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. 2024 Jan 30;9(1):e0057023.
doi: 10.1128/msphere.00570-23. Epub 2024 Jan 10.

Clinical human metapneumovirus isolates show distinct pathogenesis and inflammatory profiles but similar CD8+ T cell impairment

Jorna Sojati  1   2 , Yu Zhang  1 , John V Williams  1   3   4
Affiliations

Clinical human metapneumovirus isolates show distinct pathogenesis and inflammatory profiles but similar CD8+ T cell impairment

Jorna Sojati et al. mSphere. .

Abstract

Human metapneumovirus (HMPV) is a negative-sense single-stranded RNA virus in the Pneumoviridae family and a leading cause of acute upper and lower respiratory infections, particularly in children, immunocompromised patients, and the elderly. Although nearly every person is infected with HMPV during early childhood, re-infections occur often, highlighting difficulty in building long-term immunity. Inflammatory responses, including PD-1-mediated impairment of virus-specific CD8+ T cells (TCD8), contribute to HMPV disease severity. HMPV strains are divided into four lineages: A1, A2, B1, and B2. However, little is known about immune responses to different viral subtypes. Here, we characterize responses to four HMPV clinical isolates-TN/94-344 (A1), TN/94-49 (A2), C2-202 (B1), and TN/96-35 (B2)-in vivo in C57BL/6 (B6) mice. TN/94-49 was avirulent, while TN/94-344, C2-202, and TN/96-35 showed varying degrees of weight loss and clinical disease. Differences in disease did not correlate to virus burden in upper or lower tracts. TN/94-49 HMPV exhibited highest nose titers and delayed lung clearance. Cytokine profiles differed between HMPV isolates, with TN/96-35 inducing the broadest lung inflammatory cytokines. TN/96-35 also showed lower HMPV burden and less weight loss than other virulent isolates, suggesting a more efficient antiviral response. Interestingly, disease correlated with higher expression of T-cell chemoattractant CXCL9. All isolates elicited PD-1 upregulation and decreased IFNγ and CD107a expression in virus-specific TCD8, with little difference between HMPV subtypes. This work uncovers previously uncharacterized variations in immune responses to clinical HMPV isolates of different lineages.IMPORTANCEThis study extensively explored differences in T-cell-mediated immunity between human metapneumovirus (HMPV) clinical isolates. Much existing HMPV research has been done with strains passaged extensively in cell lines, likely acquiring mutations advantageous to in vitro replication. Clinical isolates are collected directly from human patients and have undergone <10 passages, serving as more physiologically relevant models of HMPV infection. Additionally, existing animal studies of HMPV disease mainly focus on lung pathogenesis, while HMPV infects both upper and lower airways of humans. This work highlights distinct differences in HMPV burden in upper and lower tracts between clinical isolates. Lastly, this study uniquely explores differences in host immunity between all four HMPV genetic lineages. The predominant HMPV subtype in circulation varies seasonally; thus, understanding host responses to all subgroups is critical for developing effective HMPV vaccines.

Keywords: airway immunity; human metapneumovirus; respiratory infection.

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Conflict of interest statement

J.V.W. serves on the Scientific Advisory Board of Quidel and an Independent Data Monitoring Committee for GlaxoSmithKline, neither activity involved with the work under consideration. All other authors declare no conflicts of interest.

Figures

Fig 1
Fig 1
HMPV isolates vary in pathogenesis, airway burden, and lung cytokine profiles. (A) Body weight (% on day 0) of C57BL/6 (B6) mice infected with 5E5 plaque-forming units (PFU) or mock cell lysate of each HMPV strain. Two-way ANOVA, #P < 0.001 for TN/94-49 vs. all other clinical isolates, **P < 0.01 for TN/94-344 vs. TN/96-35. (B) Clinical disease score for mice described for panel A. One point is given for each of the following criteria: hunching, huddling, fur ruffling, rapid breathing, and lethargy. Two-way ANOVA, #P < 0.001 for TN/94-49 vs. all other clinical isolates. ****P < 0.0001 for TN/94-344 vs. all other clinical isolates. (C and D) Lung (C) and nasal turbinate (D) HMPV titers by plaque assay (plaque-forming units per gram) on day 7 post-infection of mice described for panel A. LOD noted by red dashed line. (E and F) Lung (E) and nasal turbinate (F) HMPV viral RNA detection by RT-qPCR on day 7 post-infection of mice described for panel A. Data are normalized to Hprt housekeeping gene and null condition of mock-infected mouse tissue by the 2−∆∆Ct method. One-way ANOVA for (C-F), *P < 0.05, **P < 0.01, ****P < 0.0001. (G) Heat map representing protein expression levels (ng/mL) of inflammatory cytokines in lung homogenates collected on day 7 post-infection from mice described for panel A.
Fig 2
Fig 2
HMPV isolates induce dysfunctional TCD8 with high PD-1 expression and reduced effector function. (A) Frequency of total CD3+ lymphocytes (% of total live cells) in lungs of mice infected with 5E5 PFU HMPV and euthanized day 7 post-infection. (B–D) Frequency of total lung CD8+ lymphocytes (% of total CD3) (B), virus-specific (M94+) TCD8 lymphocytes (% of total CD8) (C), and PD-1+ HMPV-specific TCD8 (% of total M94+ TCD8) (D) in lungs of mice described for panel A. (E and F) Frequency of lung TCD8 expressing CD107a (E) or IFNγ (F) after 5 hours of ex vivo stimulation with HMPV M94 peptide. One-way ANOVA for panels A–F, *P < 0.05, *P < 0.05, **P < 0.01, ****P < 0.0001. (G) Representation of functionality for M94-responsive lung TCD8 cells showing % M94+ cells and % CD107a/IFNγ production in response to M94 peptide stimulation. Two-way ANOVA, **P < 0.01, ****P < 0.0001.
Fig 3
Fig 3
Characterization of virus-specific TCD8 impairment during HMPV infection. (A) Gating strategy for tetramer+, PD-1+, IFNγ+, and CD107a+ TCD8 cells. (B) Functionality plots for CD107a and IFNγ in M94-specific cells, plotted as % CD107a+ or IFNγ+ on M94 stimulation divided by % M94+ cells by tetramer staining, for mice described in Fig. 2A. (C) Positive control of lung TCD8 stimulation using PMA/ionomycin for mice described in Fig. 2A.
Fig 4
Fig 4
Virus-specific TCD8 are minimally detected in spleen tissue. (A–D) Frequency of total CD3+ lymphocytes (% of total live cells) (A), total lung CD8+ lymphocytes (% of total CD3) (B), virus-specific (M94+) TCD8 lymphocytes (% of total CD8) (C), and PD-1+ HMPV-specific TCD8 (% of total M94+ TCD8) (D) in spleens of mice described in Fig. 2A. (E and F) Frequency of spleen TCD8 expressing CD107a (E) or IFNγ (F) after 5 hours of ex vivo stimulation with HMPV M94 peptide. (G) Functionality of M94-responsive spleen TCD8 cells showing % M94+ cells and % CD107a/IFNγ production in response to M94 peptide stimulation. (H) Positive control of spleen TCD8 stimulation using PMA/ionomycin.
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