Correlation of COVID-19 Severity and Immunoglobulin Presence Against Spike and Nucleocapsid Proteins in SARS-CoV-2

Abstract

Data on the human immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins have been applied to vaccine development and diagnosing coronavirus disease 2019 (COVID-19), but little research has been done on the relationship between the human immune response and COVID-19 severity. We herein sought to determine whether there is a correlation between the immunoglobulin level and COVID-19 severity. Clinical samples were collected from 102 patients with COVID-19. Of these, 65 and 37 patients had mild and severe symptoms, respectively. An enzyme-linked immunosorbent assay using the recombinant SARS-CoV-2 nucleocapsid (N) protein, spike (S) protein, and synthetic peptides covering N and S as antigens was performed to measure the IgM and IgG levels. The correlation between the immunoglobulin level and COVID-19 severity was then analyzed. A significant difference in the level of IgG antibodies against N and of IgM antibodies against the receptor binding domain of the S protein was observed between patients with nonsevere and severe COVID-19 symptoms, and the level of IgG antibodies against N was found to be higher in patients with severe symptoms whereas the level of IgM antibodies against the S peptides was higher in patients with nonsevere symptoms. The level of specific antibodies against SARS-CoV-2 structural proteins might correlate with COVID-19 severity. If so, this fact may be useful for predicting the prognosis of the disease and in determining the appropriate treatment with greater precision.

Introduction

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive sense single-stranded RNA-enveloped virus belonging to the betacoronavirus genus and is the cause of the current coronavirus disease 2019 (COVID-19) pandemic (4). COVID-19 has a wide clinical presentation varying from asymptomaticity and mild symptoms to severe or critical symptoms requiring ventilation and extracorporeal membrane oxygenation (ECMO) (1,9,21). Patients with severe or critical COVID-19 symptoms often experience progressive respiratory failure and various complications, such as thromboembolic, cardiovascular, and neurological complications (3,12,22).

Although only 5% of patients become critically ill, these patients require prolonged intensive care, taxing the capacity of health care facilities to respond (15,21). Age is the most important risk factor of severe or critical illness along with underlying medical comorbidities, such as cardiovascular disease, diabetes mellitus, and immunosuppression (14,17). Although the risk factors associated with mortality in COVID-19 have been identified, challenges remain in precisely predicting the severity of the disease. Thus, discovery of biomarkers that can be used to predict severity may help optimize treatment of individual patients.

The structural proteins of SARS-CoV-2 consist of the spike (S) protein, envelope (E) protein, membrane (M) protein, and nucleocapsid (N) protein and its fragments. Antibodies against S proteins have been used to develop neutralizing antibodies and vaccines against SARS-CoV-1 (10,19). Furthermore, previous studies have used the S and N proteins in SARS-CoV-2 to develop a vaccine and therapy (6,18). In contrast, the correlation between the expression of antibodies to structural proteins and disease severity has not been studied in detail. The aim of this study was to investigate the relationship between the expression of antibodies against the S and N proteins in SARS-CoV-2 and disease severity using blood samples from patients with COVID-19.

Materials and Methods

Study design and patients

This study was conducted at Tokyo Metropolitan Tama Medical Center, a 789-bed public tertiary care center in Tokyo, Japan. The study institution has been admitting patients with COVID-19 since February 2020 and accepting referrals of critically ill patients from other acute care hospitals. All patients with COVID-19 diagnosed by a polymerase chain reaction (PCR) test of a nasopharyngeal swab, saliva, or sputum sample between April 1 and December 31, 2020 were initially enrolled. Of these, patients with a serum sample obtained within 28 days of symptom onset were extracted. Patients whose serum sample was not able to be stored or was obtained after 29 days from onset and patients aged <16 years were excluded.

Definition

The definition of COVID-19 severity was based on the guidelines of the Infectious Diseases Society of America. Severe illness was defined as SpO2 ≤ 94% on room air, including the requirement for supplemental oxygen. Critical illness was defined as the requirement for mechanical ventilation and ECMO and end organ damage as seen in sepsis/septic shock. All other cases were defined as mild (2). After 102 serum samples obtained from patients with COVID-19 were tested with enzyme-linked immunosorbent assays (ELISA) using the SARS-CoV-2 recombinant N and S proteins and peptides of N and S protein antigens, samples with a titer four times higher than the average titer of 10 specimens obtained from healthy (PCR-negative) volunteers were extracted for further analysis.

Clinical variables of interest and data collection

A list of potentially eligible patients and their basic demographic data were obtained from the hospital administration. After excluding patients meeting the exclusion criteria, characteristics of the patients, including comorbidity and clinical and laboratory data, were collected during hospitalization. Oxygen requirement at the time of admission and at the most critical episode during hospitalization was assessed. Outcome data on COVID-19 cases, including the all-cause mortality rate during the hospitalization and the patients' hospital discharge status, were also collected.

Recombinant proteins and peptides in SARS-CoV-2

The recombinant SARS-CoV-2 Spike protein (S), corresponding to aa 177–512, 288–512, 348–578, 387–516, and 408–664, and the recombinant SARS-CoV-2 nucleocapsid protein (N), corresponding to aa 1–120, 111–220, 1–220, 210–419, and 1–419, were prepared as described (13). Synthetic peptides covering the S and N proteins were synthesized from the receptor binding domain (RBD) of the spike protein and full-length N protein. Nine peptides covering the S protein, corresponding to aa 382–401, 397–416, 412–431, 427–446, 442–461, 457–476, 472–491, 487–506, and 502–520, and 22 peptides covering the N protein, corresponding to aa 1–20, 16.35, 31–50, 46–65, 61–80, 76–95, 91–110, 106–125, 111–130, 126–145, 141–160, 156–175, 171–190, 186–205, 201–220, 211–230, 226–245, 241–260, 256–275, 271–290, 286–305, 301–320, 316–335, 331–350, 346–365, 361–380, 376–395, 391–410, and 400–419, were synthesized by Eurofins Genetics, Inc. (Japan) (13).

Direct ELISA

Direct ELISA using the recombinant protein and peptides of the N and S proteins as coating antigens was performed to determine whether IgG antibodies were present in the sera obtained from patients with COVID-19. The Direct ELISA was conducted according to the manufacturer's protocols (Thermo) (7). Antigens are directly attached to the plate by passive adsorption, usually using a carbonate/bicarbonate buffer at pH9.0. Our previous study showed that N and S proteins bound tightly to the polystyrene surface of microplates in the alkaline conditions without loss of any antigenicity of these proteins (13).

Triplicate 100 μL aliquots, each containing either the recombinant protein or the peptides in 0.05 M carbonate buffer (pH9.0), were placed on 96-well plates, incubated for 1 h at room temperature, then washed three times with PBS containing 0.1% Tween 20 between each of the following steps: blocking the wells with SuperBlock™ Blocking Buffer in PBS (Thermo Scientific) (150 μL/well) for 1 h at room temperature; adding serum 100 μL/well (1:10,000 dilution in PBS containing 0.1% Tween 20 for IgG detection) (1:100 dilution in PBS containing 0.1% Tween 20 for IgM detection) for 1 h at room temperature; adding 100 μL/well of goat anti-human IgG(H;L)-HRP (1:10,000 dilution) (SouthernBiotech) or goat F(ab′)₂ anti-human IgM-HRP (1:10,000 dilution) (SouthernBiotech) for 30 min at room temperature; and adding 50 μL TMB Peroxidase ELA Substrate Kit (BIO-RAD) for 30 min.

After 30 min incubation, 50 μL 1 M sulfuric acid was added to stop the peroxidase reaction. Absorbance at 450 and 620 nm was measured as a reference using Infinite F50 microplate reader (TECAN, Switzerland).

Statistical analysis

Categorical variables were compared using the χ ² test or Fisher's exact test between mild and severe/critical illness. All tests for significance were two-tailed, and p < 0.05 indicated statistical significance. The institutional review board at Tokyo Metropolitan Tama Medical Center approved the study. The requirement for the patients' informed consent was waived because doing so would not adversely affect the patients' rights or welfare. All analyses were performed using Stata version 15 (StataCorp, College Station, TX).

Results

Clinical characteristics of patients with COVID-19

During the study period, 356 patients were hospitalized for COVID-19. Among these, serum samples of 117 patients were able to be preserved. Fifteen patients, whose serum sample was received after 29 days of disease onset, were excluded, leaving 102 patients for the final enrollment. Table 1 shows the demographic characteristics and clinical outcomes of these patients. Their median age was 60 years (range: 18–94 years), 59.8% (61/102) were male, and their median body mass index was 24.6 (range: 15.0–48.9). Common underlying diseases included hypertension (34.3%) and diabetes mellitus (24.5%). Among patients with severe (n = 34) and critical illness (n = 3), four (4/102, 3.9%) were intubated, and two (2/102, 1.9%) received ECMO. Eight patients (8/102, 7.8%) died during hospitalization after COVID-19 was diagnosed, and 94 patients survived.

Table 1.

Demographic Characteristics of the COVID-19 Patients

Characteristics	Overall (N = 102)	Mild illness (N = 65)	Severe and critical illness (N = 37)
Age, years, median (range)	60 (16–94)	55 (16–94)	65 (32–87)
Male	61 (59.8)	34 (52.3)	27 (73.0)
Japanese race	93 (91.2)	60 (92.3)	33 (89.2)
Body mass index, median (range)	24.6 (15–48.9)	24.2 (15–34)	24.2 (18.7–48.9)
Comorbidities/past medical history
Congestive heart failure	2 (2.0)	1 (1.5)	1 (2.7)
History of myocardial infarction	7 (6.9)	5 (7.7)	2 (5.4)
History of asthma	9 (8.8)	7 (10.8)	2 (5.4)
Chronic lung disease	4 (3.9)	1 (1.5)	3 (8.1)
Active malignancy	8 (7.8)	4 (6.2)	4 (10.8)
Diabetes mellitus	25 (24.5)	13 (20.0)	12 (32.4)
Hypertension	35 (34.3)	18 (27.7)	17 (46.0)
Peptic ulcer disease	2 (2.0)	1 (1.5)	1 (2.7)
Cerebrovascular disease	5 (4.9)	1 (1.5)	4 (10.8)
Chronic liver disease	1 (1.0)	0 (0)	1 (2.7)
Chronic kidney disease	8 (7.8)	6 (9.2)	2 (5.4)
With renal replacement	2 (2.0)	2 (3.1)	0 (0)
Without renal replacement	6 (5.9)	4 (6.1)	2 (5.4)
Connective tissue disease	1 (1.0)	1 (1.5)	0 (0)
Steroid use before COVID-19 infection	1 (1.0)	1 (1.5)	0 (0)
Obesity by physician confirmed	2 (2.0)	1 (1.5)	1 (2.7)
Dementia	5 (4.9)	4 (6.2)	1 (2.7)
Died during hospitalization	8 (7.8)	0 (0)	8 (21.6)

Data are presented as n (%) unless otherwise specified.

COVID-19, coronavirus disease 2019.

Recombinant protein and peptides in SARS-CoV-2

Table 2 and Supplementary Table S1 showed the number of samples positive for IgM/IgG against recombinant N/S protein and IgM/IgG antibodies against the peptides of N/S. The IgG level against recombinant N protein differed significantly between patients with nonsevere symptoms and those with severe or critical symptoms. The IgG antibody level against the N protein (1–220, 11–220, 210–419, and 1–419) was also higher in patients with severe or critical symptoms than in those with nonsevere symptoms. The level of IgM antibodies against synthetic peptides covering the S protein (aa 397–416, 412–431, 427–446, 457–476, and 487–506) was higher in patients with nonsevere symptoms than in those with severe symptoms.

Table 2.

Number of Samples with Positivity for IgG Against Recombinant N Protein and IgM Antibodies Against the Peptides of S Protein (N = 102)

	Mild illness (N = 65)	Severe and critical illness (N = 37)	p
IgG against recombinant SARS-CoV-2 N protein fragment
1–120 aa	1 (1.5)	1 (2.7)	1.00
111–120 aa	2 (3.1)	9 (24.3)	0.002
1–220 aa	7 (10.8)	17 (45.9)	<0.001
210–419 aa	5 (7.7)	11 (29.7)	0.009
1–419 aa	10 (15.4)	19 (51.4)	<0.001
IgM antibodies against the peptides of S fragment
382–401 aa	5 (7.7)	0	0.16
397–416 aa	8 (12.3)	0	0.03
412–431 aa	8 (12.3)	0	0.03
427–446 aa	8 (12.3)	0	0.03
442–461 aa	6 (9.2)	0	0.08
457–476 aa	6 (9.2)	0	0.08
472–491 aa	6 (9.2)	0	0.08
487–506 aa	8 (12.3)	0	0.03
502–520 aa	3 (4.6)	3 (8.1)	0.67

Data are presented as n (%) unless otherwise specified.

SARS-CoV-2, severe acute respiratory syndrome coronavirus.

The level of IgM against recombinant N protein, of IgM/IgG against recombinant S protein, of IgG antibodies against the peptides of the S protein, and of IgM/IgG antibodies against the peptides of the N protein did not differ significantly by disease severity. Of 67 patients with nonsevere symptoms, 14 (20.9%) were positive for IgM against the S protein peptides (397–416, 412–431, 427–446, 442–461, 457–476, 472–491, or 487–506 aa), whereas all the patients with severe symptoms were negative. Among the patients with nonsevere symptoms, IgM against the S peptides (397–416, 412–431, 427–446, 457–476, 472–491, or 487–506 aa) was positive only in patients whose samples were collected on days 2–7 from symptom onset.

Discussion

This study investigated the relationship between the expression of antibodies against the S and N proteins in SARS-CoV-2 and COVID-19 severity. IgG against recombinant N protein and IgM antibodies against synthetic peptides covering the S protein showed a relationship with disease severity, indicating that these antibodies are worth considering as biomarkers for predicting the severity of COVID-19.

This study demonstrated that the level of IgM antibodies against the RBD of the S protein was higher in patients with nonsevere symptoms than in those with severe and critical symptoms. The S protein occurs on the surface of SARS-CoV-2. The RBD of the S protein mediates the interaction with angiotensin-converting enzyme 2 when attaching to the host cell. The S protein is reported highly immunogenic (20), and patients with COVID-19 had an early response to the S protein antigen than to the N protein antigen (11). A previous study demonstrated that the titer of IgG and IgA against the S protein was higher in survivors than nonsurvivors (8).

Although the S-IgG level did not differ between the severe and nonsevere groups in our study, the elevated S protein-IgM titer in the nonsurvivor group might have provoked an immune response and induced the production of neutralizing antibodies, suggesting that the presence of an early immune response might have a beneficial impact on the treatment of patients with COVID-19. Patients with positivity for IgM against the S protein peptides (412–431, 427–446, 472–491, and/or 487–506 aa) within 7 days after symptom onset are unlikely to experience deteriorating symptoms (Supplementary Table S2). Crystallography revealed that the SARS-CoV-2 RBD interfaced with human ACE2 through a strong bridge between R426 on the side loop in the RBM and E329 from the human ACE2. F486 contributed to enhancing hACE2 recognition and may have facilitated the bat-to-human transmission of SARS-CoV-2 (16).

Although no specific region of the antibody against the N protein was shown to be predominantly associated with the severity of COVID-19, the level of IgG antibody against the N protein was elevated in patients with severe and critical illness. The N protein consists of 419 amino acids that regulate viral RNA replication, transcription, and synthesis, and is thought to play an important role in localization and interaction with the host cell nucleolus (6). The severity of COVID-19 is reportedly due to the cytokine storm rather than the virus itself, and the N protein might be involved in the pathway for TNF-α production, which triggers the cytokine storm (5).

This study has some limitations. First, only one-third of the patients hospitalized with COVID-19 were able to be investigated, and nonhospitalized patients with mild symptoms were not included. This limitation may have led to a selection bias. Second, the antibody titer might have been affected by the timing of the serum sample collection because the duration from symptom onset to serum collection differed in each patient. Third, antibodies against parts of the S protein outside the RBD region were not investigated.

In summary, the level of immunoglobulin against the SARS-CoV-2 structural proteins and severity of the disease may correlate in patients with COVID-19. This finding may be useful for predicting the prognosis, which might enable early intervention and improvements in the outcomes of patients with COVID-19.

Footnotes

Authors' Contributions

Y.T., S.O., and T.K. designed the study protocol. A.T. and Y.T. collected the patient data. S.O. and N.M. conducted ELISA assay. A.K., Y.T., and S.O. performed the data analysis and drafted the first version of the article. A.K. and Y.T. revised the article, and all the authors contributed to the final version of article.

Acknowledgment

We are indebted to James R. Valera for his assistance in editing the article.

Author Disclosure Statement

All the authors declare no conflicts of interest and have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest.

Funding Information

This research was supported by AMED under Grant No. JP20he0622015.

Supplementary Material

Supplementary Table S1

Supplementary Table S2

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