Broadly Reactive SARS-CoV-2-Specific T-Cell Response and Participation of Memory B and T Cells in Patients with Omicron COVID-19 Infection

January 2022 onward, India witnessed a sudden increase in Omicron COVID-19 infections, having a mild course that prompted us to identify the key host factors/immune molecules modulating disease course/outcomes. The current study evaluated the percentages of lymphocyte subsets by flowcytometry, SARS-CoV-2 specific T-cell immune response by ELISPOT, estimation of plasma cytokine/chemokine levels on a Bio-plex Multiplex Immunoassay System and anti-SARS-CoV-2 IgG levels by enzyme-linked immunosorbent assay in 19 mild Omicron infected patients, 45 mild SARS-CoV-2 (2020) patients and 36 uninfected controls from India. Natural killer cells, B and memory B cells were high in vaccinated and total Omicron-infected patients groups compared to the mild SARS-CoV-2 (2020) patient group, while CD8+ T cells were high in total Omicron-infected patients group compared to the uninfected control group (p < 0.05 each). Omicron-infected patients had T-cell response against SARS-CoV-2 whole virus, S1 proteins (wild type and delta variant) in 10 out of 17 (59%), 10 out of 17 (59%), and 8 out of 17 (47%), respectively. The current study of Omicron-infected patients elucidates broadly reactive antibody, T-cell response, and participation of memory B and T cells induced by vaccination/natural infection. The limited effect of Omicron's mutations on T-cell response is suggestive of protection from severity. Pro-inflammatory IL-6, IFN-γ, chemokines CCL-2, CCL-3, CCL-4, CCL-5, and IL-8 as potential biomarkers of Omicron infection may have future diagnostic importance. The cellular immune response data in Omicron-infected patients with parental Omicron lineage could serve as a starting point to define the readouts of protective immunity against circulating Omicron subvariants.


Introduction
All viruses change over time, which causes little to no impact on the virus's properties, such as its transmission, associated disease severity, performance of vaccines, therapeutic medicines, diagnostic tools, or other public health and social measures [1].SARS-CoV-2 has consistently mutated over the course of the pandemic, resulting in variants that are different from the original SARS-CoV-2 virus [2].The emergence of these variants of interests and variants of concerns (VOCs) during late 2020 posed an increased risk to global public health and the ongoing response to the COVID-19 pandemic.
Alpha, Beta, Gamma, and Delta were reported as VOCs causing increased risk to global public health [3].Since November 26, 2021, when WHO designated the variant B.1.1.529a VOC as Omicron, researchers around the world have been fiercely carrying out studies to have a better understanding of various aspects of Omicron [3,4].Omicron has more than 30 mutations in the Spike protein and 15 in the receptor binding domain (RBD) of the Spike protein that have immune evasive potential [5].The emergence of Omicron has caused a sharp increase in new COVID-19 infections worldwide, even in vaccinated individuals, raising concerns about immune escape.Omicron was swiftly recognized as being significantly more transmissible than Delta, though it caused less severe disease than Delta, globally [6].Despite the fact that Omicron poses a lower risk of death and serious illness than the previous SARS-CoV-2 variants, extremely high levels of transmission have resulted in a significant increase in hospitalization, continue to place tremendous strain on health-care systems in the majority of countries, and may cause significant morbidity, especially in vulnerable populations [6,7].
Epidemiological and other immunological studies could shed light on factors contributing toward the increased number of people testing positive with this variant.Studies on the epidemiology of SARS-CoV-2 Omicron subvariants have shown that the Omicron lineages have advanced mutations in the entire genome, including the spike and RBD regions.All of these heightened mutations have the ability to affect the biological characteristics of the Omicron lineages, causing immunological escape and increased transmissibility compared to the earlier VOCs [8].
There are immunological studies highlighting on the factors, mostly antibodies, contributing toward the increased number of people testing positive with Omicron.Deletions, substitutions, or mutations in the RBD and ACE2 interaction regions may reduce the neutralizing activity of vaccineinduced mAbs, convalescent plasma, and serum, as well as have an effect on antibody binding [9].The BA.2, BA.2.12.1, and BA.4/5 variants were almost resistant to therapeutic mAbs, casirivimab, idevimab, sotrovimab, cilgavimab, and evusheld in contrast to their substantial neutralizing activity against the previous VOCs [10].These results collectively indicate that further research into humoral and cellular immune response in Omicron subvariants is necessary.The effectiveness of previous SARS-CoV-2 infections and the effectiveness of vaccines toward reinfection with Omicron are partially explored.The SARS-CoV-2 Omicron variant has continued to evolve with increasing immune escape due to lower neutralizing action by mAbs, vaccination, and past SARS-CoV-2 infection [8,11].
Cumulatively, these reports suggest that there is a need to explore more about humoral and cellular immune response due to substitutions/deletions/insertions in Omicron subvariants.Thus, a potential threat has been generated due to the emergence of the Omicron variant to public health and economy.
The Omicron variant had been confirmed in 149 countries as of January 6, 2022 [12].India experienced a sudden increase in COVID-19 cases (120% increase) with the Omicron variant since January 2022, with the severity of the disease being lesser than that observed with other VOCs [12,13].Initial reports have highlighted a drastic reduction in the neutralization efficacy of infection and a decrease in the vaccine-elicited antibodies against Omicron.However, is Omicron capable to escape cellular immune responses?And if yes, then to what extent is not yet clear [14].T-cell responses are a key platform to clear viral infections, to induce B-cell activation for generating antibodies, and to help in providing protection from disease by eradicating virus-infected cells.If mutations in Omicron result in T-cell escape, it could also limit the protection provided by T cells.Six further Omicron subvariants, BQ.1, BA.2.75, CH.1.1,XBB, XBB.1.16,and XBF, are reported as variants under monitoring [15].Considering the scenario, it is important to identify the key host factors/ immune molecules that modulate the disease course and outcome of patients with Omicron COVID-19 infection that seemingly escapes neutralizing antibodies.In order to better understand the dynamics and diversity of cellular immune responses, greater focus should be placed in the future on how mutations affect particular T-cell immune responses.It is important to understand SARS-CoV-2 specific T-cell immunity in the individuals infected with Omicron lineages, as well as in possible future SARS-CoV-2 variants.With this background, the percentages of peripheral lymphocyte subsets (flow cytometry), T-cell response (ELISPOT), and cytokine profile (Bioplex Multiplex platform) were assessed in 19 mild Omicron COVID-19 patients, 45 mild SARS-CoV-2 (2020) patients, and 36 uninfected controls from Pune, Maharashtra, India.[16,17].The participants were categorized into two groups, vaccinated individuals with breakthrough infections (n = 15, COVISHIELD (ChAdOx1 nCoV-19), COVAXIN(BBV152), and Pfizer (BNT162b2 mRNA) vaccine) and unvaccinated individuals with 1st time infection (n = 4).Detailed characteristics of the Omicron-infected patients are listed in Table 1.The samples from Omicron-infected individuals were collected on 9 (2-17) days post-onset days of symptoms.Thirty-six healthy individuals from the blood donation camps organized by Sassoon General Hospital, Pune, were recruited as uninfected healthy controls for comparison.The uninfected healthy controls were negative for anti-SARS-CoV-2 IgG antibody, as using commercial enzyme-linked immunosorbent assay (ELISA (COVID Kavach-Anti-SARS-CoV-2 IgG Antibody Detection ELISA, M/s Cadila Healthcare Limited, Ahmedabad)) were only included.The healthy controls were unvaccinated.

Materials and Methods
Out of 36 uninfected subjects, we have immune phenotyping data of 18 uninfected subjects, whereas levels of plasma concentrations of cytokines, chemokines, and growth factors were carried out in all 36.The immune phenotyping data of the Omicron-infected patient group (n = 19) were compared with anti-SARS-CoV-2 IgG antibody-negative uninfected controls (n = 18) and mild COVID-19 (2020) Indian patients (n = 45).Blood samples from the mild COVID-19 (2020) patient group were collected in the acute phase during hospitalization (≤6 days).Anti-SARS-CoV-2 2 Journal of Immunology Research  All the Omicron-infected cases had a milder course of infection and recovered eventually.M, male; F, female; NA, not-available; NA * , not applicable; HTN, hypertension/hypotension (blood pressure).

Journal of Immunology Research
IgG antibody and PRNT levels were determined using S1 RBD, N protein, COVID KAWACH ELISA in the plasma of 19 patients with Omicron infection [13,14], and the details are provided in Supplementary 1.
The immune cell profile and T-cell response were correlated with the IgG antibody levels against anti-SARS-CoV-2 S1-RBD, N protein, and whole virus inactivated antigen.
A schematic representation of the study outline is provided in Supplementary 2.

Flow Cytometry Analysis
Briefly, PBMCs were incubated with fluorochrome-tagged antihuman monoclonal antibodies for 30 min in the dark.After washing, the cells were fixed with 2% paraformaldehyde.The cells were acquired on FACS Aria II (BD Biosciences, San Jose, CA, USA).For each experiment, 50,000 events were acquired within the lymphocyte gate with appropriate isotype control.Data were analyzed using FACS Diva software (Becton Dickinson, San Jose, CA, USA), and results are expressed as the percentage of positive cells in the gated population.The gating strategy is depicted in Figure 1.

SARS-CoV-2-Specific T-Cell ELISPOT Assay. SARS-
CoV-2 specific T-cell response in terms of IFN-γ release by ELISPOT assay was performed in Omicron COVID-19 cases (n = 19).ELISPOT assay was carried out as previously described [18][19][20].To estimate the number of SARS-CoV-2 specific IFN-γ secreting spot-forming cells (SFCs), PBMCs were stimulated with gamma-irradiated SARS-CoV-2 whole virus antigen, recombinant S1 protein (wild type) (SARS-CoV-2(2019-nCoV) spike S1(D614G), His Recombinant Protein, Sino Biological, USA) and recombinant S1 protein (delta variant) (SARS-CoV-2 Spike S1 (E154K, L452R, E484Q, D614G, P681R His Recombinant protein Sino Biological, USA)).Wells without any antigen served as negative controls, while those with 10 µg/mL of phytohemagglutinin (Sigma-Aldrich, USA) served as positive controls.All assays were performed in triplicates.The IFN-γ SFCs were counted on an ELISPOT reader, customized software (AID GmbH, Strassberg, Germany), and were expressed as the number per 10 5 cells.The cutoff level for SFCs was set as the average number of SFCs in the negative control wells.Results with high background readings or with no PHA responses were excluded.The number of SFCs in unstimulated wells was subtracted from the number in the antigen-stimulated wells in each subject category for comparison.
2.5.Software and Statistical Analysis.The statistical analyses were performed using IBM SPSS Statistics 25 software (SPSS Inc., Chicago, IL, USA).The Mann-Whitney U test was used for the comparison among the study groups.The mean of triplicate experiments in ELISPOT assay was considered for the analysis.Levels of all analytes were analyzed after log 10 transformation of the observed concentrations of individual cytokines/chemokines/growth factors.Receiver operating characteristic (ROC) analysis was performed using GraphPad Prism 8 software (GraphPad, San Diego, CA, USA).All the data are expressed as median (range).A p-value of <0.05 was considered significant.The dot plots were generated on GraphPad Prism 8 software (GraphPad, San Diego, CA, USA).

Characteristics of the Study Population.
The characteristics of the study groups are depicted in Table 1.Most of the study patients were harboring BA.1.1.529VOC, while three were infected with BA.1, a subvariant of Omicron.The Omicron COVID-19 patients were grouped based on their vaccination status.The study population consists of the following   2, Figure 2).However, the percentages of NK and NKT-like cells were comparable among total Omicron COVID-19 patients and uninfected control groups (Table 2, Figure 2).2) (p<0:05 in each).Further, the homeostatic distribution of CD4 + and CD8 + T cells based on CD62L and CD45RA expression in terms of naïve (CD62L + CD45RA + )/memory (CD62L + CD45RA − )/ effector memory (CD62L − CD45RA − )/terminally differentiated (CD62L − CD45RA + ) subsets were analyzed among the study groups.
In CD4 + Th-cell compartment, the percentage of the central memory population was found to be significantly high in the vaccinated Omicron COVID-19 patient group compared to the uninfected controls (p<0:05) (Table 2, Figure 3).The percentages of CD4 + T EMRA cells were significantly low in the total Omicron-infected patient group and in the vaccinated Omicron COVID-19 patient group compared to the uninfected controls (p<0:05 in each) (Table 2, Figure 3).However, the percentages of naïve and effector CD4 + memory T cells were comparable among unvaccinated total and vaccinated Omicron COVID-19 patient groups and uninfected controls (Table 2, Figure 3).
In the CD8 + T-cell compartment, the percentages of naïve and central memory populations were significantly higher, while the effector memory population was significantly lower in vaccinated and total Omicron COVID-19 patient groups compared to the uninfected control group (p<0:05 in each) (Table 2, Figure 3).The percentages of CD8 + T EMRA cells were comparable between Omicron infected (both vaccinated and total) and uninfected control groups (Table 2, Figure 3).

Correlation between Anti-SARS-CoV-2 IgG Antibody
Levels and CD8 + Terminally Differentiated T and Memory B Cells Frequencies.A lack of correlation was observed between anti-SARS-CoV-2 IgG antibody levels against the whole virusinactivated antigen and the percentages of CD4 + naïve cells (R = −0.4995,p ¼ 0:0294).A positive correlation was observed between anti-SARS-CoV-2 IgG antibody levels against the whole virus-inactivated antigen and the percentages of CD8 + terminally differentiated T cells (R = 0.5034, p ¼ 0:028) and memory B cells (R = 0.6127, p ¼ 0:0053) (Table 3, Supplementary 1).4).Notably, a single Pfizervaccinated Omicron-infected patient and one of the four unvaccinated Omicron-infected patients had spontaneous IFN-γ responses in unstimulated as 40.6 and 24 SFCs/10 5 cells, respectively.Hence, the data of these two were excluded from analysis, and the data of only 17 Omicron COVID-19 patients were taken into account.

Discussion
This investigation is one of the few to examine clinical and immunological profiles of individuals infected with Omicron SARS-CoV-2 from India, a major site for COVID-19 infection.All the enrolled symptomatic patients, whose samples were collected between the time frame 2-17 PODs, were experiencing similar symptoms at the time of sampling, and hence their immune responses were expected to be consistent.Liu et al. [25] have reported neutralizing antibody activities from two doses of ChAdOx1 nCoV-19, mRNA-1273, or MVC-COV1901 immunizations followed by a booster dose of mRNA-1273, which were able to induce detectable neutralizing antibodies against the Omicron variant.They have suggested that in addition to neutralizing titers, T-cell response may play a role in vaccine effectiveness [25].

Journal of Immunology Research
In another study by Dimeglio et al. [26], where the authors compared the concentrations of binding antibodies before breakthrough infections with Delta or Omicron SARS-CoV-2 variants and suggested that infections with the Omicron variant can occur despite high binding antibody concentrations.
Our T-cell data are in line with the aforementioned recommendations and with the related report by Keeton et al. [27], suggesting that Omicron's mutations have a limited impact on the T-cell response irrespective of vaccination or prior infection status and may still offer significant protection from severe disease.Camilla Mattiuzzi et al. [28] reported that lower severity of illness caused by the SARS-CoV-2 Omicron variant and the efficacy of vaccination contributed to lower SARS-CoV-2 pathogenicity.The detected reduction in the percentages of CD4 + Th cells and rise in the percentages of CD8 + Tc cells in the total Omicron COVID-19 patients' group and in the vaccinated Omicron COVID-19 patients group could be suggestive of a skewed immune response toward a more cytotoxic cellular immune response.This could be representing a scenario of the immune system's overall balance due to the disease/vaccination status.In a similar way, the detection of higher CD8 + T cells, CD8 + naïve T-cell subsets along with broadly reactive vaccine-generated T-cell response in vaccinated individuals of the Omicroninfected group could be associated with a milder course of infection.Observed higher B and B memory cells, crucial to prolonged protection after vaccination, and higher NK and NKT-like cells responsible for a robust adaptive immune response could be the molecules for disease resolution.
Further key finding of our data is that both vaccinated and total Omicron-infected patients displayed similar T, B, and NK cells immune responses following infection.Overall, this cumulative scenario has led to the generation of an Omicroninduced immune response that has resulted in the manifestation of a mild course of infection irrespective of the vaccination status of the patients.
Antibodies and memory B cells are considered correlates of protection against various viral infections [29].However, anti-SARS-CoV-2 IgG antibodies may not be serving the purpose as evident from the reports of reinfection in seropositive recovered/ breakthrough infections [30,31].A lack of correlation between anti-IgG antibody levels against an antigen and the percentages of CD4 + naive T cells could be related to different types of immune responses generated and also to the factors responsible for the complexity of the immune system.B cells are primarily accountable for producing anti-IgG antibodies against a particular antigen.CD4 + naive T cells are a subset of T cells and are vital for directing and controlling the immune response to different infections.These discrete immune system components can react to different stimuli independently.SARS-CoV-2 could trigger B cells to produce anti-IgG antibodies.On the other hand, memory B cells may be in charge of generating anti-IgG antibodies upon re-exposure if the person has already been exposed to the antigen.Nevertheless, it is possible that the secondary immune response does not involve CD4 + naive T cells as much.This could result in no association between the two parameters.Thus, the diversity in immune mechanisms, immunological memory, and antigen specificity  The area under the curve (AUC) values of the above analytes were greater than 0.9 (p<0:0001), which is indicative of higher diagnostic value.The high sensitivity and specificity of the analytes' cutoffs suggest that they can potentially act as biomarkers of Omicron infection.16 Journal of Immunology Research could be the factors responsible for the lack of correlation between SARS-CoV-2 specific anti-IgG antibody levels and the percentages of CD4 + naive T cells in this study.CD4 + T EMRA cells are a subset of memory T cells that have previously encountered a pathogen and are ready to respond more quickly upon reinfection.However, their precise role in various viral infections can vary.The behavior of CD4 + T EMRA cells can be complex and context-dependent.They can play both beneficial and detrimental roles in different viral infections.Anft et al. [32] have reported lower frequencies of terminally differentiated T-cell subsets in patients with both severe and critical COVID-19 disease.Further, a report by Jae Jung et al. [33] showed lower expression of T EMRA cells in COVID-19 convalescent patients for 10 months, indicating that it may take some time for the immune system to normalize postinfection with COVID-19.Thus, the current data of lower phenotypic CD4 + T EMRA cells during Omicron COVID-19 infection is an expected phenomenon.A positive correlation of IgG antibody levels against the whole virus-inactivated antigen and the percentages of CD8 + terminally differentiated T and memory B cells (Table 3) in the currently studied patients suggest the need to look for multiple correlates of protective immunity against COVID-19.
Our ELISPOT data displayed functional SARS-CoV-2 specific T-cell response irrespective of the recall antigen, indicating the presence of vaccine-induced broad T-cell immunity in vaccinated and total Omicron-infected patients.It could have implications for understanding the robustness of immunity following natural infection and/or vaccination.The emergence of pro-inflammatory cytokines IL-6 and IFN-γ and chemokines CCL-2, CCL-3, CCL-4, CCL-5, and IL-8 as potential biomarkers of Omicron infection might have future diagnostic importance [24] Wong et al. [34] reported in a Chinese population that IFN-γ, IL-12, IL-1β, and IL-6 can induce hyper-innate inflammatory response due to invasion of the respiratory tract by SARS-CoV, leading to the activation of Th1-cell-mediated immunity by the stimulation of NK and cytotoxic T lymphocytes.In a similar line, our study demonstrated elevated levels of IL-6 and IFN-γ, which might induce hyper-innate inflammatory response and might be leading to a higher percentage of NK cells in both vaccinated and total Omicron-infected individuals.It is important to note that CCL5/RANTES is a chemokine important for T-cell homing and migration during acute virus infections.[35,36].Elevated levels of CCL-5 in our study might have a similar role in the group of Omicron-infected patients.
The absence of antigen-specific immune cell profile data and immune response data of vaccinated mild SARS-CoV-2 (2020) patients could be taken as the limitations of our study.Despite that, our study demonstrated some key findings on immune response in patients with Omicron infection.

Conclusion
In summary, our study in Omicron-infected SARS-CoV-2 patients elucidates broadly reactive antibodies as well as T-cell response and participation of memory B and T cells induced by vaccination or natural infection, which could be contributing toward protection from severe COVID-19.The current data of cellular immune response in Omicroninfected patients with parental Omicron lineage may form the basis to define the readouts of protective immunity against circulating Omicron subvariants.

3. 2 . 2 .
Percentages of B and Memory B Cells.The percentages of B and memory B cells were significantly high in both in

FIGURE 1 :
FIGURE 1: Gating strategy to distinguish different lymphocyte populations by flowcytometry.PBMCs from the study participants were stained with panels of fluorochrome-labeled antibodies to assess the frequency and immune profile.The numbers in the histogram are the mean of the cell population representing for the study group.(a) Lymphocytes and NK cells (CD3 − CD56 + ), NKT-like (CD3 + CD56 + ) cells profile; (b) B(CD19 + ) cells and memory B (CD19 + CD27 + ) cells profile; (c) T-helper (CD3 + CD4 + ), T-cytotoxic (CD3 + CD8 + ) cells profile, memory Th and Tc cells, CD4 + naïve T cells, CD4 + T central memory cells effector cells, T EMRA cells.
2.1.Ethical Approval Declarations.The study was approved by the Institutional Ethical Committee for Research on Humans, based on the guidelines set by the Indian Council of Medical Research, New Delhi.Informed consent had been obtained from all participants.2.1.1.Study Subjects.The current study was carried out between December 2021 and February 2022.The enrolled Omicron COVID-19 patients (n = 19) were mostly the foreign returnees (UAE, South/West/East Africa, Europe, Middle East, USA, and UK) and were confirmed to be positive for Omicron by next-generation sequencing

TABLE 1 :
Characteristics of the study population.

Table 2
+ , CD8 + T Cells and Memory T-Cell Subsets.The percentage of CD4 + Th cells was significantly lower, while CD8 + Tc cells were significantly higher in the total Omicron COVID-19 patients group and the vaccinated Omicron COVID-19 patients group compared to the total mild SARS-CoV-2 patients (2020) group and uninfected control (Table2, Figure

TABLE 5 :
ROC characteristics of the cytokines analysis in Omicron COVID-19 patients.-infected patients and uninfected controls.Levels of all analytes were analyzed post-log 10 transformation of the observed concentrations of individual cytokine/chemokine/growth factors.The Mann-Whitney U test was used for numerical data for comparisons among the study groups.A p-value of <0.05 was considered significant.ROC analysis was performed using GraphPad Prism 8 software, AUC, area under the curve.