Antibodies Specific to Rheumatologic and Neurologic Pathologies Found in Patient with Long COVID

Abstract

The SARS-CoV-2 virus can cause hyperstimulation of the immune system, sometimes leading to the production of various autoantibodies and increased levels of interferons and interleukins in blood plasma. Background/Objectives: Only a few studies are currently focusing on the dynamics of immunological indices after any transferred infectious disease encountered by an organism for the first time. The attention of researchers and clinicians is captured by the dynamics of antibody titers and immunologic markers (interferons and interleukins), as well as the correlation of immunologic indices with changes in the symptomatology of long COVID. This paper discusses the association of antibodies against various autoantigens with rheumatological and neurological manifestations of COVID-19. Our study patient was a 36-year-old man diagnosed with polyneuropathy, which developed after COVID-19. We conducted a dynamic follow-up of the patient for two years. Methods: The blood plasma samples collected were analyzed by ELISA for different autoantigens, IFN-γ, and a variety of interleukins. Results: An association between rheumatologic and neurologic markers in patients with long COVID symptoms was considered. Antibody titers for myelin basic protein (MBP), double-stranded DNA (dsDNA), single-stranded DNA (dsDNA), and IFN-γ, IL-1, IL-6, and IL-10 levels significantly increased during the posthospital period when the patient reported persistent symptoms of long COVID, with complaints decreasing after the symptoms were resolved. Conclusions: The findings of this study shed light on the dynamic alterations of immunological factors, and elucidate the mechanism by which SARS-CoV-2 infection disrupts immunotolerance and eventually restores equilibrium, leading to the rheumatological pathology. Significantly, the notable rise in antibody titers for various autoantigens was transient and did not lead to the progression of autoimmune pathology.

1. Introduction

Some viruses are known to be risk factors for the development of various rheumatological, neurodegenerative, and autoimmune diseases [1,2]. Similar to some other viral infections, SARS-CoV-2 can also lead to the development of various disorders. The term “long COVID” has been widely used to refer to the long-term effects of COVID-19 [3].

One of the peculiarities of COVID-19 is that the virus can penetrate the central nervous system (CNS), since SARS-CoV-2 is a neurotropic virus [4,5]. Patients diagnosed with COVID-19 tend to experience rheumatological, neurological, and neuropsychiatric disorders six months after infection, with patients hospitalized in the intensive care unit (ICU) having an even higher risk of morbidity [6]. Various rheumatological and neurological symptoms develop during the acute phase in about a third of all infected patients [7]. The most common manifestations of COVID-19 include pain, fatigue, myalgia, dysosmia, dysgeusia, and headache [8,9]. More severe and less common complications include cerebral circulatory disorders, seizures, meningoencephalitis and Guillain–Barré syndrome, neuromyelitis optica spectrum disorder, and multiple sclerosis [10,11,12].

Since viruses are known to trigger autoimmune pathologies, studying the relationship between autoimmunity and COVID-19 is of particular concern. The SARS-CoV-2 virus was found to cause hyperstimulation of the immune system, resulting in various autoantibodies being synthesized [13]. We presented a comprehensive examination of the autoantibodies detected in COVID-19 in detail in a previous review [14]. This paper focuses on the association of antibodies against various autoantigens with rheumatological and neurological manifestations of long COVID in a patient who was followed up with for two years.

Our investigation of the antibodies associated with autoimmune and rheumatologic diseases took into account antibodies for double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), histones, nucleosomes, and rheumatoid factor. COVID-19 disease not requiring hospitalization in the ICU has been shown not to cause the development or enhancement of autoimmune reactions associated with the production of anti-DNA and DNA-hydrolyzing antibodies [15]. Rheumatoid factor is represented by autoantibodies leading to a sustained inflammatory cascade and tissue damage [16]. Antibodies for histone proteins can be detected in various immunologic disorders, including systemic lupus erythematosus (SLE) [17]. These characteristics have not been described in the literature in patients with long COVID.

In some cases, COVID-19 can trigger a hyperinflammatory state characterized by increased levels of cytokines, leading to increased inflammation [18]. A hallmark of cytokine storms is the abnormal release of certain cytokines into the blood, primarily interleukin-2 (IL-2) and interleukin-7 (IL-7) [19]. Some patients with severe COVID-19 have been found to exhibit elevated levels of proinflammatory cytokines (for example, tumor necrosis factor α (TNF-α), interleukin-1 (IL-1), and interleukin-6 (IL-6)) [20]. Elevated levels of interferon-β (IFN-β)—type I and interferon-λ (IFN-λ)—type III tend to be found in blood samples of convalescent patients with COVID-19 [21].

During infection with the virus, various molecules are released, including IFN. Circulating IFN can lead to the development of autoantibodies to IFN. Therefore, in this work, the dynamics of alpha-interferon autoimmunity antibodies were analyzed. The main property of such antibodies is their ability to bind IFN-alpha and neutralize its biological activity. The significance of this phenomenon is still a subject of debate [22,23].

Typically, the close monitoring of hospitalized patients ceases upon their discharge from the hospital. Posthospital follow-up is limited to only a few consultations with subspecialists. The present study involved monitoring a patient for two years after a COVID-19 event. During this time, there were ten follow-up checkpoints involving a medical examination and blood sampling. A correlation of anamnesis with plasma immunologic indices was performed in order to assess the dynamics of the indices. Such an approach allows one to assess the extent to which immunologic parameters reflect the patient’s history of rheumatologic and neurologic symptoms.

The findings of this study are expected to provide insights into the mechanism of the immune response to severe viral infection. At the beginning of this study, we hypothesized that the rheumatological and neurological symptoms observed in a patient with latent autoimmune diabetes in adults (LADA diabetes) who had severe COVID-19 would be associated with autoimmune-related changes which would not end within two or three years and may lead to the development of chronic autoimmune pathology.

2. Case Report and Methods

The study was approved by the Local Ethics Committee of the Institute of Chemical Biology and Fundamental Medicine (Protocol N8 from 15 August 2020). The patient signed an informed consent form, and informed consent was also obtained from the SLE patients involved.

The patient being focused on in our study is a 36-year-old male of Caucasian race with latent autoimmune diabetes in adults (LADA diabetes) comorbidities. LADA diabetes is an autoimmune disease characterized by an activated infiltrate of immune cells in the islets of the pancreas [24]. LADA is defined by age at onset and the presence of at least one islet autoantibody (GADA and/or IA-2A) [25] and was diagnosed before the COVID-19 pandemic. At the end of 2020, the patient was hospitalized in the ICU with acute pulmonary insufficiency. SARS-CoV-2 infection was diagnosed by reverse transcription polymerase chain reaction (RT-qPCR). After three weeks, he was released from the hospital with signs of improvement. However, over the following six months, he reported experiencing various rheumatologic and neurological symptoms, including insomnia, memory impairment, decreased visual acuity, accommodation disorders, neuropathic pain, numbness in the extremities, and unstable blood pressure. An unmotivated decrease in body weight was also noted within two months after the disease. Ten months after SARS-CoV-2 infection, the patient was vaccinated with Sputnik V. Over the course of two years following the COVID-19 outbreak, the patient underwent continuous monitoring and willingly provided blood samples for testing on a monthly to quarterly basis.

The patient exhibited a nervous system lesion concurrent with the onset of rheumatologic and neurological symptoms during his COVID-19 infection, with symptoms enduring for six months. We characterize this condition as long COVID [3]. The condition was stabilized by administering anticonvulsants, therapeutic doses of selective serotonin and norepinephrine reuptake inhibitors, and antihypertensive drugs. The drugs were given after the patient was discharged from the hospital. Over the course of therapy, the patient experienced gradual progress.

Vacuum tubes with an anti-coagulation compound (EDTA) were used to collect fasting venous blood. The antibody, interferon, and interleukin titers were determined by ELISA using the following kits: “Vector-best” (Russia, Novosibirsk): D-5505, A-8656, A-8658, A-8654, A-8760, A-8774, A-8766, A-8768, A-8772, A-8752; “Euroimmun” (Lübeck, Germany): EA 1560-9601 G, EA 1574-9601 G; Cloud-Clone Corp. (Wuhan, China): AEA539Hu, PAA421Hu01. The assay was performed according to the manufacturer’s instructions. The result was presented as the mean value of the results from a series of three independent experiments.

The findings were compared with the results obtained from a group of patients with SLE (n = 18) and a group of healthy donors (n = 22). The diagnosis of SLE was made in accordance with national (Russian Association of Rheumatologists) and international (EULAR and ACR) recommendations based on physical and laboratory tests [10.1080/03009740802419073]. The recruitment of patients and healthy donors was done at the Institute of Clinical Immunology (Novosibirsk, Russia). All patients with SLE were women, with the healthy donors represented by 8 men and 14 women. The median age of the participants was not statistically significantly different between the groups studied.

3. Results

This study aimed to examine the dynamic changes in the immunological parameters of blood plasma over a two-year period following hospitalization in the intensive care unit due to a complicated course of SARS-CoV-2. The date of the onset of the first COVID-19 symptoms was chosen as the zero-time point. Following the COVID-19 pandemic, the level of antibodies against the SARS-CoV-2 virus experienced a gradual increase for approximately 5–6 months (reaching 392 BAU/mL). Subsequently, a gradual decrease in the antibody titer was observed. After ten months, the patient was vaccinated with the Sputnik V (Gam-Covid-Vac) vaccine, which caused an increase in the IgG antibody titer to 798.0 BAU/mL (Figure 1A).

3.1. Antibodies for MBP and MOG

Antibodies for myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG) are assumed to be associated with inflammatory diseases of the CNS and some rheumatologic pathologies. These antibodies target myelin sheath proteins and are likely to play a role in neuronal demyelination [26]. A significant (2-fold) increase in the anti-MBP-IgG titer was observed in our patient six months after COVID-19, followed by a subsequent decrease in the titers of these antibodies. The anti-MOG-IgG titer was virtually constant throughout the two years of the study (Figure 1B).

Among the cohort of healthy donors, the average values of MBP and MOG antibody titers were determined to be 0.11 ± 0.04 and 0.06 ± 0.03, respectively. After a two-year period following COVID-19, the antibody titer of our patient still exceeded the average value of this indicator among healthy donors, with this excess being statistically insignificant and possibly a permissible variation from the norm.

3.2. Antibodies Specific to Rheumatological and Autoimmune Diseases

Antibodies for cell nuclear components are considered diagnostic markers for a number of rheumatological and autoimmune diseases [27,28,29]. Therefore, we focused the study on dsDNA, ssDNA, histone, nucleosome, and rheumatoid factor antibodies. According to the manufacturer’s instructions, a dsDNA and ssDNA antibody titer value of 25 RU/mL should be considered a threshold value. As is shown in Figure 2, our donor was found to have a significant increase in dsDNA antibodies (ten times the threshold value) six months after COVID-19 and a subsequent decrease in the titers of these antibodies. A similar increase in the ssDNA antibody titer, three times the threshold value, was observed. According to the manufacturer’s instructions for the ELISA kit for rheumatologic factor (Vector-Best, Novosibirsk, Russia), values greater than 20 RU/mL should be considered positive. In our case, the range of rheumatoid factor values was determined to be below the threshold values within two years after COVID-19. The titer of antibodies to histones was not zero, but did not exceed 20 RU/mL. Antibodies to nucleosomes were not detected.

For comparison, in the group of healthy donors, the values of antibody titers characteristic of rheumatological diseases did not exceed the threshold values. In patients with SLE, the titers of antibodies to dsDNA and ssDNA were significantly increased. For example, in our case, the titers were identified to be 117.7 [41.2, 200.6] units/mL and 130.0 [36.2, 97.2] units/mL, respectively. These data are consistent with those obtained earlier in our laboratory [30]. In patients with SLE, the rheumatological factor titer value was 48.0 [15.5, 163.5] units/mL. Hence, in the posthospital phase, the patient displayed increased dsDNA and ssDNA titers, indicative of SLE, with these titers subsequently decreasing without any accompanying rheumatological or autoimmune manifestations.

3.3. Dynamics of Interferons, Cytokines, and Their Corresponding Antibodies

The interferon system is a powerful barrier against viral infections [31,32,33]. The titers of cytokines (such as IL-6, IL-10, and IL-2R) were found to be elevated in most patients with COVID-19 during hospitalization compared to healthy controls [34,35]. Previous research [36] has provided evidence of a relationship between heightened titers of IL-6 and IL-10 and the development of respiratory failure, immunological dysregulation, coagulopathy, and mortality associated with COVID-19.

Our interest was in studying the dynamics of IFN-γ, comprising a number of interleukins (IL-1, Il-2, IL-6, IL-10), in the posthospital period. Figure 3A shows that the concentration of IFN-γ—type II was quite low immediately after the patient’s discharge from the hospital, followed by a significant increase lasting up to 5–6 months. During this period, rheumatologic and neurologic symptoms of long COVID were resolved. Then, IFN-γ levels were observed to decrease to the initial values. The second maximum of IFN-γ was observed after 15 months (Figure 3A). The average IFN-γ value in the healthy donors was observed to be 0.7 [0.0, 47.1] ng/mL, while in patients with SLE, the value was measured at 20.4 [0.0, 63.2] ng/mL. The overall dynamics of the interleukins exhibited a comparable profile to that of IFN-γ.

The concentrations of IL-1 and IL-6 displayed bimodal distribution, with the highest values observed during the fourth to fifth month and the eleventh month. The IL-10 concentrations also had two peaks: during the third to fourth month and the eleventh month (Figure 3B,C). In the group of healthy donors, the concentrations of IL-1, IL-6, and IL-10 were 15.7 [4.3, 43.7] ng/mL, 30.0 [22.3, 39.2] ng/mL, and 15.7 [4.3, 43.8] ng/mL, respectively. In the patients with SLE, these values were 15.9 [6.0, 34.8] ng/mL, 45.4.0 [32.5, 62.7] ng/mL, and 11.7 [8.2, 20.3] ng/mL, respectively. The IL-2 concentrations gradually decreased during the posthospital period and did not exceed 20 ng/mL during the entire follow-up period. In the patients with SLE, the value of this parameter was 2.9 [0.3, 12.0] ng/mL, and in the healthy donors, it was 3.1 [0.2, 10.3] ng/mL.

Our study revealed no significant variations in interleukin levels between the healthy donors and the patients with SLE. It is probable that this parameter is more susceptible to the impact of different environmental factors rather than the progression of autoimmune pathology.

Figure 3 shows that the level of ⍺-IFN—type I autoimmunity antibodies ranged from 0.0 to 17.0 ng/mL during the follow-up period. No correlation was found between this parameter and changes in symptoms. For comparison, the value of this parameter in the patients with SLE was 5.4 [3.9, 7.2] ng/mL, and in the healthy donors, it was 4.0 [2.6, 6.1] ng/mL.

Thus, an increase in IFN-γ, IL-1, IL-6, and IL-10 was detected during the posthospital period, characterized by rheumatologic and neurological manifestations. During the stabilization of the patient’s condition, a decrease in these indicators was observed. The patient received his first dose of the Sputnik V vaccine (Gam-COVID-Vac) 9.7 months after the onset of COVID-19, followed by the second dose 10.4 months later. While these account for the growth of BAU antibodies targeting the S-protein, the possibility that the repeated increase in IFN-γ, IL-1, IL-6, and IL-10 levels may also be associated with the vaccination process cannot be excluded.

4. Discussion

The COVID-19 pandemic made it possible to collect blood samples from donors who encountered the SARS-CoV-2 virus for the first time. Our research team had the opportunity to diagnose a new coronavirus disease at its earliest stage. We managed to differentiate COVID-19 from other diseases by using the RT-qPCR method. This work contributes to the collection of publications that specifically examine the dynamics of immunological parameters following initial exposure to any infectious disease. Furthermore, we successfully examined the evolution of immunological markers over time, illustrating how infection with SARS-CoV-2 disrupts immunotolerance and eventually establishes a stable balance.

The SARS-CoV-2 virus infection has been found to cause a substantial number of rheumatologic, autoimmune, and CNS disorders [37]. Neurological and rheumatologic disorders can be associated with the destruction of the myelin sheaths of neurons. Consequently, this investigation analyzed the dynamics of antibodies possibly involved in the process of demyelination, focusing on antibodies targeting MBP and MOG. A rise in the titer of antibodies for MBP was observed in the blood plasma of our patient, remaining elevated for five months post COVID-19, followed by a decline. The data demonstrate a significant correlation with the presence of rheumatologic and neurological symptoms associated with long COVID that were reported by the patient over five months.

Only a limited number of positive anti-MOG-IgG results have been documented in the literature for patients diagnosed with acute disseminated encephalomyelitis (OREM), acute hemorrhagic leukoencephalitis [38], or Myelin Oligodendrocyte Glycoprotein-Associated Disorder (MOGAD) [39] that developed after COVID-19. The anti-MOG-IgG titer of our patient remained unchanged for two years following his COVID-19 infection.

We evaluated the dynamics of the following antibodies associated with nuclear autoantigens: antibodies to dsDNA, ssDNA, histones, nucleosomes, and rheumatoid factor. A significant increase in the titers of antibodies for dsDNA and ssDNA was identified in the posthospital period when the patient presented complaints related to long COVID. With the complaints resolved, a decrease in the titers of these antibodies was noted. During the acute period, the levels of these antibodies were similar to those found in patients with SLE, yet our patient did not exhibit any rheumatological symptoms. Consequently, the presence of high antibody titers for dsDNA and ssDNA should be approached with caution. Our patient and the healthy group of donors did not exhibit any significant changes in the titers of antibodies for histones, nucleosomes, and rheumatoid factor for two years after COVID-19.

This study also examined the dynamics of IFN-γ, IL-1, IL-2, IL-6, IL-10, and ⍺-interferon autoimmunity antibodies and assessed their relationship with patient complaints. An increase in IFN-γ, IL-1, IL-6, and IL-10 concentrations was first observed during the posthospital period, coinciding with rheumatologic and neurological complications. A second increase in the concentration of IFN and ILs was observed after the administration of two doses of Sputnik V. The results obtained are consistent with the literature data, demonstrating that the IFN-γ response gradually decreases over time after COVID-19 and sharply increases after vaccination and the administration of a booster dose [40].

In summary, we analyzed the correlation between various autoimmune and rheumatologic markers and nervous system lesions following SARS-CoV-2 infection. During the posthospital period, when the patient reported long COVID symptoms, a significant increase was determined in the titers of MBP, dsDNA, and ssDNA antibodies, as well as elevated levels of IFN-γ, IL-1, IL-6, and IL-10. These markers showed a decrease after the symptoms were resolved.

After vaccination with Sputnik V, an increase in the titers of IFN-γ, IL-1, IL-2, IL-6, IL-10, and ⍺-IFN autoimmune-related antibodies was observed, but not in other parameters. An increase in serum chemokine levels has been documented and described not only after immunization with SARS-CoV-2 vaccines [41,42,43], but also after various other vaccinations [44,45].

It should be emphasized that the significant increase in antibody titers for a number of autoantigens was not accompanied by the further development of rheumatologic and autoimmune pathology, and eventually decreased after the resolution of the corresponding symptoms.

During the first 8 months, the patient showed a significant increase in his concentrations of dsDNA, ssDNA, and anti-MBP antibodies. A single measurement of these parameters in the blood can give a false impression of the development of an autoimmune complication after infection. However, long-term follow-up showed a decrease in titers, and two years after infection, the patient was not diagnosed with advanced autoimmune disease. We have shown that the parameters eventually decline and stabilize, but after a severe infection requiring hospitalization, this process takes at least a year. Unfortunately, the monitoring of patients usually ends after recovery from an infection, and the development of autoimmune complications is possible years later. We have shown that a considerable amount of time is needed for the parameters to stabilize and that, after a severe infection, further monitoring of the patient, lasting at least one year, is necessary for the diagnosis of the onset of autoimmune complications. Thus, the hypothesis presented to us was not fully confirmed, and over the three years following the infection the patient has not complained of any rheumatological, autoimmune, or neurological disorders.

Limitations of the study. This study is based on the case of one patient. This study includes a fairly long period of observation for the patient. The patient kindly provided his blood for two years after infection with the SARS-CoV-2 virus, and 11 samples of his blood were collected. Unfortunately, collecting a large number of blood samples from one patient is a very difficult task and we were not able to observe other patients during such a long period of time. However, the dynamics of various blood parameters after a coronavirus infection over a long period of observation have not been described in the literature and our work demonstrates such data for the first time, albeit limited to one patient.

The second limitation of this study is the presence of comorbidities in the patient, such as LADA diabetes. We do not exclude the possibility that the presence of comorbidities could have affected the severity of the SARS-CoV-2 infection, as well as the dynamics of the blood parameters during the study period.