Middle East Respiratory Syndrome Coronavirus Infection Elicits Long-lasting Specific Antibody, T and B Cell Immune Responses in Recovered Individuals

Abstract

Background: The Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic zoonotic betacoronavirus and a global public health concern. Better undersetting of the immune responses to MERS-CoV is needed to characterize the correlates of protection and durability of the immunity and to aid in developing preventative and therapeutic interventions. Although MERS-CoV-specific circulating antibodies could persist for several years post-recovery, their waning raises concerns about their durability and role in protection. Nonetheless, memory B and T cells could provide long-lasting protective immunity despite the serum antibodies levels.

Methods: Serological and flow cytometric analysis of MERS-CoV-specific immune responses were performed on samples collected from a cohort of recovered individuals who required intensive care unit (ICU) admission as well as hospital or home isolation several years after infection to characterize the longevity and quality of humoral and cellular immune responses.

Results: Our data showed that MERS-CoV infection could elicit robust long-lasting virus-specific binding and neutralizing antibodies as well as T- and B-cell responses up to 6.9 years postinfection regardless of disease severity or need for ICU admission. Apart from the persistent high antibody titers, this response was characterized by B-cell subsets with antibody-independent functions as demonstrated by their ability to produce tumor necrosis factor α (TNF-α), interleukin (IL)-6, and interferon γ (IFN-γ) cytokines in response to antigen stimulation. Furthermore, virus-specific activation of memory CD8+ and CD4+ T cell subsets from MERS-recovered patients resulted in secretion of high levels of TNF-α, IL-17, and IFN-γ.

Conclusions: MERS-CoV infection could elicit robust long-lasting virus-specific humoral and cellular responses.

Keywords: MERS-CoV; T cells; antibodies; coronaviruses; immunity; longevity.

Figure 1.

Antibody response in MERS survivors. A, Serum samples from MERS recovered (R) individuals (n = 21) and healthy donors (H) (n = 10) were tested by ELISA for anti-N, -S1 and -RBD IgG and titers were determined as EC₅₀. Neutralizing antibodies (nAbs) in serum samples were determined using pseudovirus assay and titers were determined as IC₅₀. B–E, Binding antibody titers and nAbs are reported against time of sample collection post-recovery. Mean is shown in B–E panels for each group. Abbreviations: ELISA, enzyme-linked immunosorbent assay; IC₅₀, half maximal inhibitory concentration; IgG, immunoglobulin G; MERS, Middle East respiratory syndrome; N, nucleocapsid; RBD, receptor binding domain; S1, spike subunit 1.

Figure 2.

B-cell response in MERS survivors. PBMCs were isolated from MERS recovered individuals as well as healthy donors by lymphoprep density gradient centrifugation, and incubated with either (A) MERS-S1 conjugated to PE or (B–D) MERS-S1 protein and brefeldin A. A, Representative FACS plots of MERS-S1 specific non-class switched and memory B cells, and summary data showing the percentage of these specific B cell subsets in live cells in each cell subset from MERS recovered (R) individuals (n = 10) and healthy donors (H) (n = 4). Representative FACS plots of TNF-α, IL-6 and IFN-γ expression in stimulated and nonstimulated (B) memory B cells, (C) plasmablast B cells, and (D) plasma cells, and summary data showing TNF-α, IL-6, and IFN-γ expression in live cells in each B-cell subset from MERS recovered (R) individuals (n = 10) and healthy donors (H) (n = 4). Data are shown as mean ± SD for each group. Statistics were calculated by t test. *P < .05, **P < .005, ***P = .0005. Supplementary table shows the levels of these cell subsets from total lymphocytes population. Abbreviations: FACS, fluorescence-activated cell sorting; IFN-γ, interferon γ; IL-6, interleukin 6; MERS, Middle East respiratory syndrome; ns, not significant; PBMC, peripheral blood mononuclear cell; PE, phycoerythrin, SD, standard deviation; TNF-α, tumor necrosis factor α.

Figure 3.

Single-, double- and triple-cytokine producing memory B cells. Representative FACS plots of single-, double- and triple-cytokine–producing cells in stimulated and nonstimulated (A) memory B cells (CD19⁺CD38⁻CD27⁺CD24⁺), (C) plasmablast B cells (CD19⁺CD38⁺CD27⁺CD24⁻CD138⁻) and (E) plasma B cells (CD19⁺CD38⁺CD27⁺CD24⁻CD138⁺) from MERS recovered (R) individuals and healthy donors (H). Bar graphs represent percentage of summary data showing single-, double-, and triple-cytokine expression in B-cell subsets (B) memory B cells, (D) plasmablast B cells, and (F) plasma B cells in MERS recovered (R) individuals and healthy donors (H). Data in histograms are shown as percentages of induced cytokines from restimulated cells after subtracting levels produced by unstimulated cells from each individual. Data are shown as mean ± SD for each group. Statistics were calculated by t test, *P < .05. Abbreviations: FACS, fluorescence-activated cell sorting; IFN-γ, interferon γ; IL-6, interleukin 6; MERS, Middle East respiratory syndrome; SD, standard deviation; TNF-α, tumor necrosis factor α.

Figure 4.

Memory T-cell response in MERS recovered individuals. PBMCs were isolated from MERS recovered individuals as well as healthy donors by lymphoprep density gradient centrifugation, and ex vivo re-stimulated with MERS-CoV S overlapping peptide pool. A, Representative FACS plots of TNF-α, IL-17, and IFN-γ expression from stimulated and non-stimulated CD3⁺CD8⁺CCR7⁺CD45RA⁻ central memory, CD3⁺CD8⁺CCR7⁻CD45RA⁻ effector memory and CD3⁺CD8⁺CCD7⁻CD45RA⁺ TEMRA T cells from MERS recovered individuals and healthy donors. B, Summary data showing the expression of TNF-α, IL-17, and IFN-γ among live CD8⁺ memory T-cell subsets in MERS recovered (R) individuals (n = 10) and healthy donors (H) (n = 4). C, Representative FACS plots of TNF-α, IL-17 and IFN-γ expression from stimulated and non-stimulated CD3⁺CD4⁺CCR7⁺CD45RA⁻ central and CD3⁺CD4⁺CCR7⁻CD45RA⁻ effector memory T cells from MERS recovered individuals and healthy donors. D, Summary data showing the expression of TNF-α, IL-17, and IFN-γ in live CD4⁺ memory T-cell subsets in MERS recovered (R) individuals (n = 10) and healthy donors (H) (n = 4). Mean is shown for each group. Statistics were calculated by t test, *P < .05. Supplementary table shows the levels of these cell subsets from total lymphocytes population. Abbreviations: FACS, fluorescence-activated cell sorting; IFN-γ, interferon γ; IL-17, interleukin 17; MERS, Middle East respiratory syndrome; ns, not significant; SD, standard deviation; TNF-α, tumor necrosis factor α.

Figure 5.

Single-, double-, and triple-cytokine producing memory T cells. Bar graphs represent percentage of single-, double- and triple-cytokine–producing (A) central memory CD8⁺ T cells (CD3⁺CD8⁺CCR7⁺CD45RA⁻), (B) effector memory CD8⁺ T cells (CD3⁺CD8⁺CCR7⁻CD45RA⁻), (C) terminally differentiated effector CD8⁺ T cells (CD3⁺CD8⁺CCR7⁻CD45RA⁺), (D) central memory CD4⁺ T cells (CD3⁺CD4⁺CCR7⁺CD45RA⁻), and (E) effector memory CD4⁺ T cells (CD3⁺CD4⁺CCR7⁻CD45RA⁻). Data in histograms are shown as percentages of induced cytokines from re-stimulated cells after subtracting levels produced by unstimulated cells from each individual. Data are shown as mean ± SD for each group. Statistics were calculated by t test, *P < .05. Representative FACS plots of single-, double-, and triple-cytokine–producing cells in stimulated and nonstimulated from MERS recovered (R) individuals and healthy donors are shown in Supplementary Figure 3. Abbreviations: FACS, fluorescence-activated cell sorting; IFN-γ, interferon γ; IL-17, interleukin 17; MERS, Middle East respiratory syndrome; SD, standard deviation; TNF-α, tumor necrosis factor α.