Previous COVID-19 vaccination modulates type I interferon and natural killer cell responses during SARS-CoV-2 infection

Previous COVID-19 vaccination modulates type I interferon and natural killer cell responses during SARS-CoV-2 infection | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 3 September 2025 V1 Latest version Share on Previous COVID-19 vaccination modulates type I interferon and natural killer cell responses during SARS-CoV-2 infection Authors : Luca Maddaloni , Valentina Tirelli , Ginevra Bugani , Letizia Santinelli , Matteo Fracella 0000-0002-2885-2382 , Mario Picozza , Eugenio Cavallari , Giancarlo Ceccarelli 0000-0001-5921-3180 [email protected] , Guido Antonelli 0000-0002-2533-2939 , Claudio Mastroianni , Carolina Scagnolari 0000-0003-1044-1478 , and Gabriella D’Ettorre Authors Info & Affiliations https://doi.org/10.22541/au.175692133.39680121/v1 178 views 124 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract The impact of the SARS-CoV-2 vaccine on innate immunity is not well understood. However, it has played a pivotal role in reducing COVID-19 severity and mortality. Recent findings have revealed that vaccine efficacy is influenced not only by the effective activation of adaptive immunity, but also by the modulation of innate immunity. This study evaluates the natural killer (NK) cell response and its relationship with type I interferon (IFN-I) gene expression in SARS-CoV-2-infected patients who had previously received the anti-spike vaccine, as well as in unvaccinated patients. Vaccinated individuals showed a higher frequency of NK NK CD56dim CD16- cells and increased IFN-α2 and IFN-ω mRNA expression (p < 0.05). By contrast, unvaccinated patients displayed a predominance of NK CD56dim CD16+ cells and reduced IFN-I gene expression (p < 0.05). A positive correlation was found between IFN-I levels and the frequency of NK CD56dim CD16- cells and a negative correlation between IFN-I levels and NK CD56dim CD16+ cells. Furthermore, despite having more comorbidities, vaccinated patients had faster SARS-CoV-2 clearance, which reinforces the immunological advantage conferred by vaccination. Together, these findings suggest that the SARS-CoV-2 vaccine can modify the innate immune response by enhancing the NK cell response and increasing the magnitude of IFN-I production during SARS-CoV-2 infection. Previous COVID-19 vaccination modulates type I interferon and natural killer cell responses during SARS-CoV-2 infection Luca Maddaloni 1 , Valentina Tirelli 2 , Ginevra Bugani 3 , Letizia Santinelli 3 , Matteo Fracella 4 , Mario Picozza 2 , Eugenio Nelson Cavallari 5 , * Giancarlo Ceccarelli 5 , Guido Antonelli 6 , Claudio Maria Mastroianni 3 , Carolina Scagnolari 4,7 , Gabriella d’Ettorre 3 . 1 Department of Molecular Medicine, Sapienza University of Rome, Italy ( [email protected] ) 2 Core Facilities, Istituto Superiore Di Sanità, Rome, Italy ( [email protected] ; [email protected] ) 3 Department of Public Health and Infectious Diseases, Sapienza University of Rome, Italy ( [email protected] ; [email protected] ; [email protected] ; [email protected] ) 4 Department of Molecular Medicine, Laboratory of virology, Sapienza University of Rome, Italy ( [email protected] ; [email protected] ) 5 Department of Public Health and Infectious Diseases, Sapienza University of Rome, Italy; Azienda Ospedaliero Universitaria Policlinico Umberto I, Rome, Italy ( [email protected] ; [email protected] ) 6 Department of Molecular Medicine, Microbiology and Virology Laboratory, Sapienza University of Rome, Italy ( [email protected] ) 7 Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Italy*Corresponding author: Giancarlo Ceccarelli ( [email protected] ; https://orcid.org/0000-0001-5921-3180) Abstract The impact of the SARS-CoV-2 vaccine on innate immunity is not well understood. However, it has played a pivotal role in reducing COVID-19 severity and mortality. Recent findings have revealed that vaccine efficacy is influenced not only by the effective activation of adaptive immunity, but also by the modulation of innate immunity. This study evaluates the natural killer (NK) cell response and its relationship with type I interferon (IFN-I) gene expression in SARS-CoV-2-infected patients who had previously received the anti-spike vaccine, as well as in unvaccinated patients. Vaccinated individuals showed a higher frequency of NK CD56 dim CD16 - cells and increased IFN-α2 and IFN-ω mRNA expression (p < 0.05). By contrast, unvaccinated patients displayed a predominance of NK CD56 dim CD16 + cells and reduced IFN-I gene expression (p < 0.05). A positive correlation was found between IFN-I levels and the frequency of NK CD56 dim CD16 - cells and a negative correlation between IFN-I levels and NK CD56 dim CD16 + cells. Furthermore, despite having more comorbidities, vaccinated patients had faster SARS-CoV-2 clearance, which reinforces the immunological advantage conferred by vaccination. Together, these findings suggest that the SARS-CoV-2 vaccine can modify the innate immune response by enhancing the NK cell response and increasing the magnitude of IFN-I production during SARS-CoV-2 infection. Keywords: COVID-19 vaccine, NK cells, IFN-I, innate immunity, SARS-CoV-2 Introduction In the context of the COVID-19 pandemic, the production and distribution of vaccines were critical in limiting the rate and severity of SARS-CoV-2 infection, as well as hospitalization and mortality 1-2 . Anti-SARS-CoV-2 vaccination can induce both antibody production and specific T-cell responses 3 , but an intriguing novel aspect is its ability to modulate components of innate immunity, such as natural killer (NK) cells 4 . Some recent studies have shown that NK cells are activated by anti-spike (S) vaccination or SARS-CoV-2 natural infection 5-6 , and that they are more active after re-infection 7 . In addition, NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) activity, which plays a key role in COVID-19, has been reported to be triggered by antibodies induced by natural SARS-CoV-2 infection or anti-S vaccination 8 .Among the components of innate immunity, interferons (IFNs) are able to stimulate NK cell activation and significantly suppress SARS-CoV-2 replication 9-12 . Data from the literature and from our previous studies have shown that severe COVID-19 can occur with either low and excessive type I/III IFN and IFN-stimulated genes production 13-16 . It is noteworthy that severe forms of COVID-19 have been associated with dysregulation of IFN due to viral evasion mechanisms, temporal defects in IFN production, host genetic factors or the development of anti-IFN neutralizing autoantibodies 17-21 . Recent data also suggest that these cytokines could be important during SARS-CoV-2 vaccination, with a positive correlation observed between the IFN gene signature and the level of anti-S antibody production following vaccination 22 . We have previously observed that COVID-19 patients who had been vaccinated against SARS-CoV-2 exhibit higher IFN-I-mRNA levels than unvaccinated patients and show more effective and faster resolution of infection 23 .Given the emerging novel immunological effects of anti-S vaccination 3 , and the role of NK cells in antibody-mediated SARS-CoV-2 clearance 8 , we investigated how prior anti-S vaccination might affect the NK cell response during SARS-CoV-2 infection. Therefore, we aimed to evaluate the NK cell response in SARS-CoV-2 patients previously vaccinated with the anti-spike vaccine and in unvaccinated patients. Specifically, NK cell subsets (CD56 dim CD16 + , CD56 dim CD16 - , CD56 bright CD16 + and CD56 bright CD16 - ) were investigated in peripheral blood mononuclear cells (PBMCs) from SARS-CoV-2 infected patients. To gain further insight into the role of IFN changes during anti-S vaccination, we also extended our previous study 23 , by investigating the relationship between NK cell response and IFN-α and IFN-ω mRNA levels . Materials and methods Participants This study was conducted at the Division of Infectious Diseases, Department of Public Health and Infectious Diseases, Umberto I Hospital of the Sapienza University of Rome (Italy), and included 69 outpatients with PCR test confirmed SARS-CoV-2 infection from nasopharyngeal swabs from 21 April to 10 December 2021. Patients informations were obtained from electronic medical records in the hospital’s electronic information system. All patients were high risk individuals treated with monoclonal antibodies in single administration (casirivimab 1200 mg + imdevimab 1200mg or bamlanivimab 700mg + etesevimab 1400mg). The following variables were included in the study: age, sex, vaccination status against SARS-CoV-2 and Charlson Comorbidity Index 24 . The study was performed following the Institutional Review Board (Department of Public Health and Infectious Diseases, Sapienza, University of Rome) and the Ethics Committee (Sapienza, University of Rome) approval and the signing of informed consent by all study participants .Blood samples collectionFresh peripheral blood samples were collected by venipuncture into Vacutainer tubes containing a gel separator for serum or EDTA (BD Biosciences, USA) from SARS-CoV-2-infected patients at least 48 hours after diagnosis and before monoclonal antibody treatment. The first were centrifuged at 3000 rpm for 10 min, the serum was collected and stored at -80°C. The latter were centrifuged at 1500 rpm for 10 min to isolate plasma (stored at -80°C) and further processed by Ficoll gradient centrifugation (Lympholyte; Cedarlane Labs, Canada) to obtain PBMCs. PBMCs were washed twice in phosphate-buffered saline (PBS) and stored in fetal bovine serum (FBS) supplemented with 10% dimethyl sulfoxide (DMSO) in liquid nitrogen until use. Bioassay for detection of neutralizing antibodies (nAbs) against IFN-I A neutralization assay was performed to assess the presence of autoantibodies that bind to the active site of IFN and block its biological activity 25 . The biological system used in this assay is based on the detection of the cytopathic effect of murine encephalomyocarditis virus (EMCV) on the A549 cell line, as previously described 20 . Neutralizing autoantibodies (nAbs) against IFN-α2a (Roferon-A, Roche, Switzerland), IFN-α2b (Intron, Merck & Co., USA), IFN-ω (PBL Assay Science, USA) were measured in the decomplemented sera as previously reported 25 .Anti-spike antibody titer quantificationType G immunoglobulins (IgGs) against SARS-CoV-2 spike protein were determined in serum from infected patients using a commercial assay (LIAISON® SARS-CoV-2 TrimericS IgG, DiaSorin, Italy). The anti-S antibody titers provided by the assay are expressed as binding antibody units per ml (BAU/ml), ranging between 4.81 and 2080 BAU/mL. Values 2080 BAU/mL) were automatically diluted with a factor of 1:10 using the LIAISON® TrimericS IgG Diluent Accessory.Immunophenotyping by Multiparametric Flow Cytometry assayThawed PBMCs were washed twice with PBS and the distribution of cellular phenotypes was evaluated by Miltenyi Biotec flow cytometer-MACSQuant Analyzer (eight fluorescence channels, three lasers; Miltenyi Biotec, Germany). After defining cell viability by excluding dead cells using Viobility 488/520 Fixable Dye (Miltenyi Biotec), cellular phenotypes were obtained employing different fluorochrome-labeled anti-human monoclonal antibodies: CD3-PerCP-Vio770, CD16-PE-Vio770 and CD56-PE (Miltenyi Biotec). NK cell subsets were identified as (CD3 - CD56 + ) and were further subdivided into CD56 dim CD16 + , CD56 dim CD16 - , CD56 bright CD16 + and CD56 dim CD16 - . A minimum of 10 5 events were acquired for each sample. Gating strategies and data analysis were performed using FlowJo v10.10 (Becton Dickinson, USA). Representative flow cytometry plots illustrating the gating strategy are shown in Fig. 1. Figure 1 Representative flow cytometry plots showing the gating strategy used to analyze NK cell subsets in PBMC samples. NK cells were selected via sequential gating by the exclusion of dead cells (a) and doublets (b), followed by positive gating on lymphocytes (c) and CD3 - CD56 + cells (d). Then, NK subsets were selected based on differential expression of CD56 and CD16 (e).Statistical analysisPatients’ data were presented as median (interquartile range) or number (percentage). Demographic, virological, serological, and clinical characteristics of patients were analyzed using the ”N-1” Chi-squared test. Cross-sectional data comparing vaccinated and unvaccinated patients were analyzed with the Mann–Whitney U test. The Spearman’s rho coefficient was calculated to determine the correlation between gene expression levels and cell frequencies. Statistical analyses were performed using GraphPad Prism software, version 9.4 (GraphPad Software Inc., USA) and a p-value of less than 0.05 was considered statistically significant.ResultsStudy populationA total of 69 SARS-COV-2 infected patients were enrolled in this study and divided into two groups according to anti-spike vaccination status: vaccinated (n = 47) and unvaccinated (n = 22). The demographic and clinical characteristics of these two groups are shown in Table 1; the type of anti-SARS-CoV-2 vaccine and number of doses previously received are described in Supplementary Table 1. The median elapsed time since vaccination was 5 months, with an interquartile range between 3 and 7. None of the patients reported previous SARS-CoV-2 infection. None of the unvaccinated patients had detectable anti-S antibodies in their serum, whereas all vaccinated patients tested positive for these antibodies. RT-PCR SARS-CoV-2 RNA test data were available 12 days after enrolment for 40 vaccinated and 18 unvaccinated individuals. The vaccinated group had a higher rate of negative RT-PCR tests for SARS-CoV-2 than the unvaccinated group (32.5% vs 0%, p = 0.0065) after monoclonal antibody therapy (Table 1). Table 1 Demographic, virological, serological and clinical characteristics of SARS-CoV-2 infected patients. Parameters Median (IQR 25-75%) n (%) Median (IQR 25-75%) n (%) p – values Sex assigned at birth Male - 23 (48.9) - 10 (45.5) 0.758 Female - 24 (51.1) - 12 (54.5) 0.758 Age (years) 66 (60-76) - 57 (53-73) - 0.071 Charlson Comorbidity Index* 3 (3-4) - 2 (2-3) - 0.003 Anti-spike (S) antibodies test Undetectable (< 33.8 BAU/ml) - 0 (0) - 22 (100) 33.8 BAU/ml) - 47 (100) - 0 (0) < 0.0001 Follow-up RT-PCR SARS-CoV-2- test** Positive - 27 (67.5) - 18 (100) 0.0065 Negative - 13 (32.5) - 0 (0) 0.0065 Data are expressed as median (interquartile range) or number (percentage). Data were analyzed using Mann–Whitney U and ”N-1” Chi-squared test, respectively. p < 0.05 were considered statistically significant. *The Charlson Comorbidity Index predicts the mortality for a patient who may have a number of comorbidities 24 . ** Data available for 40 and 18 vaccinated and unvaccinated patients, respectively, 12 days following single administration of monoclonal antibody therapy.Frequencies of NK cell subsets in SARS-CoV-2 infected patients according to the vaccination statusThe effect of previous SARS-CoV-2 vaccination on the distribution of NK cell subsets was investigated by stratifying all SARS-CoV-2 infected patients according to their vaccination status. Unvaccinated patients had lower percentage of NK CD56 dim CD16 - cells (p < 0.0001) compared to vaccinated patients, but an increased frequency of NK CD56 dim CD16 + cells (p < 0.0001) (Figure 2a-b). The percentage of NK CD56 bright CD16 +/- cells was similar between the two groups of SARS-CoV-2 positive patients (Figure 2c-d). Figure 2 Comparison of NK CD56 dim CD16 - (a) , NK CD56 dim CD16 + (b), NK CD56 bright CD16 - (c) and NK CD56 bright CD16 + (d) cells frequencies between vaccinated and unvaccinated SARS-CoV-2 infected patients. Data were analyzed using the Mann-Whitney U-test. ****p < 0.0001. Correlation analysis between NK cells frequencies and IFNs-I expression Considering the strong association between IFN-I and NK cell activity 9-10 and given the observed differences in NK CD56 dim cells between vaccinated and unvaccinated patients, a correlation analysis was performed between IFN-I mRNA expression levels and NK cell subsets frequencies. Specifically, IFN-α2 and IFN-ω mRNA expression data were obtained from a subset of patients (n = 69) from our previous study 23 . To avoid confounding factors that could affect this analysis, we tested for the presence of anti-IFN-I nAbs in serum samples from these participants. All serum samples were negative for anti-IFN-α2a/b and IFN-ω (<10 TRU/mL). Results confirmed our previous analysis that the unvaccinated group showed reduced IFN-α2 (p = 0.0015) and IFN-ω (p = 0.003) mRNA expression levels in PBMCs compared to the vaccinated group (Figure 3a-b). A positive correlation was found between NK CD56 dim CD16 - cells and IFN-α2 (r = 0.325; p = 0.006), whereas NK CD56 dim CD16 + cells showed a negative correlation with IFN-α2 (r = -0.391; p = 0.001) and IFN-ω (r = -0.261; p = 0.03). Correlation coefficients (r) and p-values (p) are shown in Figure 4a-d. Figure 3 Comparison of IFN-α2 (a) and IFN-ω (b) mRNA expression levels between vaccinated and unvaccinated SARS-CoV-2 infected patients. Data were analyzed using the Mann-Whitney U-test. **p < 0.01 Figure 4 Correlation between NK CD56 dim cells frequencies and IFNs-I mRNA expression levels. Data were analyzed using Spearman’s rank correlation test and p < 0.05 was considered statistically significant. Discussion In the context of the SARS-CoV-2 pandemic, vaccination against SARS-CoV-2 was critical in reducing severe clinical outcomes by promoting an effective and long-lasting immune response. While the stimulation of both T- and B-lymphocytes by the SARS-CoV-2 vaccine has been well described 3 , considerable interest has arisen in understanding the role of innate immunity. The ability of the innate immune system to develop a memory-like response after exposure to certain pathogens or vaccines has recently been described for several microorganisms. This phenomenon is defined as ’trained innate immunity’ 26 . Therefore, the first focus of our study was the evaluation of NK cell response in SARS-CoV-2-infected patients who had or had not previously been vaccinated against the spike protein. Vaccinated patients had a higher Charlson Comorbidity Index than unvaccinated patients, reflecting a greater risk of developing severe symptoms of the disease. These data reflect the vaccination strategy adopted during the pandemic, which prioritized the vaccination of those at greatest risk of severe COVID-19.Vaccinated patients were characterized by an increased percentage of NK CD56 dim CD16 - and a decreased percentage of NK CD56 dim CD16 + cells compared to unvaccinated patients. This difference in CD16 expression by NK cells could be attributed to the fact that this receptor is present on all peripheral blood CD56 dim NK cells, but activation of these cells by cross-linking of CD16 with antibodies results in its loss 27-28 . Consistently, downregulation of CD16 expression on NK cells, alongside its strong positive association with degranulation, was observed in vivo following intramuscular influenza vaccination. This evidence suggests that CD16 plays a role in the early activation of NK cells following vaccination, as well as in the modulation of NK cell responses and maintenance of immune homeostasis through downregulation 29 . Loss of the CD16 receptor was also observed in recently activated nonclassical monocytes 30 . Hagemann et al. have previously demonstrated that antibodies induced by natural SARS-CoV-2 infection or vaccination trigger significant NK cell-mediated ADCC activity 8 . The function of NK cells is regulated by various cytokines, including IFN-I, which has been shown to be closely associated with the activity of these cells 9-10 . Consistent with our previous analysis 23 , the vaccinated individuals exhibited higher expression of IFN-I genes than the unvaccinated ones. Notably, none of them had nAbs against IFN-α or IFN-ω, regardless of vaccine status. These cytokines have remarkable antiviral activity against SARS-CoV-2 11-12 , although emerging SARS-CoV-2 variants have shown progressive increased IFN resistance 31 . Notably, a weak and delayed IFN-I response is one of the hallmarks of severe forms of COVID-19 32 . Furthermore, the number of blood plasmacytoid dendritic cells, which are the main circulating IFN-α-producing cell subset, is significantly reduced in severely ill unvaccinated patients hospitalized with SARS-CoV-2 33 . The increased expression of IFN-I that we observed in vaccinated individuals suggests that they mount a more robust and effective immune response to the virus than unvaccinated individuals. This is also supported by the faster SARS-CoV-2 viral clearance observed in the first group after monoclonal antibody therapy. To further clarify this, we performed a correlation analysis between NK cell subset frequencies and IFN-I mRNA expression levels. We observed a negative correlation between the frequency of NK CD56dim CD16+ cells and IFN-I-mRNA expression levels. Thus, unvaccinated patients with reduced IFN-I mRNA levels also exhibited higher frequencies of NK CD56dim CD16+ cells. This inverse relationship suggests that reduced IFN-I expression could be associated with impaired NK cell activity. This is consistent with previous findings that IFN-I plays a crucial role in promoting NK cell activation 9-10 . In this context, the increased frequency of CD56 dim CD16 + NK cells in unvaccinated individuals may indicate a lower number of activated or functional NK cells, which could be the result of poor IFN-I signaling. The higher score on the Charlson Comorbidity Index among vaccinated patients could be perceived as a limitation of this study. However, the importance and effectiveness of vaccination is emphasized by the fact that vaccinated patients clear SARS-CoV-2 more quickly, despite being frailer. Of the 47 patients who had received the SARS-Cov-2 vaccine, five had only received the first dose. Rather than weakening our findings, this detail actually enhances the robustness of our results. One limitation of the study is the lack of quantification of antibodies directed against the Nucleocapsid protein in patient sera, which would have allowed for the exclusion of previous natural SARS-CoV-2 infection. However, given that the individuals were enrolled in 2021, that unvaccinated patients did not have anti-spike antibodies in their sera and that asymptomatic SARS-CoV-2 infection is unlikely to occur in frail individuals, it is reasonable to presume that none of the patients had previously been infected with SARS-CoV-2, as they reported.In conclusion, our results suggest that SARS-CoV-2 infected patients have different innate immune responses depending on vaccination status. Notably, vaccinated patients demonstrate an early and pronounced IFN-I response, alongside phenotypic evidence of pre-activated NK cells in the early stages of infection. Conversely, unvaccinated individuals exhibit an attenuated and delayed activation of these key antiviral pathways, underscoring the immunological priming conferred by prior vaccination. 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Keywords covid-19 vaccine ifn-i innate immunity nk cells sars-cov-2 Authors Affiliations Luca Maddaloni Universita degli Studi di Roma La Sapienza View all articles by this author Valentina Tirelli Istituto Superiore di Sanita View all articles by this author Ginevra Bugani Universita degli Studi di Roma La Sapienza View all articles by this author Letizia Santinelli Universita degli Studi di Roma La Sapienza View all articles by this author Matteo Fracella 0000-0002-2885-2382 Universita degli Studi di Roma La Sapienza View all articles by this author Mario Picozza Istituto Superiore di Sanita View all articles by this author Eugenio Cavallari Umberto I Policlinico di Roma View all articles by this author Giancarlo Ceccarelli 0000-0001-5921-3180 [email protected] Umberto I Policlinico di Roma View all articles by this author Guido Antonelli 0000-0002-2533-2939 Universita degli Studi di Roma La Sapienza View all articles by this author Claudio Mastroianni Universita degli Studi di Roma La Sapienza View all articles by this author Carolina Scagnolari 0000-0003-1044-1478 Universita degli Studi di Roma La Sapienza View all articles by this author Gabriella D’Ettorre Universita degli Studi di Roma La Sapienza View all articles by this author Metrics & Citations Metrics Article Usage 178 views 124 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Luca Maddaloni, Valentina Tirelli, Ginevra Bugani, et al. 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