunized with an hMPV-specific CTL peptide than with an irrelevant-peptide vaccine (10
). As for immunomodulation, a recent report described CTL-mediated enhancement of Th1 cytokines interleukin-12 and IFN-γ in lungs and regional lymph nodes during hMPV infection (10
). The implications of these observations for hMPV pathogenesis remain to be determined.
Despite the protective role described for CTL responses in hMPV, a recent study by Alvarez and Tripp found the primary CTL response against hMPV to be delayed (2
). We did not observe a delay in primary CTLs in our study, where vigorous activity against M2-181-89
was detected 7 days after i.p. infection and 10 days after i.n. inoculation. These differences may be explained, at least in part, by differences in predictope selection between studies (2
) and differences in design. Alternatively, 51
Cr assays may have suboptimal sensitivity when used with target cells infected with live virus (2
). Interestingly, our model, using a different inoculation strategy, conferred a minor subdominant role to the previously identified epitope in M2-256-64
Our study identified H-2d-restricted epitopes in six hMPV proteins. It is possible that additional epitopes are present in the remaining three viral proteins, L, SH, and P, or responses against other epitopes in the six selected proteins were not observed with the current peptide doses but would be detected at higher peptide concentrations. It is also theoretically possible that since epitope-specific CTL selection was based on IFN-γ production, cytotoxic cells producing other cytokines exist.
In summary, we describe a novel dominant and protective CTL epitope in hMPV M2-181-89 and subdominant epitopes in N and F. Our findings demonstrate that cytotoxicity against the virus diversifies extensively during anamnestic responses. Characterization of CD8+ T-cell immunity against hMPV will contribute to the development of vaccines to protect infants and children against this important agent.