Determination of immune response persistence after SARS-CoV-2 infection by ELISA analysis of nucleocapsid specific IgG

doi:10.21203/rs.3.rs-1238197/v1

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Determination of immune response persistence after SARS-CoV-2 infection by ELISA analysis of nucleocapsid specific IgG

https://doi.org/10.21203/rs.3.rs-1238197/v1

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Objectives

The severe acute respiratory syndrome coronavirus (SARS-CoV-2) causes Corona Virus Disease-2019 (COVID-19) and a global pandemic resulting in major changes in our daily habits and social behaviors. The mechanisms and the peculiarities of immune response against SARS-CoV-2 are not yet determined. To address this question, we initiated the study aimed to determine the persistence of immune response against SARS-CoV-2.

Results

Blood samples of the registered participants (49 registered participants - 35 females and 14 males; 17-55 years old age group) with clinically documented episodes of SARS-CoV-2 infections were collected. The samples were analyzed for quantitative determination of IgG against nucleocapsid protein of SARS-CoV-2 by enzyme linked immunoassay (ELISA) method. It has been revealed that: a) 16 (32.7%) are negative; b) 5 (10.2%) samples are equivocal; and c) 28 (57.1%) samples are positive for IgG SARS-CoV-2 nucleocapsid protein. The IgG against SARS-CoV-2 nucleocapsid protein can be used as the possible marker of the immune response. The persistence of COVID-19 immune response after 6 months of the clinical episode has been revealed. Despite of the quantity decreasing of IgG against SARS-CoV-2 nucleocapsid protein with time the exact correlation specific to the decreasing tendency has not been determined.

IgG

Nucleocapsid protein

SARS-CoV-2

The severe acute respiratory syndrome coronavirus (SARS-CoV-2) causes Corona Virus Disease-2019 (COVID-19) and global pandemic that changed our daily habits and social behaviors [1]. There are 298,409,911 active cases and 5,484,286 deaths as of 6 January, 2022 [2]. The virus as well as the peculiarities of the resulting disease are currently the top research theme; furthermore, COVID-19 is still a major global health challenge. The immunological memory is the basis of the protective immunity after infections. The mechanisms and, most importantly, the period of persistence of the immunoglobulin G (IgG) antibodies, against SARS-CoV-2 after clinical episode of the infection has still not been determined [3]. To address this question, we initiated the study aimed to determine the persistence of immune response against SARS-CoV-2 through periodical quantitative detection of IgG against viral nucleocapsid protein for recovered COVID-19 cases. The nucleocapsid protein of SARS-CoV-2 is a multivalent RNA-binding protein which is critical for viral replication and genome packaging [4]. It has been reported that the detection of the IgG against nucleocapsid protein to SARS-CoV-2 is more sensitive than antibody against spike protein in COVID-19 cases [5].

In this study were used blood samples of the students and staff members of the Petre Shotadze Tbilisi Medical Academy (TMA) with clinically documented episodes of SARS-CoV-2 infection. The students and the staff members registered after being informed about the details of the study. The age distribution of the participants was between 17 and 55. 35 participants were female, and 14 were male. By July 1st, 2020 the number of registered participants is fifty. Blood samples were collected at the university facilities. Analysis was conducted in the university laboratory for quantitative determination of IgG against nucleocapsid protein of SARS-CoV-2 by enzyme linked immunoassay (ELISA) method.

The NovaLisa SARS-CoV-2 (COVID-19) IgG ELISA diagnostical kit (NovaTec Immunodiagnostica GmbH, Germany) for in vitro diagnostic has been used. According to the manufacturer the determined positive results correspond to a specificity of 99.53% and the analytical sensitivity is defined as the probability of the assay of scoring positive in the presence of the specific analyte. The study has been performed according to the ELISA kit specific protocol; the absorbance was measured at 450 nm wavelength. For the calculation of results there were used the manufacturer’s guideline: sample absorbance value x 10 / cut-off control absorbance value. The results were interpreted as following: a) positive (> 11); b) equivocal (9 – 11); and c) negative (< 9).

It has been revealed that: a) 16 (32.7%) out of 49 samples are interpreted as negative for IgG for nucleocapsid protein of SARS-CoV-2; b) 5 (10.2%) out of 49 samples are interpreted as equivocal for IgG for nucleocapsid protein of SARS-CoV-2; and c) 28 (57.1%) out of 49 samples are interpreted as positive for IgG for nucleocapsid protein of SARS-CoV-2. All samples interpreted as negative and equivocal correlate with clinically documented episode of COVID-19 on October-November, 2020. Although 3 out of 28 positive samples interpreted as positive for IgG for nucleocapsid protein of SARS-CoV-2 correlate with clinically documented episode of SARS-CoV-2 infection on November, 2020.

Subjects were grouped according to time passed since resolution of infection and symptoms disappearance (Table 1) to observe the dynamics of IgG presence. IgG was detected in all 7 patients who have had infected one month before sampling. There were no samples of subjects infected 2 months before sampling, and only one patient with 3-month latency, who also had a positive result. 2 out of 3 patients were positive in 4-month group; 4 out of 6 in 5-month group; 9 out of 14 in 6-month group and 5 out of 18 were positive in subjects infected 6 to 8 months ago. In the latter group There were only single patients in 8- and 9-month groups, which tested negative and positive respectively. Percentage distribution of positive, equivocal and negative results between groups is shown in Figure 1.

Table 1

Presence of SARS-CoV-2 nucleocapsid IgG in subjects grouped according to time (months) since infection.
	1	3	4	5	6	7-9
Positive	7 (100%)	1 (100%)	2 (67%)	4 (67%)	9 (64%)	5 (28%)
Equivocal	0	0	0	1	2	2
Negative	0	0	1 (33%)	1	3	11

Findings suggest that IgG against SARS-CoV-2 nucleocapsid protein can be used as the possible marker of the immune response. The persistence of COVID-19 immune response after 6 months of resolution of infection and symptoms disappearance has been revealed. Despite of the quantity decreasing of IgG against SARS-CoV-2 nucleocapsid protein with time, the exact correlation specific to the decreasing tendency has not been determined. The samples with negative and equivocal results for IgG against nucleocapsid protein of SARS-CoV-2 should be further investigated for the IgG against spike, envelope or even membrane proteins, as well as neutralizing antibodies. More details concerning the persistence of immune response against SARS-CoV-2 should be obtained through the increasing the number of participants as well as further determination of IgG against nucleocapsid protein of SARS-CoV-2 in newly obtained blood samples of the registered study participants. Furthermore, obtaining and analyzing new samples from the same subjects could provide a clearer understanding of the antibody dynamics.

Limitations

The study has been performed on a small number of participants.
The study specific inclusion criterium has been defined as “clinically documented episodes of SARS-CoV-2 infection” without further explanation, i.e., onset of concrete symptoms.
The confirmation of infection by RT-PCR in swabs wasn’t been available for all participants of the study, therefore these data including CT values has been omitted.
The determination of IgG against SARS-CoV-2 nucleocapsid protein at the moment of resolution of infection and symptoms disappearance has not been performed. Therefore, the performed study is qualitative and presents the relative proportion of positive results in small cohort over time.

Ethics approval and consent to participate

The present study has been approved by the Bioethics Committee of the Petre Shotadze Tbilisi Medical Academy. All procedures performed in the present study were in accordance with the Helsinki Declaration of 1975, as revised in 2013. The participants were informed about the design and objectives of the study; all participants provided informed consent for inclusion by their registration for participation in the study.

Consent for publication

Not applicable.

Availability of data and material

Data sharing is not applicable to this article as no datasets were generated or analysed during the current study.

Competing interests

The authors declare that they have no competing interests.

Funding

The study has been funding by the Petre Shotadze Tbilisi Medical Academy.

Author’s contributions

SI performed experimental activities and data analysis; TS organized and performed samples collection; ML organized technical issues of experimental activities; EK contributes study performance and manuscript preparation. All authors read and approved the final manuscript.

Acknowledgements

Authors acknowledge participants for their interest and participation in the present study.

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Version 1

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You are reading this latest preprint version

Determination of immune response persistence after SARS-CoV-2 infection by ELISA analysis of nucleocapsid specific IgG

Status:

Version 1

Abstract

Objectives

Results

Figures

Introduction

Methods

Results

Discussion

Declarations

Ethics approval and consent to participate

Consent for publication

Availability of data and material

Competing interests

Funding

Author’s contributions

Acknowledgements

References

Status:

Version 1