Recovery from the Middle East respiratory syndrome is associated with antibody and T-cell responses

Abstract

The Middle East respiratory syndrome coronavirus (MERS-CoV) causes a highly lethal pneumonia. MERS was recently identified as a candidate for vaccine development, but most efforts focus on antibody responses, which are often transient after CoV infections. CoV-specific T cells are generally long-lived, but the virus-specific T cell response has not been addressed in MERS patients. We obtained peripheral blood mononuclear cells and/or sera from 21 MERS survivors. We detected MERS-CoV-specific CD4⁺ and CD8⁺ T cell responses in all MERS survivors and demonstrated functionality by measuring cytokine expression after peptide stimulation. Neutralizing (PRNT₅₀) antibody titers measured in vitro predicted serum protective ability in infected mice and correlated with CD4⁺ but not CD8⁺ T cell responses; patients with higher PRNT₅₀ and CD4⁺ T cell responses had longer intensive care unit stays and prolonged virus shedding and required ventilation. Survivors with undetectable MERS-CoV-specific antibody responses mounted CD8⁺ T cell responses comparable with those of the whole cohort. There were no correlations between age, disease severity, comorbidities, and virus-specific CD8⁺ T cell responses. In conclusion, measurements of MERS-CoV-specific T cell responses may be useful for predicting prognosis, monitoring vaccine efficacy, and identifying MERS patients with mild disease in epidemiological studies and will complement virus-specific antibody measurements.

Figure 1. Convalescent sera transfer protects mice from MERS-CoV infection

(A) Mice received 75 µl of patient serum intravenously (i.v.) 12 hours before MERS-CoV infection. One hour prior to infection, mice sera were collected and PRNT₅₀ assays were performed as described in Procedures. (B) Relationship between PRNT₅₀ in human sera and in mouse recipients of transferred sera. (C) To obtain virus titers, lungs were homogenized at day 3 p.i. and titered on Vero 81 cells. Titers are expressed as PFU/g tissue. n= 3 mice/group/time point. LOD-limit of detection (Left). Relationship between PRNT₅₀ in mouse sera and viral titers in mouse lungs (Right).

Figure 2. Virus-specific T cell responses are detected in all MERS survivors

PBMCs from healthy donors and MERS patients were stimulated with MERS-CoV structural protein-specific peptide pools for 12 hours in the presence of brefeldin A. Frequencies of MERS-CoV-specific CD4 (A, B) and CD8 (C, D) T cells (determined by IFN-γ intracellular staining) are shown. (E) Summary of total T cell responses against all four peptide pools is shown. (F) Relationship between T cell and neutralizing antibody responses is shown.

Figure 3. Human PBMC-derived MERS-CoV-specific T cells are highly functional

(A, C) PBMCs were stimulated with MERS-CoV structural protein-specific peptide pools. Frequency and percentage of cells expressing IFN-γ and TNF are shown. (B, D) PBMCs were stimulated with the N (B) or ME (D) peptide pools. CD4 (B) or CD8 (D) T cells were then analyzed for the indicated phenotypic markers.

Figure 4. Identification of MERS-CoV-specific T cell epitopes in mice and patients

(A) Single cell suspensions were prepared from the lungs of MERS-CoV infected DR2 and DR3 transgenic mice, and stimulated with peptides for 5–6 hours in the presence of brefeldin A. (B, C, D) DR2 or DR3-restricted patient PBMCs were stimulated with peptide pools or individual peptides for 12 hours in the presence of brefeldin A. (E) Patient PBMCs were stimulated with the ME peptide pool or individual peptides for 12 hours in the presence of brefeldin A. Frequencies of MERS-CoV specific T cells (determined by IFN-γ intracellular staining) are shown.

Figure 5. Relationship between MERS-CoV-specific T cell and neutralizing antibody responses and disease variables and severity

(A) Relationship between T cell and PRNT₅₀ responses and time p.i. when samples were obtained. (B, C) Relationship between T cell (B) and PRNT₅₀ (C) responses and co-morbidity (Co-Morbidity vs None), ventilator status, sex and age. (D, E) Relationship between T cell and PRNT₅₀ responses and the duration of virus shedding (D) and length of ICU stay (E).