Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)-Specific T Cells and Antibodies in Coronavirus Disease 2019 (COVID-19) Protection: A Prospective Study.

BACKGROUND: During the ongoing coronavirus disease 2019 (COVID-19) pandemic, many individuals were infected with and have cleared the virus, developing virus-specific antibodies and effector/memory T cells. An important unanswered question is what levels of T-cell and antibody responses are sufficient to protect from the infection. METHODS: In 5340 Moscow residents, we evaluated anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoglobulin M (IgM)/immunoglobulin G (IgG) titers and frequencies of the T cells specific to the membrane, nucleocapsid, and spike proteins of SARS-CoV-2, using interferon gamma (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assay. Additionally, we evaluated the fractions of virus-specific CD4+ and CD8+ T cells using intracellular staining of IFN-γ and interleukin 2 followed by flow cytometry. We analyzed the COVID-19 rates as a function of the assessed antibody and T-cell responses, using the Kaplan-Meier estimator method, for up to 300 days postinclusion. RESULTS: We showed that T-cell and antibody responses are closely interconnected and are commonly induced concurrently. Magnitudes of both responses inversely correlated with infection probability. Individuals positive for both responses demonstrated the highest levels of protectivity against the SARS-CoV-2 infection. A comparable level of protection was found in individuals with antibody response only, whereas the T-cell response by itself granted only intermediate protection. CONCLUSIONS: We found that the contribution of the virus-specific antibodies to protection against SARS-CoV-2 infection is more pronounced than that of the T cells. The data on the virus-specific IgG titers may be instructive for making decisions in personalized healthcare and public anti-COVID-19 policies. Clinical Trials Registration. NCT04898140.

Interplay between NOD1 and TLR4 Receptors in Macrophages: Nonsynergistic Activation of Signaling Pathways Results in Synergistic Induction of Proinflammatory Gene Expression.

Interactions between pattern-recognition receptors shape innate immune responses to pathogens. NOD1 and TLR4 are synergistically interacting receptors playing a pivotal role in the recognition of Gram-negative bacteria. However, mechanisms of their cooperation are poorly understood. It is unclear whether synergy is produced at the level of signaling pathways downstream of NOD1 and TLR4 or at more distal levels such as gene transcription. We analyzed sequential stages of human macrophage activation by a combination of NOD1 and TLR4 agonists (N-acetyl-d-muramyl-l-alanyl-d-isoglutamyl-meso-diaminopimelic acid [M-triDAP] and LPS, respectively). We show that events preceding or not requiring activation of transcription, such as activation of signaling kinases, rapid boost of glycolysis, and most importantly, nuclear translocation of NF-κB, are regulated nonsynergistically. However, at the output of the nucleus, the combination of M-triDAP and LPS synergistically induces expression of a subset of M-triDAP- and LPS-inducible genes, particularly those encoding proinflammatory cytokines (TNF, IL1B, IL6, IL12B, and IL23A). This synergistic response develops between 1 and 4 h of agonist treatment and requires continuous signaling through NOD1. The synergistically regulated genes have a lower basal expression and higher inducibility at 4 h than those regulated nonsynergistically. Both gene subsets include NF-κB-inducible genes. Therefore, activation of the NF-κB pathway does not explain synergistic gene induction, implying involvement of other transcription factors. Inhibition of IKKß or p38 MAPK lowers agonist-induced TNF mRNA expression but does not abolish synergy. Thus, nonsynergistic activation of NOD1- and TLR4-dependent signaling pathways results in the synergistic induction of a proinflammatory transcriptional program.

The influence of glatiramer acetate on Th17-immune response in multiple sclerosis.

Glatiramer acetate (GA) is approved for the treatment of multiple sclerosis (MS). However, the mechanism of action of GA in MS is still unclear. In particular, it is not known whether GA can modulate the pro-inflammatory Th17-type immune response in MS. We investigated the effects of original GA (Copaxone®, Teva, Israel) and generic GA (Timexone®, Biocad, Russia) on Th17- and Th1-type cytokine production in vitro in 25 patients with relapsing-remitting MS and 25 healthy subjects. Both original and generic GA at concentrations 50-200 µg/ml dose-dependently inhibited interleukin-17 and interferon-γ production by anti-CD3/anti-CD28-activated peripheral blood mononuclear cells from MS patients and healthy subjects. This effect of GA was reproduced using purified CD4+ T cells, suggesting that GA can directly modulate the functions of Th17 and Th1 cells. At high concentrations (100-200 µg/ml), GA also suppressed the production of Th17-differentiation cytokines (interleukin-1ß and interleukin-6) by lipopolysaccharide (LPS)-activated dendritic cells (DCs). These GA/LPS-treated DCs induced lower interleukin-17 and interferon-γ production by autologous CD4+ T cells compared to LPS-treated DCs. These data suggest that GA can inhibit Th17-immune response and that this inhibitory effect is preferentially exercised by direct influence of GA on T cells. We also demonstrate a comparable ability of original and generic GA to modulate pro-inflammatory cytokine production.

Glycolytic reprogramming of macrophages activated by NOD1 and TLR4 agonists: No association with proinflammatory cytokine production in normoxia.

Upon activation with pathogen-associated molecular patterns, metabolism of macrophages and dendritic cells is shifted from oxidative phosphorylation to aerobic glycolysis, which is considered important for proinflammatory cytokine production. Fragments of bacterial peptidoglycan (muramyl peptides) activate innate immune cells through nucleotide-binding oligomerization domain (NOD) 1 and/or NOD2 receptors. Here, we show that NOD1 and NOD2 agonists induce early glycolytic reprogramming of human monocyte-derived macrophages (MDM), which is similar to that induced by the Toll-like receptor 4 (TLR4) agonist lipopolysaccharide. This glycolytic reprogramming depends on Akt kinases, independent of mTOR complex 1 and is efficiently inhibited by 2-deoxy-d-glucose (2-DG) or by glucose starvation. 2-DG inhibits proinflammatory cytokine production by MDM and monocyte-derived dendritic cells activated by NOD1 or TLR4 agonists, except for tumor necrosis factor production by MDM, which is inhibited initially, but augmented 4 h after addition of agonists and later. However, 2-DG exerts these effects by inducing unfolded protein response rather than by inhibiting glycolysis. By contrast, glucose starvation does not cause unfolded protein response and, in normoxic conditions, only marginally affects proinflammatory cytokine production triggered through NOD1 or TLR4. In hypoxia mimicked by treating MDM with oligomycin (a mitochondrial ATP synthase inhibitor), both 2-DG and glucose starvation strongly suppress tumor necrosis factor and interleukin-6 production and compromise cell viability. In summary, the requirement of glycolytic reprogramming for proinflammatory cytokine production in normoxia is not obvious, and effects of 2-DG on cytokine responses should be interpreted cautiously. In hypoxia, however, glycolysis becomes critical for cytokine production and cell survival.

Synergistic interactions between NOD receptors and TLRs: Mechanisms and clinical implications.

Interactions between pattern recognition receptors (PRRs) shape innate immune responses to particular classes of pathogens. Here, we review interactions between TLRs and nucleotide-binding oligomerization domain 1 and 2 (NOD1 and NOD2) receptors, two major groups of PRRs involved in innate recognition of bacteria. Most of experimental data both in vitro and in vivo suggest that NODs and TLRs synergize with each other at inducing the production of cytokines and antimicrobial peptides. Molecular mechanisms of this synergy remain poorly understood, although several scenarios can be proposed: (i) direct interactions of signaling pathways downstream of NODs and TLRs; (ii) mutual transcriptional regulation of unique components of NOD-dependent and TLR-dependent signaling pathways; and (iii) interactions at the post-transcriptional level. Potential practical implications of NOD-TLR synergy are dual. In sepsis, where synergistic effects probably contribute to excessive proinflammatory cytokine production, blockade of NOD1, and/or NOD2 in addition to TLR4 blockade may be required to achieve therapeutic benefit. On the other hand, synergistic combinations of relatively small doses of NOD and TLR agonists administered before infection could be used to boost innate resistance against bacterial pathogens.

Reduced degranulation of NK cells in patients with frequently recurring herpes.

NK cells lyse virus-infected cells by degranulation; however, alterations in NK cell degranulation in persistent viral infections have not been directly studied. Earlier reports have documented a decrease in NK activity in patients with frequently recurring herpes (FRH). We corroborate these findings by showing that the degranulation responses of blood NK cells from patients with FRH, both during relapse and during remission, are significantly lower than those in healthy donors. The impaired degranulation was probably not caused by defective target cell recognition, since it was observed upon stimulation both with K562 cells and with a receptor-independent stimulus (phorbol 12-myristate 13-acetate plus ionomycin). We also show that the intracellular expression of perforin and CD107a by NK cells from patients with FRH is not different from that in healthy donors, thus excluding that the low NK cell degranulation in FRH is caused by a smaller size of the lytic granule compartment. We confirm previous reports on lowered NK activity in FRH patients and show that NK activity is significantly impaired only during remission, but not relapse; the causes for the discrepancy between the low degranulation and "normal" NK cell activity during relapse are discussed. In all, these data point at the deficit of NK cell degranulation in FRH. Whether this is a predisposing factor or a consequence of herpes simplex virus infection requires further investigation.