Dear Sir,
Most individuals affected by COVID-19 recovered uneventfully. However, mounting evidence reported patients with persisting symptoms (post-COVID sequelae),1 and identifying the predisposing factors is beneficial for facilitating diagnosis and advising public health strategy. We read with great interest the recent multicenter study by Rombauts et al. reporting the association of SARS-CoV-2 RNAemia with more post-COVID symptoms 6 months following recovery.2 An orchestrated series of immune responses drive the innate and adaptive immunity to eliminate replicating viruses during the disease course. Ongoing systemic inflammation marked by activated cellular immunity and cytokine production has been associated with organ damage and increased mortality.3 Therefore, coordinated immune resolution is indispensable during the recovery phase. This study aims to observe the immunological alteration following COVID-19 illness. In addition to the antibody titer measurement, peripheral blood mononuclear cells (PBMCs) were also collected from patients recovering from COVID-19 infection to map the cellular and humoral immune response comprehensively.
A total of 97 adults were recorded between August 1, 2021–October 31, 2021 and followed up till October 31, 2022. Among them, 51 had previously documented COVID-19 infection (post-infection group, PI), and 46 were otherwise healthy adults with neither previous COVID-19 infection nor SARS-CoV-2 vaccination (healthy control group, HC). One of the PI patients received one dose of the ChAdOx1 nCoV-19 (AZD1222, Oxford-Astra Zeneca) vaccine before the onset of infection, while others were unvaccinated up until the time of sample collection. There was no significant difference in the sex distribution between the two groups, although the PI cohort (63.0 ± 41.0 years) was older than the HC (44.3 ± 5.0 years; Table 1). The PI cohort contracted the disease between May to August 2021, where the predominant circulating virus in Taiwan was the Delta variant. Twenty and 13 patients received oxygen supplementation (classified as a severe disease) and mechanical ventilation (classified as a critical disease) during the disease course, respectively. The remaining 18 patients reported mild symptoms. Peripheral blood samples from the PI cohort were collected on average 116 days following COVID-19 diagnosis (mean 116.59 ± 13.25 days; median 113 days; ranging from 94 to 154 days).
Table 1.
Clinical characteristics of 51 participants with previous COVID-19 and 46 healthy control subjects.
| Healthy control | Post-infection | p-value | |
|---|---|---|---|
| n = 46 | n = 51 | ||
| Sex: Female (n; %) | 19 (41.3) | 17 (33.3) | 0.417 |
| Age (Mean; SD) | 44.31 (4.95) | 63 (14) | <0.001 |
| Body mass index (Mean; SD) | 23.31 (3.02) | 24.73 (4) | 0.633 |
| Comorbidities | |||
| Coronary artery disease (n; %) | 0 (0) | 17 (33.3) | <0.001 |
| Diabetes mellitus (n; %) | 5 (10.9) | 11 (21.6) | 0.156 |
| Hypertension (n; %) | 9 (19.6) | 17 (33.3) | 0.126 |
| Cancer (n; %) | 3 (6.5) | 3 (5. 9) | 1 |
| Chronic kidney disease (n; %) | 0 (0) | 3 (5. 9) | 0.244 |
| Chronic viral hepatitis (n; %) | 2 (4.3) | 3 (5. 9) | 1 |
| Liver cirrhosis (n; %) | 0 (0) | 1 (1.9) | 1 |
| Autoimmune disease (n; %) | 3 (6.5) | 0 (0) | 0.103 |
| Asthma/COPD (n; %) | 0 (0) | 2 (3.9) | 0.496 |
Intergroup comparison was performed by independent sample t-test for age and body mass index; Χ2 test for sex, coronary artery disease, diabetes mellitus, and hypertension; and Fisher’s exact test for cancer, chronic kidney disease, chronic viral hepatitis, liver cirrhosis, autoimmune disease, and asthma/chronic obstructive pulmonary disease.
SD, standard deviation; COPD, chronic obstructive pulmonary disease.
Immunity protection that occurs naturally after developing COVID-19 wanes gradually over time, but persistent circulating antibodies were still detected up to 6 months after recovery from COVID-19.4 In agreement, quantification of the circulating IgG antibody from the serum (SARS-CoV-2 IgG II Quant, Abbott, cutoff value: 50.0 AU/mL) revealed a significantly higher antibody titer in the PI cohort compared to the HC group in our current study ( Fig. 1A), although there was no significant difference in early active (CD69+), proliferative (KI-67+), nor functional (IFN-γ+) B cells. (Fig. 1B; the complete list of immune cell markers are presented in Supplementary Table 1.) Multiplex assay of inflammatory cytokines revealed a lower level of interleukin (IL)-2, tumor necrosis factor (TNF)-α, interferon (IFN)-α, and IFN-γ, as well as IL-10 in our PI cohort when compared to the HC group (Fig. 1C). However, when comparing the expression of the proinflammatory cytokine: TNF-α, to the proinflammatory counterpart: IL-10, the PI cohort evidently showed a high ratio (Fig. 1D), indicating possible ongoing inflammation 4 months after acute episodes of COVID-19. Indeed, a high TNF-α level is commonly reported in sepsis,5 and an elevated TNF-α /IL-10 ratio has also been proposed to predict susceptibility to infections in burn injury.6
Fig. 1.
(A) Quantification of antibody concentration in serum of healthy control (HC) and post-COVID-19-infection (PI) cohort (adapting SARS-CoV-2 IgG II Quant from Abbott, cutoff value: 50.0 AU/mL). (B) B cell number as a percentage of total peripheral blood mononuclear cell (PBMC) count (EA: early active, CD69+; P: proliferative, KI-67+; F: functional, IFN-γ+). (C) Cytokine expression as measured in mean of fluorescence intensity (MFI). (D) Comparison of TNF-α to IL-10 ratio in HC and PI cohort. (E) Number of respective immune cells as percentage of total PBMC count. (F) Number of Th cells, Th1 cells, and Tc cells subtypes as percentage of total PBMC count (EA: early active, CD69+; P: proliferative, KI-67+; F: functional, IFN-γ+; C: cytotoxic, CD107a+). *p value < 0.05; **p value < 0.01; ***p value < 0.001.
Further analysis of the total T cell and T helper (CD4+, Th) cells revealed lower numbers in the PI cohort (Fig. 1E), possibly explaining the lower cytokine secretion in this cohort. Proliferative (KI-67+) Th cells were notably lower, while proliferative cytotoxic T cells (CD8+, Tc) were higher than in the HC cohort. There were no notable differences in the Th1 cell subsets, total NK cell, and NKT cell numbers between the two groups (Fig. 1E). Further classification of the cell subtypes revealed lower cytotoxic NK cells, and higher functional NKT cells in the PI cohort (Supplementary Fig. 1). Analysis based on COVID-19 severity suggested higher total NKT cell numbers in critical patients, with no difference in T and B cell subtypes (Supplementary Fig. 2).
Excessive inflammatory response, lymphopenia, and cytokine release syndrome are associated with the development of acute respiratory distress syndrome (ARDS) and fatal cases.7 Thus, a coordinated resolution of inflammation plays a pivotal role in minimizing organ damage8 and keeping a homeostatic balance to avoid vicious immune depletion.9 In fact, the level of circulating cytokines is indicative of an inflammation state. High expression of proinflammatory cytokines is correlated with severe COVID-19,7 while its restoration to a normal level is expected upon recovery to facilitate the repair of tissue.10
During the study period, none of the PI group reported reinfection. Four patients visited the outpatient clinic at least once due to respiratory-related complaints, including shortness of breath, bronchitis, and persistent cough. The clinically unperceived immune disequilibrium observed in the PI cohort suggests ongoing inflammation and immune consumption. It is intriguing to observe further the clinical impact among patients who recovered from acute episodes of COVID-19.
This study is limited by the small sample number. In addition, there were no consecutive sample collections that may explain the dynamic of immune alterations following recovery from COVID-19 infection. The cellular immunity dysregulation found in our result may not be directly correlated to the presence of memory cells and immunity against reinfection of SARS-CoV-2. Moreover, our PI patients were older, and with more profound cardiovascular comorbidities, thus cautious data extrapolation should be considered.
In summary, our post-COVID-19 infection cohort showed an attenuated cytokine secretion along with lower total T cell and Th cell numbers compared to healthy controls. In addition, the cohort showed a high TNF-α /IL-10 ratio possibly indicating persistent immune dysregulation. While the recovered individuals reported no obvious post-COVID sequelae, the findings should increase cautiousness to occult immune exhaustion, and further study on ongoing chronic inflammation is warranted.
Ethical statement
Ethical approval (N202107069) was obtained from Taipei Medical University Joint Institutional Review Board. Written informed consent was obtained from each participant before enrollment.
Funding
The study was financially supported by Wan Fang Hospital Taipei Medical University, Taiwan (111-wf-eva-36) and the National Science and Technology Council, Taiwan (111-2314-B-038-147).
Declaration of Competing Interest
The authors hereby declare no competing interest.
Footnotes
Supplementary data associated with this article can be found in the online version at doi:10.1016/j.jinf.2023.02.025.
Appendix A. Supplementary material
. List of markers used to determine immune cell population.
. Number of NK cells and NKT cells as percentage of total PBMC count (EA: early active, CD69+; P: proliferative, KI-67+; F: functional, IFN-γ+; C: cytotoxic, CD107a+). * p value < 0.05; ** p value < 0.01; *** p value < 0.001.
Number of respective immune cells as percentage of total PBMC count (EA: early active, CD69+; P: proliferative, KI-67+; F: functional, IFN-γ+; C: cytotoxic, CD107a+). * p value < 0.05; ** p value < 0.01; *** p value < 0.001.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
. List of markers used to determine immune cell population.
. Number of NK cells and NKT cells as percentage of total PBMC count (EA: early active, CD69+; P: proliferative, KI-67+; F: functional, IFN-γ+; C: cytotoxic, CD107a+). * p value < 0.05; ** p value < 0.01; *** p value < 0.001.
Number of respective immune cells as percentage of total PBMC count (EA: early active, CD69+; P: proliferative, KI-67+; F: functional, IFN-γ+; C: cytotoxic, CD107a+). * p value < 0.05; ** p value < 0.01; *** p value < 0.001.