Abstract
Introduction
In December 2019, the outbreak of an unknown pneumonia was reported in Wuhan, China; on March 12, 2020, the World Health Organization (WHO) declared a pandemic due to the global spread of COVID-19 and the death of thousands of people due to the Coronavirus disease. 1 Corona Virus Disease 2019 (COVID-19) is an infectious disease caused by the SARS-COV-2 virus that causes acute respiratory syndrome (ARDS). Although most people with this disease have no symptoms, some patients with this virus experience serious problems. 2 SARS coronavirus is a Crown-like RNA virus with a diameter of 60 to 140 nm and is similar to SARS and MERS; the genome of the coronavirus encodes four important proteins, including the spike Protein (S), Nucleocapsid (N), Membrane (M) and Envelope (E). The main receptor of SARS-COV-2 virus is Angiotensin-converting enzyme 2 (ACE2) and SARS-COV-2 Receptor Binding Domain (RBD) spike protein can enter the cell after binding to the ACE2 receptor. The presence of ACE2 receptor on the surface of the heart, kidney, intestine and endothelial cells makes these organs vulnerable to COVID-19. After the invasion and multiplication of the virus, the host cell is killed and the virus is released. Also, in the human body, APC (Antigen Presenting Cell) detects the virus, delivers them to cytotoxic T lymphocytes, and activates NK cells; innate and adaptive immunity and high amounts of cytokines and chemokines are produced in the body. 3 Currently, the most effective way to fight SARS-COV-2 infection is vaccination and the RBD region of the SARS-COV-2 virus has been identified as the most important region for the construction and function of the vaccine; thus, this part is targeted in different vaccines. More than eight types of Anti-SARS coronavirus vaccines have been licensed for use by priority groups under an emergency license. These vaccines include Pfizer-BioNTech, The Sinopharm vaccine, Sputnik V Russian, AstraZeneca, and Moderna mRNA vaccine.4–6
It should be noted that in different vaccines, the amount of neutralizing antibody indicates the effectiveness of the vaccine. The higher the amount of Anti-RBD, the more immunity is created in vaccinated people. Due to the short duration of immunity, it is necessary for people to receive the third or fourth dose of vaccines, which can in turn increase the amount of Anti-RBD titer in a short period of time after injection. 7
In addition to the variable function of vaccines, a decrease in the effectiveness of vaccines may be observed due to the genetic changes of the virus or the occurrence of unwanted side effects such as Allergic, Myocarditis, Thrombosis and Thrombocytopenia.
7
One of the factors that reduces the effectiveness of the vaccines is the anti-idiotype network.
8
The anti-idiotype network was introduced in 1974 by Niels Jerne. He explained that sometimes the vertebral region of antibodies is known as self-antigen in the human body; against this region, a new antibody is made, which is called an Anti-idiotype antibody.
8
This anti-idiotype antibody is specific for the first antibody, and the paratope region of the anti-idiotype antibody is exactly the same as the epitope region of the first antibody antigen(Figure 1). In some research, anti-idiotype antibodies are used as antigens for making vaccines. It should be noted that the Anti-idiotype of the antibody has the ability to bind to the receptors of the body cells to which the antigen of the first antibody binds; this causes pathological complications, especially after a longer period of time.
9
Investigations have shown that some epitopes on the surface of SARS-COV-2 are similar to autoantigens in the human body; an example to illustrate the point is S protein (RBD), which is a protective antigen of the SARS-COV-2 virus. This antigen binds to ACE2 on the cell surface and can disrupt ACE2 signaling by various mechanisms. The anti-idiotype anti-RBD antibody can have the same effect on ACE2 as the SARS-COV-2 S protein can.
10
In people receiving three doses of the COVID-19 vaccine, there is a risk of making anti-idiotype anti-RBD antibodies due to the high level of Anti-RBD, and thus there is a risk of autoimmune diseases. The mechanism of how antiidotype works in the body. In this form, Ab2 is the anti-idiotype antibod.
Many studies have shown an association between autoimmune diseases and SARS-COV-2.11,12 Nevertheless, no studies have investigated autoantibodies in people who have injected three doses of the COVID-19 vaccine yet. In the present study, we intend to examine the development of anti-idiotype antibodies against RBD and ANA in people receiving three doses of SARS-COV-2 vaccine. It seems that with the activation of the anti-idiotype system, an antibody is made against the anti-RBD, which is similar to the spike antigen in terms of its appearance and the idiotype region. Moreover, it has the possibility of binding to ACE2 on the surface of normal tissue cells. The existence of the anti-idiotype has been mentioned in various articles; hence, the risk of autoimmune disease caused by the production of anti-idiotype has been raised and some reactions after COVID-19 vaccination have been attributed to the production of this antibody. In some studies, the presence of anti-idiotype in patients with COVID-19 has been investigated, yet no studies have examined the presence of anti-idiotype and ANA in COVID-19 vaccinated people so far. In this study, we aim to be the first to study the presence of Anti-idiotype antibodies and ANA in people who have received three doses of the COVID-19 vaccine and check that at least 4 months have passed since the injection of their third dose. The results of this study have an effective role in determining the level of concern about the possibility of autoimmune diseases after COVID-19 vaccination.
Materials and methods
This study was conducted on 180 people who had received three doses of the COVID-19 vaccine and at least 4 months had passed since the injection of the third dose. Usually, the production of Anti nuclear antibody happens some time after the start of an autoimmune reaction. After Anti idiotype antibody binds to the cell surface, inflammation occurs and this inflammation leads to the destruction of the body’s cells, and as a result, the antigens of the cell nucleus become available to the immune system and antibodies are made. The important point is that it takes time for the antibody to be made and a detectable amount to be produced. If we immediately check the production amount of this antibody, even if Anti idiotype antibody has started to be made in the body, the amount of produced antibody cannot be detected and the test result will be negative. For this purpose, information of the people who visited Tajrish Martyrs Vaccination Center in Tehran between April 22nd and August 22nd for receiving the COVID-19 vaccine was checked. Inclusion criteria were (1); the age range of 18 to 65 years (2); no symptoms of COVID-19 (3); no history of COVID-19 within the 4 months prior to sampling (4); having a negative COVID-19 PCR test (5); no history of autoimmune diseases (6); no family history of autoimmune diseases; and (7) no suspicious symptoms. Autoimmune diseases were considered based on the opinion of a rheumatologist. People with a history of COVID-19 within the 4 months before the sampling time, or with a positive COVID-19 PCR test, and also those with or suspected of autoimmune diseases were excluded from the study.
345 people were randomly selected from among those who were eligible to enter the study based on the double admission code. These people were contacted and 180 people declared their willingness to participate by signing a written consent form. 5 cc of blood was taken from participants at the immunology laboratory of the Paramedical Faculty (Iran-Tehran) and after separating the serum, the samples were frozen at minus 80°. This study has been approved by the ethics committee of Shahid Beheshti University of Medical Sciences and has the ethics code number IR.SBMU.RETECH.REC.1400.1180.
Anti-RBD anti-idiotype Assay
Due to the fact that this kit was not commercially available, the materials were first purchased, and then the Anti-idiotype Anti-RBD test kit was designed based on the competitive ELISA method. Recombinant RBD antigen (Rekom Biotech, RAG0074, Spain) with a concentration of 1.33 mg/mL and Anti-RBD antibody (Bio-Rad, AHP3017, USA) with a concentration of 1 mg/mL were purchased. Recombinant RBD antigen was diluted to 1/2660 and coated with a concentration of 0.5 μg/mL in the bottom of the well, and blocking was done according to the protocol; 13 afterwards, the Anti-RBD antibody was added to the well. This concentration was set up in such a way that after the addition of the Anti-RBD Antibody, these Antibodies would bind to the RBD antigen from only one epitope. A conjugated solution (RBD antigen of SARS-Cov-2 virus attached to peroxidase enzyme) was prepared. Anti-RBD antibody with a concentration of 8 ng/mL was added to all wells. This antibody is bound to the RBD antigens coated on the bottom of the well from its own epitope. Then, the conjugate solution was added to the wells at the same time as the serum of the people being injected with the SARS-COV-2 vaccine. PBS was used as a negative control; the competition was established between the conjugated RBD antigen and the Anti-idiotype antibody (if present) to bind to the free epitope of the antigens coated on the bottom of the plate. After 30 min of incubation, the wells were washed with washing buffer (phosphate buffer and 0.05% Tween diluted 1 to 10) in five steps. After drying, dye solution (tetramethylbenzidine and hydrogen peroxide) was added to all the wells, and incubation was done again for 15 min. To stop the enzyme activity, finally, stop solution (1 normal hydrochloric acid) was added and the absorbance was read with an ELISA reader at a wavelength of 450 nm (reference wavelength of 630 nm). The following formula was used to calculate the cut-off: Cut-off = average optical absorption of negative controls +0.05; based on this formula, an absorbance of less than 0.7 means a positive anti-idiotype antibody, and a higher absorbance was considered as a negative sample in terms of the presence of an Anti-idiotype antibody.
Measuring a quantitative amount of Sars-Cov anti IgG RBD
ELISA kit (Pishtaz Teb, PT-SARS-COV2-RBD-IgG-96. IRAN) was used to measure a small amount of SARS-COV-Anti IgG RBD. This kit is designed based on the indirect ELISA method in which the wells of the plate are covered by the antigens of the RBD region of the SARS-COV-2 virus. After washing through adding an anti-IgG antibody that is attached to the HRP enzyme, if there is an Anti-SARS-COV-2 RBD antibody of IgG type, labeled anti-human IgG is also attached to it. After washing, dye solution (tetramethyl benzidine) was poured into the wells. The intensity of the blue color is proportional to the complex formed in the wells. Enzyme activity was stopped by using a solution (STOP one normal hydrochloric acid) and the blue color changed to yellow and was read at a wavelength of 450 nm with the help of an ELISA reader. With the help of 0, 5, 10, 25, 50, and 100 standards, standard graphs were drawn and IgG values were calculated by ELISA reader. For this test, values higher than 12 RU/ML were considered positive for Anti-RBD IgG antibody; the higher this number is, the higher the amount of antibody in the person’s serum.
Measuring amount of Anti-Nuclear Antibody (ANA)
ANA-HEp2 quantitative kit (Aesku, ANA-HEP2 3119, Germany) was used to measure the antibody against nuclear antigens. This kit is based on the indirect ELISA method that can measure the amount of IgG class antibodies against Hep-2 cell nuclear antigens in human serum. The bottom of the wells is covered with the antigens obtained from the lysis of HEp-2 cells. In each well, the presence of IgG antibodies against RNP, sm RNP, smSSB/La, SSA/Ro, scl70, centromere B, Jo1, and dsDNA antigens can be identified. The diluted samples were poured into the wells. If there are antibodies against the core antigen, these antibodies will bind to the antigens on the bottom of the well. After washing through adding an Anti-IgG antibody that is attached to the HRP enzyme, if there is an antibody against RNP, sm RNP, smSSB/La, SSA/Ro, scl70, centromere B, Jo1, dsDNA antigens, labeled anti-human IgG also binds to it. After washing, dye solution (tetramethyl benzidine) was poured into the wells and the intensity of the blue color is proportional to the complex formed in the wells. Enzyme activity was stopped by using a solution (STOP one normal hydrochloric acid) and the blue color changed to yellow and was read at a wavelength of 450 nm with the help of an ELISA reader. With the help of 100, 30, 10, 3, 0, and 300 standards, a standard graph was drawn and IgG values were calculated by ELISA reader. For this test, values higher than 50 RU/ML were considered as positive IgG antibodies against RNP, sm RNP, smSSB/La, SSA/Ro, scl 70, centromere B, Jo1, and dsDNA antigens.
Results
Examining the type of injected vaccines and its relationship with the level of ANA and Anti-idiotype antibody.
Examining the type of injected vaccines and its relationship with the level of ANA and Anti-idiotype antibody.
Relationship between Anti-idiotype antibody and Anti-Nuclear Antibody (ANA).
The results related to the measurement of the quantitative amount of SARS-COV-Anti IgG
Discussion
In 1983, Plotz announced that one of the reasons for the occurrence of autoimmune diseases after contracting some viral diseases is the Anti-idiotype antibody network. 9 For example, the Anti-idiotype antibody against enterovirus coxsackievirus B3 in mice could bind with myocyte antigens and cause autoimmune myocarditis. 14 It is noteworthy that in SARS-COV-Disease, the average Anti-IgG RBD of people who had a history of corona infection was higher than that of people who were not infected with corona. First, the normality of the variable distribution of Anti IgG RBD in each of the variable subgroups of the history of corona infection was checked by the Shapiro-Wilk test, and the results of this test showed the absence of normality of the variable distribution. Next, the non-parametric Mann-Whitney test was used to compare the variable distribution of Anti IgG RBD between groups. The p-value of the test was less than 0.05 indicating a significant difference in the distribution of the Anti IgG RBD variable between people with a history of Corona and those without it.
The antigen of protecting virus is protein S (RBD). Protein S can disrupt ACE2 signaling by disrupting mechanisms and also causing the production of inflammatory cytokines by directly acting on toll-like receptors. 10 The Anti-idiotype of the antibody can have the same effect as the S protein of SARS-COV-2 on ACE2. 15 In the study of Arthur JM and his colleagues (2021), the presence of Anti-idiotype Anti-RBD antibodies in the serum of hospitalized patients with COVID-19 was studied. The results of that study determined that 93% of patients who needed to be hospitalized due to COVID-19 had Anti-idiotype Anti-RBD antibodies in their serum. 16 In our study, it was found that 14% of people, who received three doses of the COVID-19 vaccine and 4 months passed after their third dose injection, had Anti-idiotype Anti-RBD antibodies and this percentage was much lower compared to people with a severe form of COVID-19 (93). The importance of studying the Anti-idiotype of the Anti-RBD antibody is due to the capacity of this antibody in causing autoimmune diseases, so in some articles published in reputable journals, the need to measure this antibody in people receiving the COVID-19 vaccine has been emphasized. 17
It should be noted that one of the key features of autoimmune diseases is the production of autoantibodies. Although the main mechanisms of autoimmune disease are complex and still not fully understood, scientists believe that one of the mechanisms of autoimmune disease is molecular mimicry, which is carried out by infectious pathogens. 18 Infectious viruses, having antigens similar to the antigens of the human body, cause cross reactions and upset the tolerance balance of the immune system 19 like the relationship between Epstein-Barr Virus (EBV) and lupus disease. 20 Studies have shown that some epitopes on the surface of SARS-COV-2 are similar to autoantigens in the human body. For example, Anand et al. and colleagues have reported that the S1/S2 antigen on the surface of SARS-COV-2 is similar to the sodium channel surface antigens of human epithelial cells. Moreover, the similarity of surface antigens of SARS-COV-2 and three proteins named DAB1, AIFM, and SURF1 that exist in the pre-Btzinger complex of the human brain stem (pre-Btzinger C) may contribute to respiratory failure in COVID-19. The results of these studies indicate the possibility that SARS-COV-2 may induce cross-reactivity with host autoantigens and this problem can provide an explanation for different clinical manifestations and injuries related to different organs and systems after SARS-COV-2 infection.
In our study, it was found that only 2.7% of people had positive ANA 4 months after the injection of the third dose of the COVID-19 vaccine, while this percentage is much smaller compared to that of the studies conducted on people with COVID-19. Pascolini et al. reported that Antinuclear Antibodies (ANA), Anti-cytoplasmic neutrophil antibodies (ANCA), and antiphospholipid syndrome (APL) were present in 33 patients with SARS-COV-2. These results showed that antibodies were detected in 45% of people with SARS-COV-2 and these people had more severe clinical manifestations and more serious problems. Also, the results show that ANA positivity was 33%, anticardiolipin antibodies (IgGand/IgM) 24%, and antib2-glycoprotein-I antibodies (IgGand/IgM) 9%; ACN was negative in all patients.11,12
Conclusion
It seems that the possibility of the production of autoantibodies and the occurrence of autoimmune diseases following the infection or vaccination of COVID-19 is one of the serious challenges in predicting the conditions after COVID-19. Although the production rate of Anti-idiotype Anti-RBD antibodies is very high in people with severe forms of COVID-19, our study showed that this antibody is seen in a smaller percentage of people receiving three doses of the COVID-19 vaccine. Fortunately, the percentage of ANA is also much lower compared to that of people with severe forms of COVID-19. However, this study cannot definitively eliminate the concern about the production of autoantibodies following the COVID-19 vaccination. Yet, this study specifies to some extent that the production pattern of Anti-idiotype antibody and ANA in the conditions of vaccination is completely different from the conditions of COVID-19 infection; further studies are warranted to confirm and find the causes of this difference.
Footnotes
Acknowledgments
We would like to thank all the participants in this study.
Authors’ contributions
Conceptualization and Supervision Methodology, Writing – original draft, and Writing – review: H.Ch, H.B; Investigation, Formal Analysis, Writing – original draft, and Writing – review: H.Ch,H.B,F.P.
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.
Ethical statement
This article was approved at Shahid Beheshti University of Medical Sciences with code of ethics (IR.SBMU.RETECH.REC.1400.1180).
