key: cord-0255828-rrykpl0f
authors: Binayke, Akshay; Zaheer, Aymaan; Dandotiya, Jyotsna; Gupta, Sonu K; Mani, Shailendra; Tripathi, Manas; Madan, Upasna; Shrivastava, Tripti; Kumar, Yashwant; Pandey, Anil K; Rathore, Deepak K; Awasthi, Amit
title: Proinflammatory innate cytokines and metabolomic signatures predict T cell response in active COVID-19
date: 2022-04-18
journal: bioRxiv
DOI: 10.1101/2022.03.11.483930
sha: 389171faf1f909fa0fb0840a886f4bed3f11b303
doc_id: 255828
cord_uid: rrykpl0f

The underlying factors contributing to the evolution of SARS-CoV-2-specific T cell responses during COVID-19 infection remain unidentified. To address this, we characterized innate and adaptive immune responses with metabolomic profiling longitudinally at three different time points (0-3, 7-9, and 14-16 days post-COVID-19 positivity) from young mildly symptomatic active COVID-19 patients infected with the ancestral virus in mid-2020. We observed that anti-RBD IgG and viral neutralization are significantly reduced against the Delta variant compared to the ancestral strain. In contrast, compared to the ancestral strain, T cell responses remain preserved against the delta and omicron variants. We determined innate immune responses during the early stage of active infection in response to TLR 3/7/8 mediated activation in PBMCs and serum metabolomic profiling. Correlation analysis indicated PBMCs-derived proinflammatory cytokines, IL-6, IL-1β, and IL-23, and the abundance of plasma metabolites, pyroglutamate, itaconate, and azelaic acid, were predictive of a robust SARS-CoV-2-specific Th1 response at a later stage (two weeks after PCR positivity). These observations may contribute to designing effective vaccines and adjuvants that promote innate immune responses and metabolites to induce long-lasting anti-SARS-CoV-2 specific T cells response. Key Message Early proinflammatory innate cytokine response is critical for a robust T cell response during COVID-19. We report a set of plasma metabolites that significantly correlate with the innate and adaptive immune responses. Graphical Abstract

innate and adaptive immune responses with metabolomic profiling longitudinally at three 4 different time points (0-3, 7-9, and 14-16 days post-COVID-19 positivity) from young mildly 5 symptomatic active COVID-19 patients infected with the ancestral virus in mid-2020. We 6 observed that anti-RBD IgG and viral neutralization are significantly reduced against the Delta 7 variant compared to the ancestral strain. In contrast, compared to the ancestral strain, T cell 8 responses remain preserved against the delta and omicron variants. We determined innate 9 immune responses during the early stage of active infection in response to TLR 3/7/8 mediated 10 activation in PBMCs and serum metabolomic profiling. Correlation analysis indicated PBMCs-11 derived proinflammatory cytokines, IL-6, IL-1β, and IL-23, and the abundance of plasma 12 metabolites, pyroglutamate, itaconate, and azelaic acid, were predictive of a robust SARS-CoV- (1) (2) (3) . Although variants of concern (VoCs) acquired multiple mutations 28 to evade the humoral immunity generated through vaccination or natural infection or both, T cell 29 immunity remained largely preserved against variants of SARS-CoV-2 (3-6). Importantly, 30 memory T cells' response against SARS-CoV-2 persists for a long time since antigen exposure 31 and thus protects from severe infection upon re-exposure to SARS-CoV-2 and its variants (7). 32 The frequency and magnitude of antigen-specific T cell response could potentially affect the 33 clinical manifestations of . Therefore, it is critical to understand the factors that 34 shape the magnitude of T cell responses during SARS-CoV-2 infections. 35 Antigen-presenting cells (APCs) provide secondary signals in the form of cytokines that are 36 critical in modulating the T cell activation and differentiation (11). However, the phenotype of 37 cytokines secreted by innate immune cells in generating a comprehensive T cell response is not 38 clearly understood in COVID-19. Systems biology studies have shown that proinflammatory 39 cytokines, IL-6, IL-1β, and TNF-α, originating from the SARS-CoV-2 infected lungs, but not 40 from the peripheral blood cells, contribute to COVID-19 severity (12). Therefore, studying the 41 systemic levels of cytokines to predict the T cell response may prove misleading. There are 42 limited reports of cellular innate immune responses and their correlation with adaptive immunity. 43 Few recent longitudinal studies have documented the early innate and adaptive immune 44 responses during mild 14) ; however, no reports identify a correlation of an early 45 innate immune response with the generation of T cell response in active COVID-19. To address 46 this, we used TLR 3, 7, and 8 agonists to stimulate PBMCs isolated within three days of PCR 47 diagnosis of mild-COVID-19 patients and measured the range and magnitude of innate cytokine 48 release, which were further correlated with the evolution of virus-specific T cell response during 49 active Since an extensive interplay is reported between plasma metabolites and immune cell activation 51 in , metabolic analysis in the plasma samples of longitudinal active 52 COVID-19 patients pointed out a set of metabolites significantly correlate with the innate and 53 adaptive immune responses. 54 5 Our results reveal that the antiviral TLR-specific proinflammatory cytokine activity by PBMCs, 55 along with a plasma metabolic signature, works in a coordinated fashion to generate a robust T 56 cell immune response against COVID-19 infection. The graphical abstract was prepared using Biorender.com. correlated with the SARS-CoV-2 specific innate and T cell immune responses (Fig. 1A) . 141 To study the kinetics of the antibody response against the SARS-CoV-2, we first investigated the 142 IgG response against the SARS-CoV-2 RBD protein using Enzyme-linked immunosorbent 143 assays (ELISAs). Anti-RBD (ancestral) IgG was increased significantly (in 85% of the patients) 144 such that the median relative fold change of IgG response increased three-fold by V2 and V3 145 compared to V1 (Fig. 1B) . Anti-RBD IgG response was found to be decreased against delta 146 compared to ancestral RBD proteins, as indicated by their ratio (Fig. 1C-E) . While the median 147 NAb titers increased significantly by V2 and V3 as compared to V1 against the ancestral virus 148 ( Fig. 1F) , NAb titers against the delta were found to be considerably decreased at V2 and V3 as 149 10 compared to the ancestral virus (p<0.0001, Fig. 1H ). We observed that even by V3, only 33% 150 (7/21) individuals had detectable levels of NAbs against the Delta variant ( Fig. 1G) , suggesting 151 that the humoral immune protection against VoCs in active COVID-19 patients infected with 152 ancestral strain is broadly inadequate.

Spike-specific T cell response in COVID-19 patients: 154 Next, we studied the initiation of the antigen-specific T cell response against ancestral, delta, and to the ancestral response, the geometric mean of the AIM+ in CD4+ response was reduced by 190 31% against delta (p=0.0162) and 58% against Omicron spike (p=0.0005) (Fig. 4A) . However, 191 the functional profile remained broadly comparable among the variants (Fig. 4 B-D) . We also 192 did not find any significant differences between the functionality of the antigen-specific T cells 193 as determined by the expression of intracellular cytokines such as IFN-γ, IL-2, and TNFα ( Fig. 4 194 E). A pairwise comparison further revealed that the CD4+ AIM response was preserved entirely 195 in 30% of samples stimulated with delta spike peptides and 15% stimulated with the omicron 196 spike peptides (Fig. 4 F, G) . 197 The geometric mean antigen-specific CD8+ AIM response was reduced by 3 to 5 fold in the 198 delta and omicron variants, respectively (Fig. 4 H) . However, the functional phenotypes of the in plasma samples from V1 as well as V3 (Fig. 7 A-B) . Notably, the metabolites such as 259 pyroglutamate, itaconate, and norvaline which are involved in the glutathione metabolism, citrate 260 cycle, and amino acid biosynthesis, correlated significantly with the early and late-stage immune 261 responses (Fig. 7) . Acknowledgment: 319 We acknowledge the funding through the Mission COVID Suraksha grant 320 (BT/CS0010/CS/02/20) -from BIRAC, Department of Biotechnology, for enabling this study. 321 We thank all the volunteers who donated their blood samples for this study. We sincerely thank 322 the staff and students of the Translational Health Science and Technology Institute (THSTI;   323 Faridabad, India) for helping to ensure the smooth conduct of the study. 

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