Highlights•

Moderate and severe/critical groups had high inflammation at presentation.

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Inflammation did not normalise for the severe/critical at 2 weeks.

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Mild and moderate groups had good T and B memory responses at 2 weeks.

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Severe/ critical group had an exhausted immune response at 2 weeks.

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By 24 weeks, Severe/ critical group had good memory B and T cell responses.

AbstractBackground

: Limited data on short versus long-term changes in adaptive immune response across different COVID-19 disease severity groups.

Methods

: A multi-center prospective study of 140 adult patients with COVID-19 (a total of 325 samples) were analysed for inflammatory markers and lymphocyte subsets at presentation, weeks 2 and 24.

Results

: Inflammatory markers at presentation were higher in the critical/severe than in moderate and mild. A predominance of memory B cell response in the mild and moderate group was noted by week 2. In contrast, the immune system in the severe/critical was dysfunctional with expansion of exhausted CD8+T cells and atypical memory B cells. By 24 weeks, there was a possible trend of normalization.

Conclusion

: There was substantial difference in the degree of inflammation and distribution of different B and T cell subsets in the different disease severity groups. Despite the initial dysfunctional immune response in the severe/critical group, a comparable memory B and CD8+T cell responses to the mild group was achieved at 24 weeks.

Key words

Introduction:

The spectrum of COVID-19 disease ranges from asymptomatic infection to severe respiratory syndrome with systemic inflammation (Huang, Wang, et al. 2020). Patients with severe disease usually have significantly elevated inflammatory markers such as high C-reactive protein (CRP) and a significant increase in the ferritin and lactate dehydrogenase (LDH) levels compared to mild cases (Chen et al. 2020; Velavan and Meyer 2020). Moreover, evaluation of lymphocytes especially T cells and subsets can help in assessing the severity and progression of COVID-19 disease, particularly in hospitalized patients (Zhang et al. 2020; Huang, Berube, et al. 2020).

There are some recent studies that have looked at different aspects of longevity of immunity to COVID-19 (Peluso et al. 2021; Dan et al. 2021; Shuwa et al. 2021). Some looked at the kinetics of CD4+T cells, CD8+T cells and B cells over 8 months in hospitalized versus non-hospitalized patients (Dan et al. 2021). Memory B cells persisted for 8 months while memory CD4+ (effector memory, TEM and central memory, TCM) and CD8+ (terminal differentiated effector memory, TEMRA) declined with half-life of ∼ 4 and 6 months (Dan et al. 2021). Another study examined the immunological differences between different groups (mild, moderate, and severe) of COVID-19 infection and compared acute versus overall convalescent group (from all initial groups) (Shuwa et al. 2021). It has shown that patients with severe disease had reduced transitional B cells and increased double negative compared to other groups among other alterations, but all of these have reverted to normal at 6 months post infection (Shuwa et al. 2021). In contrast, the cytotoxic changes among CD8+T cells persisted at 6 months in the convalescent patients (Shuwa et al. 2021). Peluso et al has examined immune response in non hospitalised versus hospitalised patients at 2 months, 4 months and 8 months. They have shown that specific CD4 and CD8+T cells were detected even at 8 months in both examined groups (Peluso et al. 2021).

There is limited literature that have looked at short versus long-term changes in the immune T and B subsets across the different groups of COVID-19 in relation of their inflammatory milieu. Moreover, none of these has examined the long-term immune effect among the different groups to find out if either the mild or severe has developed different memory pattern. Therefore, the aim of this prospective study was to assess changes of T and B subsets in different severity groups of COVID-19 infection at presentation and track their changes at 2 weeks and at 24 weeks post infection.

Methods:

Study design:

Total of 140 community-acquired of COVID-19 cases were recruited in the study in the period between June 1st-November 30th 2020. All patients met the WHO case definition which is symptomatic patient with a positive SARS-CoV-2 Nucleic Acid Amplification Test. Inpatients were all new positive cases getting admitted through emergency department and recruited after consenting. Outpatients were mainly healthcare workers or their relatives who came to the family medicine & public health department who tested positive and consented to participate. All patients recruited from this clinic were mild cases. However, there were six mild cases (using WHO criteria) admitted for different reasons such as managing dehydration, colitis, bowel ischemia, and a premature rupture of membranes in a pregnant woman. At presentation, all patients were stratified into four groups: mild, moderate, severe, and critical based on WHO classification (Table 1). Serum and whole blood samples were collected for biochemical tests (CRP, LDH, and ferritin) and lymphocyte subsets, respectively.

The tests were performed at presentation (day 0) and at 2 weeks after diagnosis. In addition, other time points were included: on day 7 (moderate category) and on days 3 and 7 (severe and critical categories). Moreover, lymphocyte subset testing was performed for the mild and severe/critical group at 24 weeks post presentation. Data sheet was designed to collect relevant patient's demographic, clinical, laboratory and radiological data as presented in tables 2 and 3.

Laboratory parameters:

Cobas s311 analyser was used to measure CRP, LDH and ferritin. DuraClone IM T and B cell subsets tubes were used for assessment of different subsets. Used clones for each CD markers are listed in table 4.

A total of 100,000 events were collected. Briefly, 100μl of blood was added to the tube containing the desired cocktail of antibodies and incubated for 20 minutes at room temperature. 100μl of lysing solution optilyse-B or Versalyse was added according to the manufacture's (Beckman coulter) recommendation. This was followed up with a wash step. Acquisition of samples and analysis was done using Navios and Kaluza version 2.1. Gating strategies included are illustrated in Supp. Figure 1 and 2. Some of CD19+ B cells, CD+4 and CD8+ T-cells subsets summarized in Table 5.

Statistical analysis:

All data were entered and analyzed using SPSS software ver. 22.0 (IBM, Armonk, NY, USA) and GraphPad Prism software v.6.0 (GraphPad Software, San Diego, CA, USA). Group comparisons were performed using Mann-Whitney U-test (2 groups comparisons), Kruskal-Wallis Anova test (for >2 groups comparisons) and chi-square test. To adjust for days from onset of symptoms, linear regression analysis was performed. Analysis of variance was used to analyze the laboratory parameters each time point. Immune cell markers were analyzed using fold changes between different severity groups (moderate/mild, severe & critical/mild, and severe & critical/moderate) and between two weeks over baseline in each severity group. P-value was adjusted for multiple comparison using false discovery rate (FDR) and data are presented in volcano plot in which significant hits were identified by fold change of 1.3 and FDR adjusted p-value <0.05.

Results:

Demographic and clinical characteristics:

From the 140 adult patients, a total of 325 samples were analysed for inflammatory markers and 147 samples were assessed for the different distribution of immune subsets.

None were vaccinated against SARS-CoV-2 or previously infected. Out of the 140 patients, 54(38.6%) had mild, 29 (20.7%) moderate and 57 (40.7%) had severe/ critical disease. Out of the severe/critical group, 28 (49.1%) had ICU admission and 25 (49%) required mechanical ventilation. On the other hand, 1 (2%) mild case had ICU admission due to bowel ischemia and 1 (3.4%) moderate case progressed over time to severe and admitted to ICU. Eleven patients (19.3%) from the severe/ critical group died within 30 days of admission. All alive patients were discharged before week 8 of their presentation to the hospital. Older age, hypertension, fever, shortness of breath, chest infiltrates, oxygen requirement, acute respiratory distress syndrome (ARDS), and mechanical ventilation showed statistically significant difference in relation to disease severity in the current study (Tables 3). Of interest, the gastrointestinal manifestations were seen mainly in patients with moderate disease. Steroids and antibiotics were used more the severe/critical group (n=50 patients, 98%) compared to other groups (p-value < 0.0001) (Table 4). Of note, some of the patients in the different severity groups were on steroids therapy prior to COVID-19 infection for different indications (e.g., hematological malignancy, solid organ transplant and connective tissue disease). Patients with moderate and severe-critical disease received steroids as part of covid-19 management. On the other hand, mild cases were given short courses of corticosteroids for different clinical indications at the time (asthma, part of induction of a pregnant woman for fetal lung maturation, etc.)

Progression of inflammatory markers;CRPLDH,and Ferritinover 2 weeks.

At the time of presentation, ferritin and CRP levels were significantly higher in severe/critical compared to mild and moderate groups. CRP and ferritin levels follow a down trend in all groups; levels normalized for the mild group, nearly normalized for the moderate but did not normalize in the severe/critical group at week two (Fig 1). On the other hand, LDH did not differ significantly between the three examined groups throughout the analysis time.

Figure 1Levels of CRP (n=325), Ferritin (n=320) and LDH (n=313) over the course of 2 weeks after diagnosis of COVID-19 infection among mild (n=37), moderate(n=23), and severe/critical cases (n=55) (Mean and SEM are plotted). Reference range: shaded area, CRP 0-8 mg/L, ferritin F:11-307 mcg/L, M: 24-336 mcg/L, LDH F: 135-214, M: 135-225 IU/L). * p-value <0.05 linear regression analysis adjusted for days from onset of symptoms.

Frequency of B andTcell subsetsamongdifferentgroupsatpresentation

We first compared fold changes of multiple immune cells between disease severity groups. The transitional B cells were 1.4-fold lower in moderate group compared to the mild group, however this was not statistically significant (FDR p-value= 0.30) (not shown). Patients in the severe/critical had lower frequency of transitional B cells compared to patients in the mild group (fold change 2.2, FDR p-value= 0.02) (Figure 2B). On the other hand, the moderate had lower frequency of CD27+CD28- TCM CD8+ T cell subsets compared to the mild group (Figure2A).

Figure 2The differential expansion and contraction of different T and B cell subsets at the time of presentation between; A) Moderate (n= 18) compared to mild (n=28), B) Severe/critical (n= 22) compared to mild and C) Severe/critical compared to moderate cases. Percentage of cells were used in the analysis. Significantly expanded subtypes are identified by FDR adjusted p-value <0.05 (Mann-Whitney U test) and fold change of above 1.3 in which red labeled subtypes are significantly upregulated and blue ones are significantly downregulated. Blue bars: downregulation fold change values, red bars: upregulation fold change values. Cells details: CD27+CD28- out of TCM (CD197+CD45RA-) CD8+ Tcell; Plasmablast (CD27+CD38+) out of CD19+ B cell; B memory (CD19+CD27+) out of CD45+ leucocyte cell; B naïve (CD19+CD27-) out of CD45+ leucocyte cell; B cell (CD19+) out of CD45+ leucocyte cell; IgM+IgD+ out of memory (CD27+ CD19+) B cell;IgM+IgD- out of memory (CD27+ CD19+) B cell; Transitional (CD24+CD38+) B cell out of CD19+ B cell; TEMRA (CD197-CD45RA+) out of CD8+ T cell and PD-1+CD57+ out CD8 T cell.

On the other hand, moderate group compared to the mild exhibited a significant expansion of B cell responses, higher percentage of circulating total B cells including total naïve (CD19+CD27-) and total memory (CD19+CD27+) B cells. The latter included both natural unswitched (CD27+IgM+IgD+) and pre-switched (CD27+IgM+IgD-) memory B cells. In addition, there was an increased frequency of antibody-secreting cells (ASC) (CD19+CD27++CD38++) (Figure 2B).Patients in the severe/critical compared to the mild group presented with higher frequency of cytotoxic CD8+ (PD-1+CD57+) and highly differentiated (cytotoxic and cytokine producers) CD8+ TEMRA (CCR7-CD45RA+) (Figure 2B).There was no statistically significant difference in the subset's distributions between the moderate and severe/critical groups (Figure 2C).

Overall, there was a predominance of memory B cell response in the moderate group, while cytotoxic T cells predominate among the severe at the onset of SARS-CoV-2 infection.

Frequency of B and T cellsubsets amongdifferent groups, 2 weeks postinfection:

At 2 weeks post presentation, lymphocyte subsets showed a lower frequency of transitional B cells and CD27-CD28+TCM CD4+ T cells in the moderate compared to the mild group (Figure 5A). The initial lower frequency of transitional B cells seen at presentation in the severe/critical compared to the mild group, was maintained and was even broaden at 2 weeks. (Figure 3B). The differential expansion of the memory B cell subsets that was seen at presentation between the moderate and mild groups was lost by 2 weeks.

Figure 3The differential expansion and contraction of different T and B cell subsets at 2 weeks between; A) Moderate(n=18) compared to mild(n=28), B) Severe/critical (n=22) compared to mild and C) Severe/critical compared to moderate cases. Percentage of cells were used for analysis. Significantly expanded subtypes are identified by FDR adjusted p-value <0.05 (Mann-Whitney U test) and fold change of above 1.3 in which blue labeled subtypes are significantly downregulated. Blue bars: downregulation fold change values. Cell details: CD27-CD28+out of TCM (CD197+CD45RA-) CD4+ Tcell; Transitional (CD24+CD38+) B cell out of CD19+ B cell.

The differential expansion of the cytotoxic and effector CD8+ T cell subsets between the severe/critical and mild groups was lost by 2 weeks. In fact, by 2 weeks, severe had more exhausted (PD-1+CD57-) CD8+ T cells (Figure 4C). There was no statistically significant difference in the lymphocyte subset's distributions between the moderate and severe/critical groups (Figure 4C). Therefore, the main finding seems to be that moderate and severe/critical groups had significant lower transitional B cells at 2 weeks compared to the mild group.

Figure 4The differential immune progression of different T and B cell subsets over 2 weeks; A-C, each comparison was made between 2 weeks over basal levels. A) Mild (n=28) B) Moderate (n=18), C) Severe/critical (n=22) cases. Percentage of cells were used in the analysis. Significantly expanded subtypes are identified by FDR adjusted p-value <0.05(Mann-Whitney U test) and fold change of above 1.3 in which red labeled subtypes are significantly upregulated and blue ones are significantly downregulated. Blue bars: downregulation fold change values, red bars: upregulation fold change values. Cell details: Transitional (CD24+CD38+) B cell out of CD19+ B cell; Plasmablast (CD27+CD38+) out of CD19+ B cell; B memory (CD19+CD27+) out of CD45+ leucocyte cell.; CD27+IgD- out of B (CD19+) cell; CD27-CD28- out of TEM (CD197-CD45RA-) CD8+ T cell; CD27-CD28- out of TEM (CD197CD45RA-) CD4+ T cell; PD-1+CD57+out CD4 T cell; B naïve (CD19+CD27-) out of CD45+ leucocyte cell; PD-1+CD57+ out CD8 T cell; CD27-CD28+out of TCM (CD197+CD45RA-) CD8+ T cell; IgM+IgD- out of memory (CD27+ CD19+) B cell.; TEM (CD197-CD45RA-) out of CD8+ T cell; CD45RA- PD-1+ out of CD8+ T cell; CD27-CD28+out of TCM (CD197+CD45RA-) CD8+ T cell; CD27+IgD+ out of B (CD19+) cell; TCM (CD197+CD45RA-) CD8+ T cell; IgM+IgD- out of naive (CD27- CD19+) B cell; CD27+CD28+ out of TEMRA (CD197-CD45RA+) CD8+ T cell; IgM-IgD- out of naive (CD27-CD19+) B cell; CD27+CD28+ out of TEM (CD197-CD45RA-) CD8+ T cell; PD1+CD57-out CD8 T cell; CD27-IgD- out of B (CD19+) cell and CD27+CD28+ out of TEMRA CD197-CD45RA+) CD8+ T cell.

Short term(2 weeks)immune kinetic post SARS-CoV-2 infection:

Expansion of memory B cells and reciprocal contraction of transitional B cells coincide with recovery in the mild and moderate groups. There was an expansion of the total memory B cells (CD19+CD27+) including both natural unswitched (IgD+CD27+) and switched (IgD-CD27+) memory B cells. On the other hand, there was a mild downregulation of transitional B cells during the recovery phase compared to the onset of acute infection (Figure 4A-B).In addition, patients in the moderate group exhibited a significant expansion of the recent naïve B (CD19+CD27-) cells that have started to lose their surface IgD including IgM-IgD- and IgM+IgD- cells. Moreover, there was a reciprocal reduction in total naïve B (CD19+CD27-) and pre-switched memory (CD19+CD27+IgM+IgD-) B cells, circulating plasmablast and an extensive reduction in transitional B subsets cells (Figure 4B).Furthermore, over the course of 2 weeks, there was an expansion of different effector /memory CD8+T cells subsets including intermediate effector CD27+CD28+TEMRA CD8+ T cells, effector TEM CD8+, TCM CD8+, as well as activated CD45RA- PD-1+CD8+ T cells. However, this was accompanied with reduction in the highly differentiated effector and cytotoxic CD4+ and CD8+T cells; highly differentiated effector and cytokine producers (CD27-CD28-) TEM CD4+ and CD8+, cytotoxic (PD-1+CD57+) CD4+T and CD8+, and (CD27-CD28+) TCM CD8+ T cells (Figure 4B).The short immune kinetic in the severe/critical group was marked with expansion of the intermediate effector CD27+CD28+TEMRA CD8+ T cells, CD27+CD28+TEM CD8+ T cells as well as exhausted PD-1+CD57-CD8+T cells. In addition, there was some B cell response including expansion of (IgM+IgD- naïve CD19+ B cell and extrafollicular response characterized by expansion of atypical memory B cells (CD19+CD27-IgD-) B cells. Like the immune response in the moderate group, there was a substantial reduction of transitional B cells at 2 weeks compared to presentation (Figure 4C).

Long term(24 weeks)immune kinetic post SARS-CoV-2 infection:

Examining trends over 24 weeks has focused on subsets with significant fold changes in the 2 weeks follow up compared to the baseline in the mild and the severe groups. This included 9 subsets; memory (CD19+CD27+) B cell, switched memory (CD19+CD27+IgD-) B cell, un-switched memory (CD19+CD27+IgD+) B cell, transitional (CD19+ CD38+CD24+) B cell, IgM+IgD- naïve (CD19+CD27-) and double negative memory (CD19+CD27-IgD-) B cell. It also included CD27+CD28+TEM CD8+ T cells, CD27+CD28+TEMRA CD8+ T cells as well as exhausted (PD-1+CD57-) CD8+T cells (Figure 4 A &C). Despite lack of significance due to the small sample size (9 in both groups) in the 24 weeks follow up, there was an observed trend in most of these subsets.In the mild group, the significant increasing trend seen at 2 weeks among different memory B subsets; total (CD19+CD27+), unswitched (CD19+CD27+IgD+) and switched (CD19+CD27+IgD-) has persisted at 24 weeks post SARS-COV-2 infection. In contrast, the decreasing trend in the transitional B cells seen initially at 2 weeks has plateaued at 24 weeks (Figure 5).Figure 5The differential immune progression of different T and B cell subsets at time; 0, 2 and 24 weeks for the different groups; mild, moderate and severe/critical(unfilled triangle) patients with COVID-19. Plotting only those with significant fold changes from figure 4 A & C. Mild (dark filled circle), at 0 and 2 (n=28), and (n=5), at 24 weeks. Moderate (unfilled square) at 0 and 2 (n=18). Severe (unfilled triangle) at 0 and 2 (n=22), and (n=4), at 24 weeks. Data shown are mean and SEM for percentage of cells from flow cytometry. Significant pvalues <0.05 (Mann-Whitney U test) between groups at each time point indicated as a, b and c. a: between mild and moderate, b: between mild and severe, c: between moderate and severe. Cell details: Transitional (CD24+CD38+) B cell out of CD19+ B cell; B memory (CD19+CD27+) out of CD45+ leucocyte cell; CD27+IgD- out of B (CD19+) cell; CD27+IgD+ out of B (CD19+) cell; CD27-IgD- out of B (CD19+) cell; IgM+IgD- out of naive (CD27- CD19+) B cell; PD-1+CD57-out CD8 T cell; CD27+CD28+ out of TEMRA (CD197-CD45RA+) CD8+ T cell and CD27+CD28+ out of TEM (CD197-CD45RA-) CD8+ T cell.In the severe/critical group, after initial drop in the frequency of circulating transitional B cells, there was an expansion of transitional B cells with a reciprocal contraction of IgM+IgD- naïve B cells during the time from 2 to 24 weeks. Moreover at 24 weeks, (CD27+CD28+ TEMRA) CD8+ T cells continued to increase while (CD27+CD28+ TEM) CD8+ T has declined back to the baseline level. These changes were accompanied with the resolution of the exhausted CD8+ T cells and atypical memory B cell subsets (Figure 5).

Discussion:

Our study demonstrated significant alterations in the frequency of different lymphocyte subsets at the different assessed time points among the examined COVID-19 disease groups. Patients with mild disease had higher transitional B cells compared to patients in moderate and severe groups. The significance of these transitional B cells is still controversial, but more evidence is accumulating to support their role in the robust humoral response against viral infection and production of neutralizing antibodies. This role is supported in recent literatures where higher frequency of transitional B cells was reported in patients with mild COVID-19 disease (Sosa-Hernández et al. 2020; Oliviero et al. 2020).

B cell response in patients with moderate disease at the time of recruitment showed massive memory B cells responses compared to the mild group comprised of germinal central derived B cells including unswitched (CD19+CD27+IgM+IgD+) memory B and pre-switched (CD19+CD27+IgM+IgD-) (Ellebedy et al. 2020), total (CD19+CD27+) memory B (Oliviero et al. 2020) and plasmablast cells. This could be explained knowing that blood was collected on a median of 2 days post symptoms for the mild group and 5 days for the moderate (Auladell et al. 2019). The outstanding expansion of memory B cell subsets seen at recruitment in the moderate group compared to the mild was not sustained at 2 weeks. This might suggest that the mild group had expanded their memory B cell subsets to an equal level (Figure 5) (Sosa-Hernández et al. 2020). In fact, this increase of the switched and unswitched memory B cells is known to be seen in patients with shorter duration of symptoms (Newell et al. 2021; Dan et al. 2021).

The origin of these newly formed memory B cell subsets could be from recruitment of immature transitional B cells and or mature naïve B cells (Zhou et al. 2020) that are differentiated into different memory subsets and plasma cells (Bemark 2015; Carrion et al. 2019) explaining the reciprocal downregulation of these cells. In contrast to the initially seen upregulation, there was a contraction of plasmablasts in the moderate group in the two weeks follow up (Figure5B) and this was thought to be in association with generation of germinal center derived long lived plasma cells (Newell et al. 2021). The expansion of different memory B cell subsets including total, un-switched and switched B memory subsets persisted at 6 months post infection is suggestive of a likely long lasting immunity in examined groups in agreement with previous studies (Dan et al. 2021).

Atypical memory B cells seen in the severe/critical group was reported previously (Oliviero et al. 2020; Woodruff et al. 2020) and found to be associated with reciprocal disappearance of transitional and naïve B cells suggesting that they could be the origin of atypical memory B cells. They are seen usually in inflammatory diseases especially chronic diseases such as systemic lupus erythematosus (SLE) among others (You et al. 2020; Ruschil et al. 2020) . It is reported to be associated with increase in the levels of inflammatory markers such as CRP and production of high level of neutralizing antibodies but not sufficient to control the disease (Woodruff et al. 2020). In fact, it was documented that severe group had higher level of SARS-CoV-2 specific antibodies that are not sufficient to compact the high inflammatory response (Shrock et al. 2020). Despite this initial formation of atypical memory B cell in the severe group, there was expansion of the total and switched memory B cells to a comparable level to the mild at 24 weeks.

After initial antigen exposure and activation, naïve CD8+T cells can differentiate into effector or central memory phenotype. There are different models to explain the pathway toward differentiation of different memory subsets. One of them is the presence of high antigen dose coupled with inflammation (Kinjyo et al. 2015; Joshi and Kaech 2008). High inflammatory milieu and high antigen dose favors the development of effector and cytotoxic CD8+T cells such as TEM and TEMRA (Rao et al. 2020) while lower dose of antigen and less inflammation direct formation of TCM CD8+ T cells (Joshi and Kaech 2008).

The mild had higher frequency of circulating effector CD8+ T cells like TEM and TEMRA (Figure 5) compared to severe group, in line with the important role of T cell response in the control of the infection in the mild group (Steiner et al. 2021). Of note, all observed fold changes over the 2 weeks in the mild group, are not massive (all fold changes are less than 5) which might suggest the prompt initial immune response was enough to handle the infection. These immune responses might explain the mild symptoms and prompt resolution of inflammatory parameters in line with previous findings(Bergamaschi et al. 2021).

Similarly, the moderate group had progressive increase in the TCM CD8+T cells (not statistically significant, not shown) as well as there was progressive expansion of intermediate effector CD27+CD28+TEMRA CD8 SARS-CoV-2 specific T cells that were efficient in handling SARS-CoV2 infection as described previously (Neidleman et al. 2020; Dan et al. 2021). This response might has contributed to the control and dampening response of the highly differentiated effector and cytotoxic (CD27-CD28- TEM) CD4+ and CD8+ T cells in this group.

Inflammatory markers including CRP, ferritin, and LDH at presentation were higher in the critical/severe group than in moderate and mild groups. CRP and ferritin have normalised for the mild and moderate but not completely in the severe/critical group at week two (Figure 1).

Possibly due to the prolonged inflammation, the severe/critical group had initial significant expansion of effector (TEMRA) and cytotoxic (CD57+PD-1+) CD8+T cells (Zheng et al. 2020; Gong et al. 2020) that was not able to control the infection leading to more inflammation. This has progressed during the two weeks into dysfunctional immune response with exhausted (PD-1+CD57-) CD8+T cell(Rha and Shin 2021; Haring, Badovinac, and Harty 2006; Tilstra et al. 2018) and atypical memory B cell formation (De Biasi et al. 2020; Zheng et al. 2020; Ruschil et al. 2020) rather than proper and typical memory B cells. At 24 weeks post SARS-CoV-2 infection, immune response has eventually recovered as frequency of atypical memory B cell and exhausted CD8+ T cell went back to their baseline level. Moreover, there was increase of the effector and cytotoxic (C27+CD28+) TEMRA CD8+T cells and different memory B cell subsets.

Our study had limitations. First, loss of follow up sampling especially at 24 weeks. Second, sequencing of the circulating variants was not available during the study; therefore, it is unclear if this is replicative with other variants. As a future work, it will be interesting to examine the immune kinetics in vaccinated versus non-vaccinated groups presenting with different severity of COVID-19 infection and replicate these finding on a bigger cohort.

Conclusion:

There was substantial difference in the degree of inflammation and distribution of different B and T cell subsets in the different disease severity groups. A good memory B and effector

CD8+T cell responses were formed in the mild and moderate groups that have persisted till 24 weeks post presentation. Despite the initial dysfunctional immune response in the severe/ critical group possibly due to the higher inflammatory milieu, there was a comparable memory B and effector CD8+T cell responses at 24 weeks with the mild group.

Conflict of interest:

The authors declare no conflict of interest.

Fund:

The study was funded by The Ministry of Higher Education and Research and Innovation in Oman (RC/COVID-MED/MICR/20/01).

Ethical approval:

Ethical approval was obtained through the institutional research committees (MREC #212 and AFMS-MREC 010/2020).

Acknowledgments:

This study was funded by The Research Council. We would like to express our great appreciation to the hospital administration(s) at SQUH, AFH and the Medical Research Centre at SQU for their full support to facilitate conduction of this study.

We are particularly grateful for the great assistance of our research assistants; Dalia Abbas, Shiji Sajimon, Nadia Thomas, Dyna Cardiel, and Zaid AlHaji. We thank all the clinical and laboratory staff in COVID-19 team and participants for their assistance.

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Appendix. Supplementary materialsArticle InfoPublication History

Accepted: July 7, 2022

Received in revised form: July 6, 2022

Received: April 12, 2022

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DOI: https://doi.org/10.1016/j.ijid.2022.07.026

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