Immune response variation in mild and severe COVID-19 patients

Sixty patients with COVID-19 infection were categorized into mild and severe groups, and their immune response was analyzed using flow cytometry and complete blood count. An observed increase in immune activation parameters, notably a higher percentage of CD4 lymphocytes co-expressing CD69 and CD25 molecules, and enhanced activity of the macrophage-monocyte cell line was noted in the mild group. Although Group 2 (severe COVID) had fewer CD4 cells, significant migration and proliferation were evident, with increased CD4CD69, CD8 HLA-DR+, and CD8CD69 lymphocytes. The CD4 to CD8 ratio in Group 1 suggested potential autoimmune reactions, while Group 2 indicated potential immunosuppression from severe infection and employing immunosuppressive drugs. Additionally, Group 2 exhibited an increased neutrophil count, hinting at possible bacterial co-infection. Group 1 showed differences in CD4RO and CD8RA lymphocyte populations, implying that cellular immunity plays a role in developing efficient postinfectious immunity. This intimation suggests that vaccination might mitigate the severity of the coronavirus infection and prevent complications, including long-term COVID-19.


INTRODUCTION
Severe acute respiratory syndrome caused by coronavirus was first detected in Wuhan, China, in December 2019.In March 2020, the World Health Organization (WHO) declared the 2019 coronavirus

Study design and participants
This prospective cohort study was conducted over an asynchronous time spanning from March 9, 2021, to December 28, 2021.Sixty patients with PCRconfirmed COVID-19 infection were categorized into infection a pandemic, which caused an overload of the healthcare system in most countries globally and led to substantial economic losses. 1 According to the WHO dashboard (data observed on August  17, 2023), there are almost 770 million confirmed cases of COVID-19 and about 7 million deaths. 2 In addition to the severe course of the infectious disease with a high mortality rate, Taleghani and Taghipour noted that many patients with COVID-19 are asymptomatic or have only mild symptoms of the disease while maintaining high contagiousness, which in turn leads to difficulties with screening, prevention, and control of the epidemic. 3he immune response associated with SARS-CoV-2 infection is central to the pathogenesis of COVID-19, involving hyperactivation of the innate immune response, formation of extracellular traps of neutrophils, and lymphocytopenia. 4,5Not all immune responses are protective, as antibodydependent enhancement of humoral immunity may contribute to SARS-CoV-2 infection.In contrast, a T-lymphocyte response in a cellmediated reaction may contribute to a cytokine storm.Chen et al. focussed on the fact that the immune hypothesis of the pathogenesis of coronavirus infection explains the high vulnerability of elderly patients. 6Because COVID-19 is pathophysiologically associated with the development of a cytokine storm, it leads to endothelial dysfunction and endotheliitis, which in turn causes the development of microvascular thrombi, ischemia, and multiple organ failure, which determines the multisystem nature of the lesion.Anka et al. also pointed out that individuals with a severe course of COVID-19 typically have eosinopenia and lymphopenia with a marked decrease in the frequency of clusters of differentiation CD4+ and CD8+ T-cells, basophils cells, and natural killers (NK) cells. 7Additionally, several studies have provided growing evidence that COVID-19 can cause an immune system to become dysregulated and result in the emergence of autoimmune disorders (especially vasculitis and arthritis, less often -idiopathic inflammatory myopathies, systemic lupus erythematosus and sarcoidosis, systemic scleroderma, and others). 8,9herefore, identifying the immunopathological effects of COVID-19 may become a potential immunotherapy target and is essential for choosing Immune response variation in mild and severe COVID-19 patients two groups based on the symptom severity: mild without hospitalization and long COVID syndrome (21 patients) and severe (39 patients), requiring hospitalization and oxygen therapy for over ten days.Sample size determination was based on prior research data on immune responses in COVID-19 patients.Using a power of 0.8, an alpha level of 0.05, and an effect size derived from preliminary data, a sample size of 60 was deemed sufficient to detect statistically significant differences between the mild and severe groups.G*Power software was utilized for these calculations.The Medical University of Bialystok Institutional Review Board approved the study on February 21, 2021 (No 1046-A).

Treatment and sample collection
Patients were administered standard treatment, which included dexamethasone and anticoagulant prophylaxis.Blood samples were collected from the severe group between the tenth and the fourteenth day of hospitalization.Blood samples were obtained for patients in the mild group through scheduled visits to outpatient clinics associated with the research institute.Specifically, 10 to 14 days post-positive PCR confirmation, patients were invited to the clinics for sample collection, ensuring parity in the sampling timeline between both groups.Samples were stored in ethylenediaminetetraacetic acid-laden tubes.

Laboratory analysis
Blood samples underwent staining by mixing 10 mL of monoclonal antibodies (Beckman Coulter, USA) with 100 mL of whole blood.These antibodies were pre-bound to fluorochromes.After a 20-minute incubation, samples were processed using the Coulter rapid no-wash whole blood lysis station.Analysis was conducted using the Beckman Coulter Fc 500 MCL flow cytometry analyzer, examining a minimum of 10 5 cells from each sample (Table 1).

Statistical analysis
Before performing the Welch t-test, data was analyzed using the Shapiro-Wilk test for normality.Results from this test indicated that the data deviated from a normal distribution, prompting the exploration of alternative testing methods.The Welch t-test was chosen due to its robustness in handling unequal variances between groups.Additionally, Levene's test for equality of variances was conducted and indicated that the two groups had unequal variances, further justifying the use of the Welch t-test.All statistical analyses were executed using the SPSS software, version 27.

RESULTS
Several immunological differences emerged during the comparative analysis of the groups under study (Table 2).In Group 1, CD3, NK cells, and The discrepancies in immune response markers, such as the eosinophil and basophil counts, and the significant difference in the total lymphocyte count, emphasize the chronic inflammatory response in patients with severe infections.The elevated number of neutrophils in Group 2 might suggest a potential bacterial infection, coupled with the state of immunosuppression. 16The lymphopenia trend in Group 2 can be a natural response to viral invasion and could also predict future complications.
The observed immunological responses and the continued use of immunosuppressive therapeutic regimens highlight their importance in counteracting excessive immune system activation.
In individuals not requiring hospitalization, specific lymphocytes suggest the development of strong postinfectious immunity, vital for herd immunity.Vaccination remains crucial in reducing COVID-19 severity, protecting against primary symptoms, and potentially safeguarding against PCC effects.The dominance of HLA-positive CD4 lymphocytes in Group 1 aligns with prior studies, 17,18 suggesting prolonged circulation in the bloodstream, indicative T lymphocytes (CD3) were prevalent when labeled with CD62L, CD4, and CD4HLA lymphocytes.In Group 2, there was a higher presence of CD4CD69, CD8 (including those positive for HLA-DR), CD8CD69 lymphocytes, CD4CD25 cell counts, and a noticeable difference in CD4RO and CD8RA lymphocyte subsets.Differences were also observed in eosinophil, basophil, and monocyte counts in standard blood examination, with a significant variation in peripheral blood lymphocyte count.Additionally, in the lymphocyte formula, the number of neutrophils was higher in Group 2, with a trend towards lymphopenia compared to Group 1.

PATIENT CONSENT
Informed consent was obtained from all individuals included in this study.

Table 1 . Monoclonal antibodies used for staining T-, basophils-, NK-, natural killer T-cells, regulatory T-cells, and monocytes.
Source: Created by the authors.VOL.2024 / ART.11 Immune response variation in mild and severe COVID-19 patients

Table 2 . Comprehensive analysis of lymphocyte differentiation clusters and general blood analysis indicators across two groups.
Note: M -median; T -modified Student's (Wilk's) test.
Immune response variation in mild and severe COVID-19 patients reaction and proliferation during PCC.Conversely, individuals recovering from COVID-19 who did not require hospitalization demonstrated a higher proportion of CD4CD45RO and CD4CD62L lymphocytes, suggesting the evolution of potent postinfectious immunity, an integral component of herd immunity.The first group's augmented CD4/ CD8 ratio may indicate a predisposition toward autoimmune events.For those with a severe infection trajectory, a diminished ratio could suggest a tilt towards immunosuppression, likely due to the intense infection and the use of immunosuppressive medications, such as dexamethasone.Notwithstanding, immunosuppressive treatments, encompassing corticosteroids, are presently deemed the most efficacious, given the hyperactivation of the immune system and the propensity to trigger autoimmune disorders.Delving deeper into the nuances of the immune response to SARS-CoV-2 infection can pave the way for novel therapeutic strategies targeting the overarching inflammatory reaction stemming from a cytokine storm's onset.Comprehensive knowledge of the humoral response elicited by vaccinations promises to optimize preventive strategies against potential viral outbreaks in the future.This is particularly relevant when considering the formulation and dosing for individuals under therapeutic immunosuppression or those experiencing the natural physiological aging process.Future investigations could focus on understanding T-cells' regulatory cytokine expression, comparing the immunological response in asymptomatic individuals, and longitudinally monitoring patients with designated control checkpoints.