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. 2023 Feb 9;18(2):e0281444. doi: 10.1371/journal.pone.0281444

Severe and mild-moderate SARS-CoV-2 vaccinated patients show different frequencies of IFNγ-releasing cells: An exploratory study

Eugenio Garofalo 1,#, Flavia Biamonte 2,3,#, Camillo Palmieri 2, Anna Martina Battaglia 2, Alessandro Sacco 2, Eugenio Biamonte 1, Giuseppe Neri 1, Giulio Cesare Antico 4, Serafina Mancuso 4, Giuseppe Foti 5, Carlo Torti 6, Francesco Saverio Costanzo 2,3, Federico Longhini 1,*, Andrea Bruni 1
Editor: Mohd Adnan7
PMCID: PMC9910754  PMID: 36757971

Abstract

Background

Despite an apparent effective vaccination, some patients are admitted to the hospital after SARS-CoV-2 infection. The role of adaptive immunity in COVID-19 is growing; nonetheless, differences in the spike-specific immune responses between patients requiring or not hospitalization for SARS-CoV-2 infection remains to be evaluated. In this study, we aim to evaluate the spike-specific immune response in patients with mild-moderate or severeSARS-CoV-2 infection, after breakthrough infection following two doses of BNT162b2 mRNA vaccine.

Methods

We included three cohorts of 15 cases which received the two BNT162b2 vaccine doses in previous 4 to 7 months: 1) patients with severe COVID-19; 2) patients with mild-moderate COVID-19 and 3) vaccinated individuals with a negative SARS-CoV-2 molecular pharyngeal swab (healthy subjects). Anti-S1 and anti-S2 specific SARS-CoV-2 IgM and IgG titers were measured through a chemiluminescence immunoassay technology. In addition, the frequencies of IFNγ-releasing cells were measured by ELISpot.

Results

The spike-specific IFNγ-releasing cells were significantly lower in severe patients (8 [0; 26] s.f.c.×106), as compared to mild-moderate patients (135 [64; 159] s.f.c.×106; p<0.001) and healthy subjects (103 [50; 188] s.f.c.×106; p<0.001). The anti-Spike protein IgG levels were similar among the three cohorts of cases (p = 0.098). All cases had an IgM titer below the analytic sensitivity of the test. The Receiver Operating Curve analysis indicated the rate of spike-specific IFNγ-releasing cells can discriminate correctly severe COVID-19 and mild-moderate patients (AUC: 0.9289; 95%CI: 0.8376–1.000; p< 0.0001), with a diagnostic specificity of 100% for s.f.c. > 81.2 x 106.

Conclusions

2-doses vaccinated patients requiring hospitalization for severe COVID-19 show a cellular-mediated immune response lower than mild-moderate or healthy subjects, despite similar antibody titers.

Introduction

Infection by Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) is characterized by the development of a complex disease (COVID-19) with a wide range of respiratory [1] and non-respiratory [2–4] symptoms, which may lead to critical illness and death of patients [5]. The immune system reacts to the virus through the innate and adaptive responses. The innate system reacts to SARS-CoV-2 by recruiting specialized immune cells, such as plasmacytoid dendritic cells and macrophages, whereas the adaptive immune system mainly comprises antibody-producing B cells, and CD4+ and CD8+ T cell endowed with helper and effector functionalities (CD4+) [6, 7]. Both humoral and cellular components of adaptive immunity play distinct and complementary roles in disease resolution and protection from infection or re-infection. In an elegant study by Sekine et al. [8], SARS-CoV-2-Specific T-Cells have been characterized in acute and convalescent unvaccinated patients with asymptomatic to mild disease. The authors reported that, while in early acute phase of SARS-CoV-2 infection CD8+ T cell populations mainly expressed immune activation and cytotoxic molecules together with inhibitory receptors, in the convalescent phase SARS-CoV-2 specific T-cells were skewed toward an early differentiated memory phenotype [8]. Therefore, the time from virus exposure determined the emergence of specific memory cells against SARS-CoV-2 [8]. Another study conducted in unvaccinated patients further confirmed that SARS-CoV-2 specific T-cells were present also during asymptomatic SARS-CoV-2 infections, with a similar initial Interferon-Gamma (IFNγ) secreting T-cell count to severe COVID-19 patients [9].

Anti-SARS-CoV-2 vaccines are the most important preventive strategy against critical forms of COVID-19 [10]. In the beginning, the vaccination with theBNT162b2 vaccine consisted of two consecutive mRNA doses administered 21 days apart [11]. The first cycle of vaccination induces a spike-specific humoral and cellular immune response that was shown to be efficient in 95% of naïve individuals [12]. However, the immune response induced by the dual doses of BNT162b2 vaccine wanes over a period of months, deeming necessary a “booster” dose [10, 13, 14], particularly in immunosuppressed patients [15].

In patients with advanced age or comorbidities, hospitalization and critical COVID-19 have been reported even if recently vaccinated [16] and the efficacy of vaccination wanes over time [14]. These patients presented a low whole blood IFNγ release, despite high anti-Spike IgG titers [16]. Another study suggests that the BNT162b2 mRNA vaccine provides a different (i.e., poorer) immune response (including IFNγ-secreting T-cell counts) in older COVID-naïve adults, as compared to younger cases [17].

Despite the growing knowledge on the role of adaptive immunity in COVID-19, from a clinical point of view there remains the urgency to define correlates of protection or risk of severe disease in vaccinated patients. In this study, we aim to evaluate the spike-specific immune responses in patients with severe or mild-moderate SARS-CoV-2 infection, after breakthrough infection following two doses of BNT162b2 mRNA vaccine.

Materials and methods

After obtaining local Ethical Approval (approval number 54/2022, 17th February 2022) and in accordance with the Declaration of Helsinki and the principles of the Good Clinical Practice guidelines, we conducted this prospective cohort study from April to May 2022, including adults which received the two BNT162b2 vaccine doses in previous 4 to 7 months. The trial was prospectively registered on clinicaltrials.gov on 19th April 2022 (registration number: NCT05338736). Written informed consent was obtained from all participants. All individual, de-identified datasets generated during and/or analyzed during the study are available from the corresponding author on reasonable request.

Population

All consecutive adult (≥18 years/old) conscious patients were screened at the emergency department and/or hospital admission if referred to our center from other hospitals without COVID-19 wards.

We enrolled 3 cohorts of 15 consecutive cases: 1) vaccinated patients with a positive SARS-CoV-2 molecular pharyngeal swab with severe to critical COVID-19, as defined by a peripheral oxygen saturation <94% in room air and therefore requiring hospitalization (severe patients); 2) vaccinated patients with a positive SARS-CoV-2 molecular pharyngeal swab with pauci- or asymptomatic COVID-19 not requiring hospitalization (mild-moderate patients) and 3) vaccinated individuals with a negative SARS-CoV-2 molecular pharyngeal swab (healthy subjects). Patients were classified in the 3 cohorts at the time of swab result. All SARS-CoV-2 infected patients must test positive for their first time to be included, whereas healthy subjects should never test positive before. Furthermore, all 45 subjects must have received the two BNT162b2 vaccine doses in the previous 4 to 7 months.

We excluded all cases with one or more of the following criteria: 1) active malignancy; 2) immunosuppressive or immunomodulant therapies; 3) organ transplantation; 4) steroid therapy since more than 10 days; 5) pregnancy; and 6) refusal to participate.

Data collection and analysis

After inclusion, demographic and clinical features were recorded. Furthermore, blood samples were collected in BD Vacutainer plasma tubes containing lithium heparin tubes (Becton Dickinson; Plymouth, UK).

Chemiluminescence immunoassay technology was used for the quantitative determination of anti-S1 and anti-S2 specific trimeric IgG antibodies to SARS-CoV-2 (LIAISON SARS-CoV-2 TrimericS IgG Assay, DiaSorin, Saluggia, Italy) and for SARS-CoV-2-specific IgM (LIAISON SARS-CoV-2 IgM Assay, DiaSorin, Saluggia, Italy), according to the manufacturer’s instructions [18]. IgG levels ≥ 13.0 AU/mL and IgM levels ≥ 1.1 INDEX were considered positive.

The frequencies of IFNγ-releasing cells were measured by Enzyme-Linked immunoSPOT (ELISpot) Plus Human IFNγkit (Mabtech AB, Stockholm, Sweden) following the manufacturer’s instructions [19]. Briefly, freshly isolated Peripheral Blood Mononuclear Cells (PBMCs) from whole blood samples were rested overnight before assay. The wells of a microplate pre-coated with the anti-IFN-γ monoclonal antibody mAb1-D1K were washed with Phosphate Buffered Saline 1X (Sigma Aldrich, St. Louis, MO, USA) and blocked with culture medium containing 10% batch tested Fetal Bovine Serum (Sigma Aldrich, St. Louis, MO, USA). As standard, 4 × 105 PBMCs were seeded per well and stimulated with SARS-CoV-2 Spike peptide pool (Mabtech AB, Stockholm, Sweden) at a concentration of 2 μg/ml for 14 h. Negative (i.e., PBMCs treated with peptide vehicle DMSO) and positive (PBMCs stimulated with monoclonal anti-CD3-2) controls were also included. After washing, the wells were developed with human biotinylated IFNγ detection antibody (1:2,000, clone 7-B6-1), followed by incubation with streptavidin-Alkaline Phosphatase and 5-bromo-4-chloro-3’-indolyphosphate p-toluidine salt/nitro-blue tetrazolium chloride-plus substrate. Spots were counted using an automated spot analyzer (BIOREADER-3000, Bio-Sys GmbH, Germany). Mean spot counts for negative control wells were subtracted from the mean of test wells to generate normalized readings, presented as spot forming cells (s.f.c.) × 106. All laboratory tests were performed in accredited laboratories of our University Hospital.

Statistical methods

Given the exploratory design of this study, we arbitrarily enrolled 15 cases per cohort. We assumed all our data as non-parametric. Continuous variables were compared with the Mann-Whitney tests for analysis of variance by ranks. Post-hoc Dunn’s test was applied for pairwise multiple comparisons when indicated. The Receiver Operating Curve (ROC) for the rate of spike-specific IFNγ-releasing was designed and the Area Under the Curve (AUC) was computed to assess the ability to discriminate severe from mild-moderate patients. We considered significant two-sided p values <0.05. Statistical analysis was performed using the Sigmaplot v. 12.0 (Systat Software Inc., San Jose, California).

Results

Demographic, anthropometric, and clinical features of the cases are reported in Table 1.

Table 1. Population characteristics.

Severe patients (n = 15) Mild-moderate patients (n = 15) Healthy subjects (n = 15) p value
Age (years) 62 [52; 73] 53 [51; 60] 51 [46; 59] 0.080
Female sex—n (%) 8 (53%) 9 (60%) 5 (33%) 0.315
Vaccine last dose (days) 154 [150; 181] 154 [151; 177] 156 [152; 171] 0.886
Comorbidities–n (%)
 Arterial hypertension 12 (80%) 8 (53%) 7 (47%) 0.143
 Diabetes 4 (27%) 2 (13%) 3 (20%) 0.660
 Obesity 4 (27%) 2 (13%) 2 (13%) 0.544
 Dyslipidemia 3 (20%) 1 (7%) 3 (20%) 0.508
 Chronic cardiac failure 1 (7%) 0 (0%) 0 (0%) 0.360
 Cerebrovascular disease 2 (13%) 0 (0%) 0 (0%) 0.123
 Chronic Kidney Failure 1 (7%) 0 (0%) 0 (0%) 0.360
 Hypo- or hyperthyroidism 1 (7%) 1 (7%) 3 (20%) 0.407
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No patients received steroids before study inclusion and none of the mild-moderate patients deteriorated to severe. All patients had their first positive SARS-CoV-2 molecular pharyngeal swab and symptoms within 36 hours before inclusion.

Fig 1 depicts the frequencies of spike-specific IFN-γ releasing cells (on the left) and the titers of IgG (on the right) measured in our cohorts of cases. In addition, a representative IFN-γ ELISPOT from one case per cohort is shown in Fig 2. The spike-specific IFNγ-releasing cells were significantly lower in SARS-CoV-2 severe patients requiring hospitalization (8 [0; 26] s.f.c.×106), as compared to COVID-19 mild-moderate patients (135 [64; 159] s.f.c.×106; p<0.001) and healthy subjects (103 [50; 188] s.f.c.×106; p<0.001). No difference was recorded between mild-moderate patients and healthy subjects (p = 0.852). Conversely, the anti-Spike protein IgG (p = 0.098) levels were similar among the three cohorts of cases. In particular, IgG (BAU/ml) titers were 1670 [124; 12300] in severe patients, 6600 [4100; 19000] in mild-moderate patients and 1470 [1020; 5040] in healthy subjects. All cases but one showed positive IgG levels. All 45 cases had an IgM titer below the positivity threshold and under the analytic sensitivity of the test and therefore they cannot be reported for quantitative comparisons.

Fig 1. The frequencies of spike-specific IFN-γ releasing cells and the titers of IgG measured in our cohorts of cases are depicted on the left and right, respectively.

Fig 1

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The bottom and top of the box indicate the 25th and 75th percentiles, respectively. The horizontal band close to the middle of the box represents the median, whereas the ends of the whiskers represent the 10th and 90th percentiles. P values within study cohorts are also reported.

Fig 2. The figure reports a representative IFN-γ ELISPOT from one case per severe, mild-moderate and healthy cohorts, from the left to the right.

Fig 2

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The ROC analysis indicated the rate of spike-specific IFNγ-releasing cells was a good parameter at discriminating correctly severe from mild-moderate patients (area under the curve: 0.9289; 95% CI: 0.8376–1.000; p< 0.0001). A diagnostic specificity of 100% was observed for s.f.c. > 81.2/106 PBMCs, a value corresponding to a diagnostic sensitivity of 66.7% (Fig 3).

Fig 3. The figure depicts the Receiver Operating Curve (ROC) of the rate of spike-specific IFNγ-releasing to discriminate mild-moderate from severe patients.

Fig 3

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Discussion

In this exploratory study of subjects with two doses of the BNT162b2 vaccine, we report that the frequencies of spike-specific IFNγ-releasing cells (i.e. the pattern of T cell response) in severe COVID-19 patients is lower than in mild-moderate patients after breakthrough SARS-CoV-2 infection and in healthy subjects. Differently, the humoral response does not differ among our three cohorts. Considering our findings, frequency of spike-specific IFNγ-releasing cells appears to be a relevant marker for severe disease risk that larger prospective studies should confirm. Other studies investigated this area, most of which looked at humoral immunological parameters, such as specific SARS-CoV-2 antibodies [16] and cytokines [20], or phenotypic characterization of lymphocyte subsets [20, 21]. The frequency of IFNγ-releasing cells is a less widespread approach, mainly due to the methodological complexity of the measurements.

Our findings are of paramount importance: in case of SARS-CoV-2 infection, the rate of IFNγ-releasing cells in dually vaccinated patients requiring hospitalization is significantly lower than patients with a pauci- or asymptomatic manifestation. ROC analysis demonstrated good diagnostic accuracy for marker T cell response, which means that it is possible to define cut-off values that can optimize diagnostic specificity or sensitivity.

Our study substantially confirms previous findings reported in unvaccinated patients. During the first wave of SARS-CoV-2 epidemic, Chandran et al. reported that the immune system reacts to SARS-CoV-2 infection with a rapid and efficient T cell response in non-severe COVID-19 patients [22]. Tan et al. reported that the early assessment of IFNγ-releasing cells was positively related with the severity and the course of the disease in 12 patients [23]. In particular, in severe COVID-19 patients the low (or even absent) quantity of IFNγ-releasing T cells in the peripheral blood could be caused by a defective induction of SARS-CoV-2 T cells [23]. Furthermore, T cell response to SARS-CoV-2 infection varies along the disease course, increasing progressively during the first 15 days after symptoms onset [23]. Of note, our study population tested positive to SARS-CoV-2 and/or showed flu-like symptoms within 36 hours from study inclusion, zeroing the possible bias of different T cell response related to the disease progression.

The role of IFNγ-releasing T cells in the immune response against SARS-CoV-2 have been extensively assessed [24]. A high IFNγ-producing T-cell activity is strongly associated with a low disease severity in acute [25] and in convalescent COVID-19 patients [6, 8, 26]. These results from unvaccinated patients are similar to our findings in vaccinated patients.

In vaccinated and severe patients, the lack of a T-cell mediated response remains to be clarified if it is associated to a reduced response of the patient to the vaccine (and therefore already present before SARS-CoV-2 infection) or if it is an inappropriate and low reaction directly to the infection. A recent study reported that dually vaccinated patients, admitted to Intensive Care Unit, lack of T-cell response despite high IgG titers [16]. The authors raised concerns regarding the assessment of the sole humoral immune response, suggesting the need to also assess the cellular response to SARS-CoV-2 at least in high-risk patients [16]. Therefore, we can formally support the speculation that the T-cell response “provides the underpinning control of serious tissue damage”, despite the presence of high IgG titers [13]. In addition, our findings suggest that the lack of a T-cell mediated response may identify patients at risk of severe disease in case of SARS-CoV-2 infection, thus benefiting of a “booster” dose of vaccine. If an uncoordinated T-cell activity and antibody responses expose vaccinated subjects to the risk of severe disease, these patients would benefit of a “booster dose” before being SARS-CoV-2 infected [16]; otherwise, after infection, the immunological status may only predict the quick development of a severe disease requiring hospitalization. Of note, this hypothesis requires further specifically designed investigations to be confirmed or not.

We also titer the IgM and IgG levels to evaluate the humoral immune response. All patients showed a negative titer of IgM, well below the analytic sensitivity of the test, and a dominance of IgG, as expected by the IgG class-switch of spike-specific memory B cells [27]. Previous studies reported that after two doses of BNT162b2 mRNA vaccine the mean antibody titer reduces over time; in particular, although in all subjects the authors recorded a high level of antibodies after three months from the vaccination [28], in a following analysis the antibodies’ titer declined over time with very low titers in subjects which received their second dose more than 150 days before [29]. In keeping with these findings [28, 29], we also recorded a detectable IgG titer in all included patients, although they received their last dose more than 150 days before. Of note, IgG titers do not correlate with the disease severity and patients’ outcomes [30], supporting our hypothesis that the cellular immune response plays a major role at this regard.

Before drawing our conclusions, some limitations deserve discussion. First, the patients’ sample might be considered small. However, this sample is even larger than a previously published study on a similar topic [16]. In addition, this study has a merely explorative aim. Second, we did not assess the possible degree of peripheral lymphopenia and characterize in detail the different response of CD4+ and CD8+ T-cells. Of note, in more severe and critically COVID-19 patients, counts of peripheral CD4+ and CD8+ T cells are reduced, while their status is hyperactivated [31]. Third, our study included patients receiving a dual dose of vaccine against SARS-CoV-2; however, it remains unclear and to be investigated if a similar cell immune response verifies also in patients which received a “booster” dose. Fourth, we investigated the Spike-specific T cell responses but not to other SARS-CoV-2 structural (membrane and nucleoprotein) and non-structural (open reading frames) proteins. Since the T cell response might be multi-antigenic and it may play a role in understanding different disease severity [9, 25], further studies specifically designed are required. Last, included patients with severe COVID-19 disease were older than both mild-moderate patients and healthy subjects. Even if not statistically significant, this should be considered as a possible confounder.

Conclusions

In conclusion, patients vaccinated with 2-dose of vaccine and developing a severe form of COVID-19 show a lower cellular-mediated immune response than mild-moderate or healthy vaccinated subjects, although antibody titers were similar.

Data Availability

All relevant data are within the paper.

Funding Statement

The authors received no specific funding for this work.

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Decision Letter 0

Mohd Adnan

25 Oct 2022

PONE-D-22-23169Hospitalized and non-hospitalized SARS-CoV-2 vaccinated patients show different frequencies of IFNγ-releasing cells: an exploratory studyPLOS ONE

Dear Dr. Longhini,

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I'd like to thank you for asking me to review this interesting paper which explains that 2-doses vaccinated patients requiring hospitalization for COVID-19 show a cellular-mediated immune response lower than non-hospitalized or controls, despite similar antibody titers.

The authors should read these published articles to deep discuss their findings:

-Coppeta L, Ferrari C, Somma G, Mazza A, D'Ancona U, Marcuccilli F, Grelli S, Aurilio MT, Pietroiusti A, Magrini A, Rizza S. Reduced Titers of Circulating Anti-SARS-CoV-2 Antibodies and Risk of COVID-19 Infection in Healthcare Workers during the Nine Months after Immunization with the BNT162b2 mRNA Vaccine. Vaccines (Basel). 2022 Jan 18;10(2):141. doi: 10.3390/vaccines10020141. PMID: 35214600; PMCID: PMC8879462.

-Coppeta L, Somma G, Ferrari C, Mazza A, Rizza S, Trabucco Aurilio M, Perrone S, Magrini A, Pietroiusti A. Persistence of Anti-S Titre among Healthcare Workers Vaccinated with BNT162b2 mRNA COVID-19. Vaccines (Basel). 2021 Aug 25;9(9):947. doi: 10.3390/vaccines9090947. PMID: 34579184; PMCID: PMC8472926.

-Sahin U., Muik A., Vogler I. BNT162b2 Induces SARS-CoV-2-Neutralising Antibodies and T Cells in Humans. Preprint. [(accessed on 11 December 2020)]. Available online: https://www.medrxiv.org/content/10.1101/2020.12.09.20245175v1

Reviewer #2: Thank you for the opportunity to review this interesting manuscript. The authors describe the immune response of hospitalized and ambulatory patients infected with COVID-19 and use healthy individuals as controls. Their main finding was a low level of IFNγ-releasing cells among hospitalized patients and a high predicting value for severe disease. While their findings are of value for future research and detecting patients at risk, there are still some major issues that should be addressed.

Major issues:

#1: Definition of study groups: the authors define the groups as hospitalized and non-hospitalized. However, there are several reasons for admission of COVID-19 patients beside their disease severity. I recommend changing the groups (throughout the paper, title, and conclusion) to severe, mild-moderate and healthy subjects.

#2: Study design: For proper validation of the results and the ability to implement them in future studies, several important issues must be addressed by the authors:

Methods:

- How were patients selected? Was it during their stay in the emergency department?

- At which point in the disease course the blood samples were drawn – was it at admission to the COVID-19 department?

- Did patients with severe disease received dexamethasone (or other steroids) for their disease prior to their inclusion in the study? If so – for how many days?

Results:

- What is the mean time from the vaccines to study inclusion for each group?

- What is the mean time from the PCR and from the first COVID-related symptoms to study inclusion for each group?

- Were any patients with mild/moderate disease deteriorated after being included in the study?

- Any significant comparison between two of the three groups should be addressed in Table 1. Currently the footnote of the table addresses this issue, but it is not mentioned in the table.

#3: discussion: While this section is properly written and presents the main findings, I believe the authors need to further discuss the following ideas:

- What is your theory for the basis of your results?

Do you think the low levels of IFNγ-releasing cells were present in these patients before being infected with COVID-19, which means it can be screened in the wider vaccinated population? On the other hand, do you think these patients had an inappropriate response to the infection (which reflects in low levels of IFNγ-releasing cells, and is also the basis for the study you cite in citation 12) and therefore have severe disease? If so, screening is relevant only among infected patients.

Based on your theory you should relate to previous studies, to the results of the healthy subjects (and their high levels of IFNγ-releasing cells) and to the possible implications in the general population.

- Previous studies describe both high and low levels of IFNγ among patients with severe disease, while this was not discussed by the authors in relation to their findings.

- Levels of IgG-S were previously shown not to correlate with disease severity and outcomes. The authors should address it to support their findings. In this regard, I recommend them to use the following paper which showed similar results for comparison: https://doi.org/10.1371/journal.pone.0268050

- The age of hospitalized patients was substantially older (p value is not everything when it’s a small number of patients). The authors should address this issue as a possible confounder or limitation.

Minor issues:

– In the end of the results, you state “A diagnostic specificity of 100% was observed for s.f.c. > 81.2 x 106…”. If I understand correctly, a lower value than 81.2 is indicative for a severe disease not higher. If so it should be changed accordingly.

Reviewer #3: Manuscript Title: Hospitalized and non-hospitalized SARS-CoV-2 vaccinated patients show different frequencies of IFNγ-releasing cells: an exploratory study

Summary:

This is a short research article that showed that hospitalized (severe) COVID-19 patients (n=15) present significantly lower level of Spike-specific T cells as compared to mild COVID-19 patients (n=15) and uninfected vaccinated controls (n=15) after breakthrough infection. In comparison, there is no significant differences for the IgG antibody titre among the three groups.

While the manuscript is simple, it might add some light on importance of cellular immunity in controlling SARS-CoV-2 infection. However there are large limitations that should be clearly highlighted.

First, this is certainly not the first manuscript that analyze T cell response in patients with severe or mild or asymptomatic SARS-CoV-2 infection. As such the introduction should acknowledge the work on T cell response published by other authors that have already showed presence of T cells not only in Covid-19 but also on asymptomatic individuals ( i.e Sekine, T. et al. Robust T Cell Immunity in Convalescent Individuals with Asymptomatic or Mild COVID-19. Cell 183, 158-168.e14 (2020), Le Bert, N. et al. Highly functional virus-specific cellular immune response in asymptomatic SARS-CoV-2 infection. J Exp Med 218, e20202617 (2021).). Furthermore, the abstract ( and discussion) does does not clarify that the analysis of Spike-specific T cell response was done after infection and not before the infection. As such the work does not provide any data about” correlate of protection from infection” but only analyze the pattern of T cell response after SARS-CoV-2 infection. This should be clearly mentioned.

As such the abstract should be changed . Authors should clarify that the analysis was done after infection. Thus “ In this study we aim to evaluate the spike-specific immune responses in patients requiring or not hospitalization for SARS-CoV-2 infection, after breakthrough infection following two doses of BNT162b2 mRNA vaccine” .

In addition the authors should also acknowledge that their work substantially confirmed in vaccinated individuals previous work that show lower level of SARS-CoV-2 specific T cells in individuals with severe COVID-19 ( I.e. Tan, A., et al. 2021. Early induction of functional SARS-CoV-2-specific T cells associates with rapid viral clearance and mild disease in COVID-19 patients. Cell Reports, 34(6), p.108728. and Chandran, A. et al. Rapid synchronous type 1 IFN and virus-specific T cell responses characterize first wave non-severe SARS-CoV-2 infections. Cell Reports Medicine 3, 100557–100557 (2022).) ).

The authors should also discuss and pointed out that in severe COVID-19, lymphocytes count is decreasing and SARS-CoV-2 T cells can be recruited at the site of inflammation and as such the quantity of circulating SARS-CoV-2 T cells can be reduced. This is why it is important to perform longitudinal early analysis ( as in Tan, A., et al. 2021. Early induction of functional SARS-CoV-2-specific T cells associates with rapid viral clearance and mild disease in COVID-19 patients. Cell Reports, 34(6), p.108728. and Chandran, A. et al. Rapid synchronous type 1 IFN and virus-specific T cell responses characterize first wave non-severe SARS-CoV-2 infections. Cell Reports Medicine 3, 100557–100557 (2022) and not only at single time when inflammatory events might be at their peak. This is why is also important to understand when their T cell analysis was performed .

The authors should also acknowledge that the analysis was focused only on Spike, but since samples were collected from donors who were infected with SARS-CoV-2, instead of looking only at Spike-specific T cell responses, it will be complimentary to also look T cells specific for other SARS-CoV-2 structural (Membrane and Nucleoprotein) and non-structural (ORFs) proteins. This will allow the authors to not only better understand the breath of the T cell responses, which is possibly different among the different cohorts, but also know the better representative magnitude of SARS-CoV-2 T cell response, as cellular immunity is contributed by more than Spike-specific T cells. Multi-antigenic T cell response might play a paramount role in preventing severe COVID-19, so it should be discussed in the context of this study.

Other minor points:

1. The authors should be clearer about the demographics of the patients recruited, particularly the number of days post-infection, number of days post-vaccination and whether the donors have recovered from COVID-19, as they were not clear on whether they are studying acute or convalescent samples.

2. Could the authors clarify on the use of steroid therapy (the statement “since more than 10 days”) or other therapy (i.e. antivirals or antibody), particularly in the “severe/critical COVID-19” cohort.

3. The study could benefit from the availability of clinical parameters describing the patients (degree of peripheral lymphopenia in hospitalized vs non-hospitalized patients, inflammatory markers, etc.)

4. It might be better for the authors to include individual datapoints on top of the box plots for the graphs plotted in Figure 1. As it seems like there are obvious outliers, and that cannot be easily inferred with the current graphs. Plotting individual datapoints will allow the readers to better appreciate the data.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

**********

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PLoS One. 2023 Feb 9;18(2):e0281444. doi: 10.1371/journal.pone.0281444.r002

Author response to Decision Letter 0

8 Dec 2022

Reviewer #1

The authors should read these published articles to deep discuss their findings:

- Coppeta L, Ferrari C, Somma G, Mazza A, D'Ancona U, Marcuccilli F, Grelli S, Aurilio MT, Pietroiusti A, Magrini A, Rizza S. Reduced Titers of Circulating Anti-SARS-CoV-2 Antibodies and Risk of COVID-19 Infection in Healthcare Workers during the Nine Months after Immunization with the BNT162b2 mRNA Vaccine. Vaccines (Basel). 2022 Jan 18;10(2):141. doi: 10.3390/vaccines10020141. PMID: 35214600; PMCID: PMC8879462.

- Coppeta L, Somma G, Ferrari C, Mazza A, Rizza S, Trabucco Aurilio M, Perrone S, Magrini A, Pietroiusti A. Persistence of Anti-S Titre among Healthcare Workers Vaccinated with BNT162b2 mRNA COVID-19. Vaccines (Basel). 2021 Aug 25;9(9):947. doi: 10.3390/vaccines9090947. PMID: 34579184; PMCID: PMC8472926.

- Sahin U., Muik A., Vogler I. BNT162b2 Induces SARS-CoV-2-Neutralising Antibodies and T Cells in Humans. Preprint. [(accessed on 11 December 2020)]. Available online: https://www.medrxiv.org/content/10.1101/2020.12.09.20245175v1

We thank the Reviewer for his/her suggestion. We included and quoted the two papers by Coppeta et al. in the Discussion section, whereas we prefer to not include the paper by Sahin, since this is a non-peer reviewed paper published as a pre-print. Therefore, this manuscript may contain some flaws requiring the revision process before publication and to consider it as scientifically valid manuscript.

 

Reviewer #2

Major issues:

1) Definition of study groups: the authors define the groups as hospitalized and non-hospitalized. However, there are several reasons for admission of COVID-19 patients beside their disease severity. I recommend changing the groups (throughout the paper, title, and conclusion) to severe, mild-moderate and healthy subjects.

We thank the Reviewer for his/her suggestion. We have completely followed her/his suggestion and cohorts have been renamed as required.

2) Study design: For proper validation of the results and the ability to implement them in future studies, several important issues must be addressed by the authors:

Methods:

- How were patients selected? Was it during their stay in the emergency department?

We have now included in the revised version of the manuscript this information.

- At which point in the disease course the blood samples were drawn – was it at admission to the COVID-19 department?

As already mentioned in the manuscript: “After inclusion, demographic and clinical features were recorded. Furthermore, blood samples were collected in BD Vacutainer plasma tubes containing lithium heparin tubes (Becton Dickinson; Plymouth, UK).”

- Did patients with severe disease received dexamethasone (or other steroids) for their disease prior to their inclusion in the study? If so – for how many days?

No patients received steroids before study inclusion. This is now specified in the results section.

Results:

- What is the mean time from the vaccines to study inclusion for each group?

The reviewer is right, we missed this information that is relevant. We have now included it in Table 1. Since we assumed data as non-parametric, we have described the time (i.e. days) from last vaccine dose in median [interquartile range].

- What is the mean time from the PCR and from the first COVID-related symptoms to study inclusion for each group?

Thanks, we have now specified this in Materials and Methods. Patients were classified in the 3 cohorts at the time of swab result that corresponds to the inclusion time.

- Were any patients with mild/moderate disease deteriorated after being included in the study?

None of the included mild/moderate patients deteriorated to severe after study inclusion. This is now specified in the revised text.

- Any significant comparison between two of the three groups should be addressed in Table 1. Currently the footnote of the table addresses this issue, but it is not mentioned in the table.

We thank the Reviewer for her/his indication. There are no significant differences among populations and footnotes have been deleted accordingly.

#3: discussion: While this section is properly written and presents the main findings, I believe the authors need to further discuss the following ideas:

- What is your theory for the basis of your results?

Do you think the low levels of IFNγ-releasing cells were present in these patients before being infected with COVID-19, which means it can be screened in the wider vaccinated population? On the other hand, do you think these patients had an inappropriate response to the infection (which reflects in low levels of IFNγ-releasing cells, and is also the basis for the study you cite in citation 12) and therefore have severe disease? If so, screening is relevant only among infected patients.

Based on your theory you should relate to previous studies, to the results of the healthy subjects (and their high levels of IFNγ-releasing cells) and to the possible implications in the general population.

We thank the Reviewer for his/her comment. We have included in the manuscript (discussion) this concept that is trivial. However, it remains difficult to state if our findings are related to the infection or a weak response to the vaccine. At this regard, we have left open the possibility and the question to a specifically designed trial.

- Previous studies describe both high and low levels of IFNγ among patients with severe disease, while this was not discussed by the authors in relation to their findings.

Following the Reviewer’s suggestion, we have included a paragraph in the discussion.

- Levels of IgG-S were previously shown not to correlate with disease severity and outcomes. The authors should address it to support their findings. In this regard, I recommend them to use the following paper which showed similar results for comparison: https://doi.org/10.1371/journal.pone.0268050

We thank the Reviewer for her/his thoughtful suggestion that has been fully included in the discussion.

- The age of hospitalized patients was substantially older (p value is not everything when it’s a small number of patients). The authors should address this issue as a possible confounder or limitation.

Done, thanks.

Minor issues:

– In the end of the results, you state “A diagnostic specificity of 100% was observed for s.f.c. > 81.2 x 106…”. If I understand correctly, a lower value than 81.2 is indicative for a severe disease not higher. If so it should be changed accordingly.

In our study, diagnostic specificity refers to the IFN-γ ELISPOT's ability to correctly define patients with mild / moderate disease, which is 100% for s.f.c. greater than 81.2/PBMCs 106. 

Reviewer #3

First, this is certainly not the first manuscript that analyze T cell response in patients with severe or mild or asymptomatic SARS-CoV-2 infection.

As such the introduction should acknowledge the work on T cell response published by other authors that have already showed presence of T cells not only in Covid-19 but also on asymptomatic individuals ( i.e Sekine, T. et al. Robust T Cell Immunity in Convalescent Individuals with Asymptomatic or Mild COVID-19. Cell 183, 158-168.e14 (2020), Le Bert, N. et al. Highly functional virus-specific cellular immune response in asymptomatic SARS-CoV-2 infection. J Exp Med 218, e20202617 (2021).). Furthermore, the abstract (and discussion) does not clarify that the analysis of Spike-specific T cell response was done after infection and not before the infection. As such the work does not provide any data about” correlate of protection from infection” but only analyze the pattern of T cell response after SARS-CoV-2 infection. This should be clearly mentioned.

Following the Reviewer suggestions, we have accordingly revised the manuscript and all suggestions have been included.

As such the abstract should be changed. Authors should clarify that the analysis was done after infection. Thus “In this study we aim to evaluate the spike-specific immune responses in patients requiring or not hospitalization for SARS-CoV-2 infection, after breakthrough infection following two doses of BNT162b2 mRNA vaccine”.

Done.

2. In addition the authors should also acknowledge that their work substantially confirmed in vaccinated individuals previous work that show lower level of SARS-CoV-2 specific T cells in individuals with severe COVID-19 ( I.e. Tan, A., et al. 2021. Early induction of functional SARS-CoV-2-specific T cells associates with rapid viral clearance and mild disease in COVID-19 patients. Cell Reports, 34(6), p.108728. and Chandran, A. et al. Rapid synchronous type 1 IFN and virus-specific T cell responses characterize first wave non-severe SARS-CoV-2 infections. Cell Reports Medicine 3, 100557–100557 (2022).)

Following the Reviewer suggestions, we have accordingly revised the manuscript.

3. The authors should also discuss and pointed out that in severe COVID-19, lymphocytes count is decreasing, and SARS-CoV-2 T cells can be recruited at the site of inflammation and as such the quantity of circulating SARS-CoV-2 T cells can be reduced. This is why it is important to perform longitudinal early analysis ( as in Tan, A., et al. 2021. Early induction of functional SARS-CoV-2-specific T cells associates with rapid viral clearance and mild disease in COVID-19 patients. Cell Reports, 34(6), p.108728. and Chandran, A. et al. Rapid synchronous type 1 IFN and virus-specific T cell responses characterize first wave non-severe SARS-CoV-2 infections. Cell Reports Medicine 3, 100557–100557 (2022) and not only at single time when inflammatory events might be at their peak. This is why is also important to understand when their T cell analysis was performed .

Following the Reviewer suggestions, we have accordingly revised the manuscript.

The authors should also acknowledge that the analysis was focused only on Spike, but since samples were collected from donors who were infected with SARS-CoV-2, instead of looking only at Spike-specific T cell responses, it will be complimentary to also look T cells specific for other SARS-CoV-2 structural (Membrane and Nucleoprotein) and non-structural (ORFs) proteins. This will allow the authors to not only better understand the breath of the T cell responses, which is possibly different among the different cohorts, but also know the better representative magnitude of SARS-CoV-2 T cell response, as cellular immunity is contributed by more than Spike-specific T cells. Multi-antigenic T cell response might play a paramount role in preventing severe COVID-19, so it should be discussed in the context of this study.

We agree with the Reviewer, and we acknowledge her/his concern as limitation of the study. Unfortunately, we do not have samples anymore to provide this adjunctive analysis. In addition, this was out of our study aim and therefore we now suggest that further studies are required at this regard.

Other minor points:

1. The authors should be clearer about the demographics of the patients recruited, particularly the number of days post-infection, number of days post-vaccination and whether the donors have recovered from COVID-19, as they were not clear on whether they are studying acute or convalescent samples.

As also required by Reviewer #2, we have included these data in the revised text.

2. Could the authors clarify on the use of steroid therapy (the statement “since more than 10 days”) or other therapy (i.e. antivirals or antibody), particularly in the “severe/critical COVID-19” cohort.

As also required by Reviewer #2, we have now specified that none of included patients received steroids before study inclusion

3. The study could benefit from the availability of clinical parameters describing the patients (degree of peripheral lymphopenia in hospitalized vs non-hospitalized patients, inflammatory markers, etc.)

We agree with the Reviewer, but these data are not available for non-hospitalized (mild-moderate) patients and control (healthy) group. This is now recognized as limitation of the study.

4. It might be better for the authors to include individual datapoints on top of the box plots for the graphs plotted in Figure 1. As it seems like there are obvious outliers, and that cannot be easily inferred with the current graphs. Plotting individual datapoints will allow the readers to better appreciate the data.

Following the Reviewer’s suggestion, we have not plotted also individual datapoints.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file. (22.9KB, docx)

Decision Letter 1

Mohd Adnan

13 Jan 2023

PONE-D-22-23169R1Severe and mild-moderate SARS-CoV-2 vaccinated patients show different frequencies of IFNγ-releasing cells: an exploratory studyPLOS ONE

Dear Dr. Longhini,

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We look forward to receiving your revised manuscript.

Kind regards,

Mohd Adnan, PhD

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

Manuscript is significantly improved by the authors. However, there are still some minor concerns raised by the reviewer. Please address these concerns and resubmit accordingly.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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Reviewer #2: Yes

Reviewer #3: (No Response)

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Reviewer #2: Yes

Reviewer #3: (No Response)

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Reviewer #2: Yes

Reviewer #3: (No Response)

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Reviewer #3: (No Response)

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Reviewer #2: Thank you for the opportunity to re-review this manuscript. The authors have made substantial changes and answered all the issues I've raised. The manuscript has substantially improved. Still there are several issues which should be addressed:

- Many minor spelling and grammar mistake should be revised. For example – in the abstract – few spaces are missing, "discriminating" should be discriminate, and so on. This occurs throughout the text.

- If I understand correctly after the revision – all included patients were diagnosed only upon hospital arrival and not before? If so you should change the sentences describing the first two groups which state "in the previous 10 days". Later you wrote that PCR was positive within 36 hours (results), please state the correct timing in all the places.

Reviewer #3: The authors have addressed my main reservations. The text is more balanced and describes objectively the results obtained.

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Reviewer #2: No

Reviewer #3: Yes: Antonio Bertoletti

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PLoS One. 2023 Feb 9;18(2):e0281444. doi: 10.1371/journal.pone.0281444.r004

Author response to Decision Letter 1

16 Jan 2023

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

References have been checked and they should be fine per style. To the best of our knowledge, no retracted manuscripts have been included.

Additional Editor Comments:

Manuscript is significantly improved by the authors. However, there are still some minor concerns raised by the reviewer. Please address these concerns and resubmit accordingly.

Concerns pointed out from Reviewer #2 have been addressed. Please, see below.

Reviewers' comments:

Reviewer #2:

Thank you for the opportunity to re-review this manuscript. The authors have made substantial changes and answered all the issues I've raised. The manuscript has substantially improved. Still there are several issues which should be addressed:

- Many minor spelling and grammar mistake should be revised. For example – in the abstract – few spaces are missing, "discriminating" should be discriminate, and so on. This occurs throughout the text.

Following the Reviewer’s comments, we have checked the entire manuscript and correct.

- If I understand correctly after the revision – all included patients were diagnosed only upon hospital arrival and not before? If so you should change the sentences describing the first two groups which state "in the previous 10 days". Later you wrote that PCR was positive within 36 hours (results), please state the correct timing in all the places.

We agree with the Reviewer that the manuscript lacked clarity. The need for a positive PCR swab in the previous 10 days was an inclusion criterion; however, in the results we have specified that all patients resulted positive within 36 hours before inclusion. We have decided to leave only the inclusion timing (36 hours) and to delete the time limit of inclusion criterion. We believe that now the manuscript has improved in its clarity.

Reviewer #3:

The authors have addressed my main reservations. The text is more balanced and describes objectively the results obtained.

Ok, thanks.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file. (15.9KB, docx)

Decision Letter 2

Mohd Adnan

24 Jan 2023

Severe and mild-moderate SARS-CoV-2 vaccinated patients show different frequencies of IFNγ-releasing cells: an exploratory study

PONE-D-22-23169R2

Dear Dr. Longhini,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Mohd Adnan, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Acceptance letter

Mohd Adnan

31 Jan 2023

PONE-D-22-23169R2

Severe and mild-moderate SARS-CoV-2 vaccinated patients show different frequencies of IFNγ-releasing cells: an exploratory study

Dear Dr. Longhini:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Mohd Adnan

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    Attachment

    Submitted filename: Response to Reviewers.docx

    Click here for additional data file. (22.9KB, docx)
    Attachment

    Submitted filename: Response to Reviewers.docx

    Click here for additional data file. (15.9KB, docx)

    Data Availability Statement

    All relevant data are within the paper.

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