Acute Viral Respiratory Infection Rapidly Induces a CD8+ T Cell Exhaustion-like Phenotype.

Acute viral infections typically generate functional effector CD8(+) T cells (TCD8) that aid in pathogen clearance. However, during acute viral lower respiratory infection, lung TCD8 are functionally impaired and do not optimally control viral replication. T cells also become unresponsive to Ag during chronic infections and cancer via signaling by inhibitory receptors such as programmed cell death-1 (PD-1). PD-1 also contributes to TCD8 impairment during viral lower respiratory infection, but how it regulates TCD8 impairment and the connection between this state and T cell exhaustion during chronic infections are unknown. In this study, we show that PD-1 operates in a cell-intrinsic manner to impair lung TCD8. In light of this, we compared global gene expression profiles of impaired epitope-specific lung TCD8 to functional spleen TCD8 in the same human metapneumovirus-infected mice. These two populations differentially regulate hundreds of genes, including the upregulation of numerous inhibitory receptors by lung TCD8. We then compared the gene expression of TCD8 during human metapneumovirus infection to those in acute or chronic lymphocytic choriomeningitis virus infection. We find that the immunophenotype of lung TCD8 more closely resembles T cell exhaustion late into chronic infection than do functional effector T cells arising early in acute infection. Finally, we demonstrate that trafficking to the infected lung alone is insufficient for TCD8 impairment or inhibitory receptor upregulation, but that viral Ag-induced TCR signaling is also required. Our results indicate that viral Ag in infected lungs rapidly induces an exhaustion-like state in lung TCD8 characterized by progressive functional impairment and upregulation of numerous inhibitory receptors.

Lung CD8+ T Cell Impairment Occurs during Human Metapneumovirus Infection despite Virus-Like Particle Induction of Functional CD8+ T Cells.

UNLABELLED: Human metapneumovirus (HMPV) is a major cause of respiratory disease in infants, the elderly, and immunocompromised individuals worldwide. There is currently no licensed HMPV vaccine. Virus-like particles (VLPs) are an attractive vaccine candidate because they are noninfectious and elicit a neutralizing antibody response. However, studies show that serum neutralizing antibodies are insufficient for complete protection against reinfection and that adaptive T cell immunity is important for viral clearance. HMPV and other respiratory viruses induce lung CD8(+) T cell (TCD8) impairment, mediated by programmed death 1 (PD-1). In this study, we generated HMPV VLPs by expressing the fusion and matrix proteins in mammalian cells and tested whether VLP immunization induces functional HMPV-specific TCD8 responses in mice. C57BL/6 mice vaccinated twice with VLPs and subsequently challenged with HMPV were protected from lung viral replication for at least 20 weeks postimmunization. A single VLP dose elicited F- and M-specific lung TCD8s with higher function and lower expression of PD-1 and other inhibitory receptors than TCD8s from HMPV-infected mice. However, after HMPV challenge, lung TCD8s from VLP-vaccinated mice exhibited inhibitory receptor expression and functional impairment similar to those of mice experiencing secondary infection. HMPV challenge of VLP-immunized µMT mice also elicited a large percentage of impaired lung TCD8s, similar to mice experiencing secondary infection. Together, these results indicate that VLPs are a promising vaccine candidate but do not prevent lung TCD8 impairment upon HMPV challenge. IMPORTANCE: Human metapneumovirus (HMPV) is a leading cause of acute respiratory disease for which there is no licensed vaccine. Virus-like particles (VLPs) are an attractive vaccine candidate and induce antibodies, but T cell responses are less defined. Moreover, HMPV and other respiratory viruses induce lung CD8(+) T cell (TCD8) impairment mediated by programmed death 1 (PD-1). In this study, HMPV VLPs containing viral fusion and matrix proteins elicited epitope-specific TCD8s that were functional with low PD-1 expression. Two VLP doses conferred sterilizing immunity in C57BL/6 mice and facilitated HMPV clearance in antibody-deficient µMT mice without enhancing lung pathology. However, regardless of whether responding lung TCD8s had previously encountered HMPV antigens in the context of VLPs or virus, similar proportions were impaired and expressed comparable levels of PD-1 upon viral challenge. These results suggest that VLPs are a promising vaccine candidate but do not prevent lung TCD8 impairment upon HMPV challenge.

Acute clearance of human metapneumovirus occurs independently of natural killer cells.

Human metapneumovirus (HMPV) is a major cause of respiratory disease. The role of NK cells in protection against HMPV is unclear. We show that while HMPV-infected C57BL/6 mice had higher numbers of functional lung NK cells than mock-treated mice, comparing NK cell-depleted and control mice did not reveal differences in lung viral titers, histopathology, cytokine levels, or T cell numbers or function. These data indicate that NK cells are not required for host control of HMPV.

Novel cis-regulatory function in ICR-mediated imprinted repression of H19.

Expression of coregulated imprinted genes, H19 and Igf2, is monoallelic and parent-of-origin-dependent. Like most imprinted genes, H19 and Igf2 are regulated by a differentially methylated imprinting control region (ICR). CTCF binding sites and DNA methylation at the ICR have previously been identified as key cis-acting elements required for proper H19/Igf2 imprinting. Here, we use mouse models to elucidate further the mechanism of ICR-mediated gene regulation. We specifically address the question of whether sequences outside of CTCF sites at the ICR are required for paternal H19 repression. To this end, we generated two types of mutant ICRs in the mouse: (i) deletion of intervening sequence between CTCF sites (H19(ICR∆IVS)), which changes size and CpG content at the ICR; and (ii) CpG depletion outside of CTCF sites (H19(ICR-8nrCG)), which only changes CpG content at the ICR. Individually, both mutant alleles (H19(ICR∆IVS) and H19(ICR-8nrCG)) show loss of imprinted repression of paternal H19. Interestingly, this loss of repression does not coincide with a detectable change in methylation at the H19 ICR or promoter. Thus, neither intact CTCF sites nor hypermethylation at the ICR is sufficient for maintaining the fully repressed state of the paternal H19 allele. Our findings demonstrate, for the first time in vivo, that sequence outside of CTCF sites at the ICR is required in cis for ICR-mediated imprinted repression at the H19/Igf2 locus. In addition, these results strongly implicate a novel role of ICR size and CpG density in paternal H19 repression.

Human metapneumovirus small hydrophobic (SH) protein downregulates type I IFN pathway signaling by affecting STAT1 expression and phosphorylation.

Type I interferon (IFN) is a key mediator of antiviral immunity. Human metapneumovirus (HMPV) inhibits IFN signaling, but does not encode homologues of known IFN antagonists. We tested the hypothesis that a specific viral protein prevents type I IFN signaling by targeting signal transducer and activator of transcription-1 (STAT1). We found that human airway epithelial cells (capable of expressing IFNs) became impaired for STAT1 phosphorylation even without direct infection due to intrinsic negative feedback. HMPV-infected Vero cells (incapable of expressing IFN) displayed lower STAT1 expression and impaired STAT1 phosphorylation in response to type I IFN treatment compared to mock-infected cells. Transient overexpression of HMPV small hydrophobic (SH) protein significantly inhibited STAT1 phosphorylation and signaling, and recombinant virus lacking SH protein was unable to inhibit STAT1 phosphorylation. Our results indicate a role for the SH protein of HMPV in the downregulation of type I IFN signaling through the targeting of STAT1.

New Approaches for Immunization and Therapy against Human Metapneumovirus.

Human metapneumovirus (HMPV) is a paramyxovirus discovered in 2001 in the Netherlands. Studies have identified HMPV as an important causative agent of acute respiratory disease in infants, the elderly, and immunocompromised individuals. Clinical signs of infection range from mild upper respiratory illness to more serious lower respiratory illness, including bronchiolitis and pneumonia. There are currently no licensed therapeutics or vaccines against HMPV. However, several research groups have tested vaccine candidates and monoclonal antibodies in various animal models. Several of these approaches have shown promise in animal models. This minireview summarizes the current therapies used to treat HMPV infection as well as different approaches for immunization.