Sustained Responses of Neutralizing Antibodies Against Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in Recovered Patients and Their Therapeutic Applicability

Abstract

Background: Zoonotic coronaviruses have emerged as a global threat by causing fatal respiratory infections. Given the lack of specific antiviral therapies, application of human convalescent plasma retaining neutralizing activity could be a viable therapeutic option that can bridges this gap.

Methods: We traced antibody responses and memory B cells in peripheral blood collected from 70 recovered Middle East respiratory syndrome coronavirus (MERS-CoV) patients for 3 years after the 2015 outbreak in South Korea. We also used a mouse infection model to examine whether the neutralizing activity of collected sera could provide therapeutic benefit in vivo upon lethal MERS-CoV challenge.

Results: Anti-spike-specific IgG responses, including neutralizing activity and antibody-secreting memory B cells, persisted for up to 3 years, especially in MERS patients who suffered from severe pneumonia. Mean antibody titers gradually decreased annually by less than 2-fold. Levels of antibody responses were significantly correlated with fever duration, viral shedding periods, and maximum viral loads observed during infection periods. In a transgenic mice model challenged with lethal doses of MERS-CoV, a significant reduction in viral loads and enhanced survival was observed when therapeutically treated with human plasma retaining a high neutralizing titer (> 1/5000). However, this failed to reduce pulmonary pathogenesis, as revealed by pathological changes in lungs and initial weight loss.

Conclusions: High titers of neutralizing activity are required for suppressive effect on the viral replication but may not be sufficient to reduce inflammatory lesions upon fatal infection. Therefore, immune sera with high neutralizing activity must be carefully selected for plasma therapy of zoonotic coronavirus infection.

Keywords: MERS-CoV; neutralizing antibody; plasma therapy.

Figure 1.

Kinetic changes of IgG antibody responses against S1 antigen of MERS-CoV in 70 participants from 12 to 36 months after symptom onset. A, Collected sera were tested by a commercial ELISA kit. The assay was semi-quantitatively evaluated by calculating a ratio of the extinction value of the patient sample over the extinction value of the calibrator. Optical density (OD) ratios < 0.7 were considered negative, ratios > 1.4 (dashed line) were considered positive, and ratios ≥ 0.7 and ≤ 1.4 were considered as intermediate. B, Relative proportion of sera with negative, positive, and intermediate OD ratio values is presented in clinical severity groups (GI ~ GIII) at the indicated time points (G-I: n = 9 ~ 18, G-II: n = 20 ~ 33, and G-III: n = 12 ~ 18). Abbreviations: IgG, immunoglobulin G; MERS-CoV, Middle East respiratory syndrome coronavirus.

Figure 2.

Correlation of anti-S1 OD ratio with anti-spike IgG titer and neutralizing activity (ppNT₅₀ and PRNT₅₀) in sera from 50 patients. A, Correlation of OD ratio values against S1 antigen with the antibody titers against spike antigen and the neutralizing titers against the pseudotyped lentivirus (ppNT₅₀) or MERS-CoV (PRNT₅₀) were assessed (left panels). Nonlinear regression curves (exponential growth) and goodness of curve fit (r² value) are presented. B, Kinetic changes of anti-S IgG titers and neutralizing activity (ppNT₅₀ and PRNT₅₀) in sera samples are presented in clinical severity groups (GI ~ GIII). Box and whiskers (min to max) plots including median (black line) and mean (+) values of each plot are presented at the indicated time points (G-I: n = 11, G-II: n = 23, and G-III: n = 16). Abbreviations: IgG, immunoglobulin G; MERS-CoV, Middle East respiratory syndrome coronavirus; OD, optical density.

Figure 3.

Quantification of spike-specific memory B cells in peripheral blood mononuclear cells (PBMCs) taken at 12 and 36 months after infection in 36 subjects. A, Representative images of B cell ELISPOT results. B, Correlation of OD ratio values against S1 antigen with anti-S IgG-secreting B cell counts were assessed by linear regression (black line) and Spearman’s rank test (r_s and P value). PBMCs were taken at 12 and 36 months after infection from 36 subjects (G-I: n = 7, G-II: n = 16, and G-III: n = 13) and applied for analysis of spike antigen-specific IgG secreting memory B cells. C, Kinetic changes of anti-S IgG-secreting B cells in PBMCs are presented. Statistical analysis was performed using one-way ANOVA, followed by the Newman–Keuls t-test for comparisons of values among the severity groups at the indicated time points. Abbreviation: ASC, antibody-secreting cells; IgG, immunoglobulin G; OD, optical density. *, P < .05; **, P < .01.

Figure 4.

Correlation of antibody levels with fever duration, viremic period, and maximum viral loads during infection period. Correlations of antibody levels (anti-S IgG titer, ppNT₅₀, and PRNT₅₀ in sera collected at 1 year after infection) with the indicated parameters observed during infection periods in 50 subjects (G-I: n = 11, G-II: n = 23, and G-III: n = 16) were assessed by linear regression (black line) and Spearman’s rank test (r_s and P value). Abbreviations: IgG, immunoglobulin G; Max., maximum.

Figure 5.

Evaluation of therapeutic efficacy of pooled sera from recovered patients in hDPP4-Tg mice. A, hDPP4-Tg mice were challenged intranasally with MERS-CoV at 2500 PFU/mouse (5 × LD50) and then treated with pooled sera (100 µL/mouse) or therapeutic mAb/3B11 (20 µg in 100 µL of PBS/mouse) four times (1 hour and 1, 2, and 3 days postinfection). The antibody titers and neutralizing activity (PRNT₅₀) of pooled sera (negative, moderate, and high titer) are summarized in Table 2. Virus-challenged mice were monitored for 14 days to evaluate survival rate (left) and body weight changes (right). The body weight data are presented as means + SD of mice in each group (CNT: n = 5, moderate, high titer, and mAb/3B11: n = 8). Significant differences between the experimental group and control group (CNT) treated with non-immune sera are indicated (**, P < .01). B, MERS-CoV viral loads were assessed by measuring PFU (left) and copy numbers of viral RNA (right) in lung tissues collected at 4days after infection. Statistical significance between the experiment group and control group was tested by using a two-tailed Student’s t-test. Abbreviation: MERS-CoV, Middle East respiratory syndrome coronavirus. *, P < .05; **, P < .01.

Figure 6.

Pathological changes in lungs of hDD4-Tg mice infected with lethal dose of MERS-CoV. A and B, Lung tissue sections collected from mice at 4 days after infection were stained with hematoxylin and eosin. Pathological scores of infected lungs (n = 6/group) (bar graphs: mean + SD, A) and representative scanned images are presented (B). Bar, 100 μm. C, Correlation of histopathological scores with viral loads (copy numbers of viral RNAs) was assessed by linear regression (black line) and Spearman’s rank test (r_s and P value). Abbreviations: CNT, control group; MERS-CoV, Middle East respiratory syndrome coronavirus.